Structure-Function Relationships of the Cytoplasmic Domains of the Cystic Fibrosis Transmembrane Conductance Regulator

Fabian S. Seibert

A thesis submitted in confonnity with the requirements for the degree of Doctor of Philosophy, Graduate Department of Eiochemistry, University of Toronto.

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The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permetbat à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microfom, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfichelnlm, de reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author' s ou autrement reproduits sans son permission. autorisation. The cystic fibrosis transrnembrane conductance regulator (Cm)is a chloride chamel, located in the apical membrane of wet epithelia. Mutations in the gene coding for CFTR result in cystic fibrosis (CF) either by disruptiag the biosynthetic processing of the protein or through the production of a functionally irnpaired channel. When studying CFTR. the molecule traditionally has been dissected into three functional units: (i) the dodeca- helical transrnembrane pore that fomthe ion conductive pathway, (ii) two cytoplasrnically located nucleotide binding folds which hydrolyze ATP to open as well as to close the channel. and (iii) a cytoplasmically located R-domain, the phosphorylation of which is required to put CFTR into an activation competent fom. The major base phosphorylating CFTR is the CAMP-dependent protein kinase (PKA). Upon hormonal induction, PKA phosphorylates several sites within the R-domain, thereby allowing a graded response to various levels of stimulation. Thus far, only the importance of perfect dibasic consensus sites for potential interactions with PKA ha been investigated in this mechanism. The present study demonsmtes that PU-mediated phosphorylation under lirniting conditions also occurs on monobasic consensus sites and contributes to CFTR activation. Additional phosphorylation is shown to be accepted by sites that lack a positively charged residue in close physical proximity. To obtain an initial idea of associations between the three functional uni& of CFTR, this thesis also addresses the importance of the previously uninvestigated cytoplasmic loops (CLs) that connect the transrnembrane helixes on the cytoplasrnically exposed side of the pore-forming unit. Reconstruction of CF-associated point mutations in al1 four CLs illustrates that many of the amino acid substitutions inhibit maturation of Cmsuggesting that correct foiding of the loops may be critical for achievernent of the correct overall conformation of the protein. In addition, the CLs contribute to CFTR function since mutations in both CL1 and CL3 significantly alter the open probability of the chloride channel. However, the loops are functionaily distinct since CL1 mutations modify the mean closed tirne of the cbannel, whereas CL3 aiterations mainly affect the mean open time. The fact that mutations in the loops influence the gating of CFTR, rather than its conductance, may suggest that despite their proximity to the pore entrante, the importance of the CLs lies in relaying regdatory stimuli to the pore as opposed to an involvement in actual ion movement. Fabian S. Seibert Department of Biochernistry University of Toronto 1997

Thesis Title: Structure-Function Relationships of the Cytoplasrnic Domains of the Cystic Fibrosis Trammembrane Conductance Regulator 'Jeder Tag ist ein neuer Anfang, ein Morgen voiler Waerme.'

Br. lmmanuel Jakobs

'Each day brings a new dawn.' ACKNOWLEDGMENTS

Bavaria and Austria are linked by a graceful mountain named Untersberg. Should you ever visit Untersberg, you may climb one side of the mountain, cross a long plateau with beautiful alpine fauna and flora, and then descend on the opposite side through a steep, wet cave. This is the most întimidating part of the tour, but once greeted by the sunlight, you are rewarded with a rnagnificent view of the Northem Alps. When my

father and 1 took this trip, we stopped on the plateau and talked about life. Maybe 1 have

reached that sarne point in my studies, so 1 would Iike to sit dom and Say thank you to everybody who has made this possible. 1 am especially grateful to my supervisors, Dr. David Clarke and Dr. Jack

Riordan, for their excellent guidance, continued support, and much patience. With his balanced, calm, but determined approach to life, Dr. Clarke never failed to disperse rny oh-so-fiequent womes. I especially know to appreciate that Dr. Clarke always looked out for me and ensured that things happened in such a way that they were beneficial to me and to my future. Dr. Riordan first introduced me to this tield of research, for which he radiates great enthusiasm. Dr. Riordan's excitement about rny data was highly rewarding

and helped to push on. Both supervisors gave me much fieedom in my approach to the

addressed problems, but at the same time, they were always willing to provide superb advice on theory and techniques. 1 was very fortunate to be allowed to see two quite different approaches to science and hope to be able to build on both. Thank you, David, thank you, Jack, for everything!

An invaluable element towards the completion of this work was Dr. Tip Loo. 1 would Iike to thank Dr. Loo for many helpful discussions and for patiently giving expert advice. In molecular biology problems anse fiequently, but have no fear, there is Dr. Loo. Also. I now believe that the 'just do it' approach really works. Over the years 1 had many colleagues whose humor and advice were a tremendous help. Cheryl Ho was a wonderful lab-mate who trîed to make sure that 1 kept life in perspective and who was always willing to assist. Dr. Xiu-Bao Chang showed me how to cut a plasmid, Dr. Zbysko Gnelczak taught me the G-A-T-C's of CFTR,and Tim Jensen first htroduced me to phosphorylation experiments. Dr. Gergely Lukasc, Dr. Abdalla Mohammed, and Tom Singer gave me the illusion that 1 was something like a real scientist by fiequently discussing their hdings with me and Dr. Gretchen Kiser is the most cheerful penon there is in the CF field. Many thanks also to Noa Alon, Dr. Richard Callaghan, Dr. Am Duhanty, Barbara Greis, Yue-Xian Hou, Dr. Norbert Kartner, Dr. Mohabir Ramjeesingh, Monique Sapiano, Rose Templeton, Dr. Iris Ziegler, and our lab- neighbors. I want to express my gratitude to Dr. John Hanrahan for allowing me to visit his laboratory and to Dr. Yanlin Jia, Dr. Paul Linsdell, Dr. Ceri Mathews, and Joe Tabcharani for giving me the very basic feel that 1 have for the patch-clarnping technique and for showing me what Montreal is al1 about.

Thank you ver-much, also, to my cornittee members, Dr. Reinhart Reithmeier and Dr. David Williams, for al1 their advice and especially for improving this thesis by critically and thoroughly reviewing it.

Finally, 1 want to Say a very special thank you to my parents and to my brother Peter. My family was the one consistent point of reference over the last few years. They were always there for me to share the ups and to buffer the downs. 1 defmitely lucked out to be part of this family. WelI, it's time to head for the cave! TABLE OF CONTENTS

ABSTRACT

TABLE OF CONTENTS

LIST OF FTGURES

LIST OF TABLES

LIST OF AIBBREVIATIONS XIV Short-forms xiv Legend of Phosphorylation Mutants xv

CHAPTTER 1: INTRODUCTION

Cystic Fibrosk (CF) Symptoms of the Disease Ainvays Pancreas Other Organs Treatments CF - a Disease of Altered Fluid Transport Physiology of the Sweat Gland Physiology of the Ainvays Search for the Biochemical Defect in CF

The CF Gene and its Gene Product Identification of the Gene Affected in CF The Gene Product - CFTR Predictions of Topology ABC-superfamily of Proteins Function of the Gene Product Conductance Properties of CFTR Expression of CFTR

Regdafion 4 CFTR Regulation by Phosphorylation CAMP-dependentprotein kinase (PU) Protein Kinase C (PKC) Additiond Kinases Phosphatases The R-domain Regulation by ATP Nucleotide Binding Folds Biochemical and Functional Findings Models of Regdation by ATP Evidence to Support ATP Models Reconciling Two Modes of Regulation

Targeting and Processing of CFTR The Phe 508 Deletion Mutation Inefficient Processing of CFTR Processing Characteristics Chaperones Degradation of CFTR Rescue of Misprocessed Molecules Synthesis of the CFTR Protein Apical Processes in CFTR Expression Apical Targeting Recycling and Recniitment

Additional Functions of the CFTR Molecule Classes of CF-causing Mutations Regulation of Sodium Conductance Regulation of the Ou~dRectifier History Activation of the Outward Rectifier Is CFTR an ATP Channel? Role in Mucin Secretion Additional Proposals

CHAITER 2: MATERIALS AND METHODS Construction of Vectors and Mutants Expression of Mutants Temperature Shift and Glycerol Exposure Protein Detection Endoglycosidase H Digestion Phosphorylation of CFTR Cyanogen Bromide Cleavage of Phosphorylated CFTR Antibody Recognition of Cyanogen Bromide Cleavage Products Ce11 Surface Labeling CFTR Proteolysis in Membrane Vesic les Iodide Efflwr Studies Patch-clamp Studies of CFTR-Expressing CHO Cells

CHAPTER 3: PKA-MEDIATED PHOSPHORYLATION OF CJTR RESIDUE SER 753 AND ITS ROLE IN CHANNEL ACTIVATION

Chapter Summary

Introduction

Results and Discussion Expression Studies 1OSA-CFTR is still Phosphorylated by PKA Localization of PKA Phosphorylation in I OSA-CFTR Mutagenesis of Possible Phosphorylation Sites within the 5.8 kDa Segment Evaluation of the Functional Effect of Ser 753 Mutagenesis

CHAPTER 4: MONOBASIC CONSENSUS SITES FOR PKA-MEDIATED PHOSPHORYLATION OF CFIX

Chapter Summary

In troduction

Results and Discussion Ongin of the -9 kDa CNBr Cleavage Product (Band 2) Mutagenesis of Monobasic Consensus Sites for PKA Interactions in the R-domain 122 Processing Characteristics of I SSA-CFTR 122 Labeling Characteristics of 15 SA-CFTR 125 Anion Permeation of ISSA-CFTR Expressing BHK Cells 130 Monobasic Consensus Sites for Interaction with PKA in NBFl 137

CHAPTER 5: DISEASE-ASSOCIATED MUTATIONS IN THE FOURTH CYTOPLASMIC LOOP OF CFïR COMPROMISE BIOSYNTHETIC PROCESSING AND CHLORIDE CHANNEL ACTIVITY 142

Chapter Summury 143

Resuh and Discussion Identification of Processing Mutants in CL4 of CFTR Functional Analysis of CLA Mutants by Iodide Efkx Patch-clamp Analysis of CL4 Mutants CHAPTER 6: CYTOPLASMlC LOOP THREE OF CFïR CONTRIBUTES TO REGUlLATION OF CHLORIDE CHANNEL ACI'IVITY

Chaptm Summury

Resulrs and Discussion Rocessing Muîants in CL3 Evaluation of CL3 Mutations by Iodide Efflux Mutationai Analysis of Residue Gly 970 Patch-clamp Analysis of CL3 Muîants Does the Rdomain Play a Role in Observed CL3 Effects?

C-R 7: N-TERMINAL CYTOPLASMIC LOOPS OF CFTR. WHAT DO THEY DO?

Results and Discussion Biochemical Analysis Functional Analysis Correlation of Observed Effect to Patient Phenotype

CHAPTER 8: DISCUSSION AND FUTURE DIRECTIONS

Discussion

Future Directions Phosphoiylation Cytoplasrnic Loops

REFERENCES LIST OF FIGURES

Fig. 1.1. Composite mode1 of ainvay epitheliai ion transport. 12 Fig. 1.2. Predicted topology of Cm 17 Fig. 1.3. Schematic representation of dibasic consensus sites for interaction with PKA. 28 Fig. 1.4. Proposed mode1 of CITR regdation.

Fig. 3.1. Promotion of CFTR expression with heavy metals and sodium butpte. Fig. 3.2. Detection of phosphorylation of 1OSA-CFTR by PKA. Fig. 3.3. In vitro PKA phosphorylation of CFTR harvested from SF9 insect cells. Fig. 3.4. Localization of residual 1OSA-CFTR phosphorylation by CNBr cleavage. Fig. 3.5. Identification of the site phosphorylated in the 5.8 kDa segment. Fig. 3.6. Functional evaluation of 1 1SA-S753A-CFTR. Fig. 3 -7. Anion conductance of l6SA-CFTR expressing cells.

Fig. 4.1 . CNBr cleavage of R-domain deletion mutants. Fig. 4.2. Schematic of monobasic consensus sites in the R-domain. Fig. 4.3. Expression of 15SA-CFTR. Fig. 4.4. CNBr cleavage of CFTR phosphoproteins. Fig. 4.5. Functional analysis of ISSA-CFTR by iodide efflux. Fig. 4.6. Application of an inactive forskolin analogue to the iodide efflux. Fig. 4.7. Functional analysis of 1SSA-CFTR by single-channel patch-clamping. Fig. 4.8. Study of monobasic PKA consensus sites in NBF 1.

Fig. 5.1. Simplified representation of CF-associated point mutations identified in CL4 148 Fig. 5.2. Evaluation of mutations in CL4 regarding their effect on processing. 150 Fig. 5.3. Effect of temperature shifi on processing of CL4 mutants. 156 Fig. 5.4. Evaluation of mutations in CL4 by iodide efflux. 159 Fig. 5 S.Single-channel activity associated with processed CL4 mutants. 162 Fig. 5.6. Mean kinetic properties of single CUmutant channels. 164

Fig. 6.1. Schernatic representation of CL3 mutations within CFTR. Fig. 6.2. immunoblots of CL3 mutants. Fig. 6.3. Temperature- and glycerol-sensitivity of CL3 mutants. Fig. 6.4. Tryptic cleavage of membrane embedded CL3 mutants. Fig .6S.Deglycosylation/regiycosylation of CL3 variants. Fig. 6.6. Functional evaluation of CL3 mutants by iodide efflux. Fig. 6.7. Surface labelling of CFTR expressing cells. Fig. 6.8. Iodide efnux measurements of cells expressing CFTR Gly 970 mutants. Fig. 6.9. Mean kinetic properties of single CL3 mutant channels. Fig. 6.10. Chlonde conductance properties of single CL3 mutant channels. Fig. 6.1 1. CL3 mutations in the 1 1S A-CFTR background.

Fig. 7.1. Schematic representation of CL 1 & 2 mutations. 205 Fig. 7.2. Processing characteristics of CFTR carrying CL 1 & 2 mutations. Fig. 7.3. Surface labeling of CL 1 & 2 CFTR variants Fig. 7.4. Temperature sensitivity of CL 1 & 2 CITR variants. Fig. 7.5. Fundional evaluation of CL 1 & 2 CFTR variants by iodide efnux. Fig. 7.6. Single channel activity associated with processeci CL 1 & 2 mutants. Fig. 7.7. Mean kinetic properties of single CL 1 & 2 mutant charnels. LIST OF TABLES

Table 1.1. Organs aEected in CF patients. 6 Table 1.2. Observed phosphorylation profiles of CFTR residues by PKA and PKC in vih-O and in vivo. 3 1

Table 5.1. Summary of processing characteristics of CL4 mutants venus the patient phenotype. 153

Table 7.1. Processing characteristics of CL 1 & 2 mutants versus patient phenotype. 222 LIST OF ABBREVIATIONS

ABC ATP-binding cassette proteins ALLN N-acetyl-L-leucinyl-L-1eucinyl-l-norleucinal AMP-PNP 5 '-adenosine( P,y-imin0)triphosphate ASF ainvay surface fluid BHK baby hamster kidney CAMP cyclic 5 '-3 ' adenosîne monophosphate CD circu1a.r dichroism CF cystic fibrosis CFTR cystic fibrosis transmembrane conductance regulator cGK cGMP-dependent protein kinase cGMP cyclic GMP CHO Chinese hamster ovary CL cytoplasmic loop CM centi Morgan CNBr cyanogen bromide DrnS 4.4'-diisothiocyanatostilbene-2'2'-disulfonic acid DMSO dimethylsuIfoxide DPC diphenylamine-2-carboxylate EL extracellular loop ENaC amiloride-sensitive sodium channel ER endoplasmic reticulum JBMX 3-isobutyl- 1-methyl-xanthine 1-v current-voltage relationship MDCK Madin Darby canine kidney 8-NjATP 8-azidoadenosine S'-triphosphate NBF nucleotide binding fold ORCC outward rectifier Po open probability PCR polyrnerase chah reaction PD potential difference PI pancreatic insufficient PKA CAMP-dependent protein kinase PKC protein kinase C PMA phorbol myristate acetate PS picos iemen PS pancreatic suficient PVDF polyvinylidene difluoride SDS sodium dodecyl sulfate PAGE polyacrylamide gel electrophoresis

xiv SPQ 6-methoxy-N-(3-suifopropyl)-quin0liaium SRP signal recognition particle TET metal-tetracycline~antiporter TM transmembrane helix

B. Legend of Phosphorylation Mutants:

1 OSA changes Ser or Thr of al1 10 dibasic PKA-consensus sites to Ala (9 are in R-domain, one just N-terminal to NBFI) in wild-type background

11SA changes S753 to Ala in 1OSA background

16SA changes al1 Ser and Thr of 5.8 kDa CNBr fiagment of the R-domain to Ala in l OSA background

15SA 1 1 SA plus 4 monobasic PKA-consensus sites of the R-domain changed to Ala (S670A, T690A, T787A, S790A)

WT4SA changes four potential phosphorylation sites in NBF 1 to Ala in wild-type background (S466/485/489/557A)

1 1SA4SA S466/485/489/557A in 1 I SA background

WT-4SE S466/485/489/5 57E in wild-type bac kground

8SE-3SE S466/485/489/557E in 8SE background

8SE changes Ser of 8 R-domain located dibasic PKA-consensus sites to Glu in wild-type background CWTER 1

Introduction Cystic Fibrosis (CF')

Cystic fibrosis (CF) is the most common fatal genetic disease among Caucasians

with an incidence of one in every 2000 live births (Quinton, 1990). Every twentieth person is a CF carrier, however, heterozygotes are asymptomatic. CF is seldomly found in blacks and almost never in Asians (Rubin and Farber, 1994).

Symptom of the Disease In 1913 Garrod and Hurley reported a consanguineous English farnily in which several children showed steatorrhoeal and died of bronchopneumonia2. Today, this report is considered to be one of the first documented case descriptions of CF, although numerous references to infants with meconium ileus and children with CF-like pancreatic and lung disease are sprinkled throughout the literature fiom as early as 1650 (Boat et al., 1989). Interestingly, Garrod and Hurley (19:2) suggested that the described defect was

inherited in an autosomally recessive manner, a theory which was disputed quite strongly

until the autosomal recessive inheritance pattern of CF could eventually be delineated unequivocally through extensive pedigree studies. Initial comprehensive descriptions of the disease were provided by Fanconi et al. (1936) and Anderson (1938) who combined

case histories and autopsies to characterize symptoms of CF and the changes it produces in various organs. Based on the microscopie features of the pancreatic tissue Anderson

( 1938) gave the disease its name, calling it 'cystic fibrosis of the pancreas'. In the decades

that followed. the clinical description of CF has become more complete as summarized

by Boat et aI. ( 1989). Ainvqs - CF is often categorized as a polyexocrinopathy (Davis and di

Sant' Agnese, 1980) because defects are expressed predominantly in exocrine organs. The sites of severest consequence are the airways, with respiratory tract disease accounting for

' Fat indigestion, resulting in passage of fat in large amounts in the feces. Acute inflammation of the walls of the mialler bronchial tubes. with ineguiar areas of consolidation due to spreading of the inflammation into peribronchiolar aheoli and the alveolar ducts. three-quarters of al1 hospital admissions and more than 90% of the morbidity and mortality in CF (Penketh et al., 1987). The lungs of CF infants appear to be structurally normal, but soon afier birth, microbial infection and build-up of thick mucus are detected (Bedrossian et al., 1976). Although this observation has ken long established, it is still unclear whether infection precedes or is a consequence of mucus build-up (Waltner et al., 1987). Generally, the initial invader is StuphyIococcus aureur, which is followed - predominately by Pseudomonas aeruginosa adherence, but other organisms can be involved as well (Warner, 1992). Bacterial infection extending beyond the ainvays is distinctly uncornmon, so that local rather than general host defense mechanisms must be compromised in the lung (Boat et al., 1989). Generally, chronic infections with the associated dammation lead to progressive loss of lung function due to endobronchitisJ. bronchiectasisJ, and parenchyma15 destruction, although a wide range of severity is observed for the pulmonary malfunction of CF patients (Quinton, 1990; Biner. 1995).

The DNA released during ce11 lysis of the inflarnmatory response will continuously enhance the viscosity of the mucus. With increasing disease, ventilation perfusion irnbalance occurs leading to hypoxia6 and udtimately pulrnonary hypertension7 (Ryland and Reid. 1975). In older patients with severe disease, bronchial artery hypertrophy8 may produce massive pulmonary haemorrhage (Hodson and Wamer, 1992). Pancreas - Pancreatic ductules are blocked by inspissations. Initial luminai obstruction and dilation of ~5esecretory acini are followed in later stages by atrophy of the exocrine glands and progressive fibrosis. The inability to release pancreatic enzymes causes maldigestion of protein and fat so that stools are bulky and foul-smelling. If

inflammation of the rnucous membrane of bronchial tubes. ' Cbronic dilation of bronchi or bronchioles as a sequel of inflammatory disease or obstruction 5 Parenchyma are the distinguishing or specific cells of a gland or organ, contained in and supported by the comective tissue hmework. Decrease below nomal levels of oxygen in blood or tissue. 7 High blood pressure in the pulrnonary circuit. ' Greater bulk through increase in six, but not in number. untreated, affected individuals fail to gain weight and growth is inhibited (Bone, 1996). The endocrine pancreas is af5ected to a smaller extent. However, the incidence of glucose

intolerance increases with age. The pancreatic status of CF patients falls into two distinct groups - pancreatic sufficient (PS) patients do not require supplernentation with digestive enzymes, pancreatic insufficient (PI) patients do. There is a general notion that pancreatic sufficiency reflects a less severe forrn of CF (Kerem et al., 1989; Welsh and Smith. 1993). Other Orgum - In most organs afTected by CF, a recurring theme is obstruction of passages by mucus, despite increasïng evidence that the mucus glands themselves are morphologically normal before onset of the secondary effects of the disease @avis and di Sant'Agnese, 1980). Although on autopsy signs of the disorder generaiiy can be seen in organs other than lungs and pancreas, clinical manifestations involving these organs occur in only 5% of al1 patients (Rubin and Farber, 1994). Affected organs and the resulting complications are summarized in Table 1.1. In addition, in 10% of al1 CF infants small bowel obstruction, known as meconium ileus, is observed, which results in failure

to pass the intestinal contents that have accumulated Ni utero in the immediate postpartun periad. This is generally not due to malfunction of the intestine itself but rather due to

failure of pancreatic enzyme secretion and Iack of digestion of intraluminal contents in

urero (Boat et al., 1989). Although intestinal problems are fairly uncornmon later on in

life, it has been suggested that an intestinal effect may be responsible for the hi& occurrence of the disease. Possibly, CF heterozygotes withstand secretory diarrhea better than normal individuals, thus having a selective advantage (Gabriel et al., 1994). However, experimental evidence to support this theory is limited (Cuthbert et al., 1995). An almost ubiquitous fmding is male infertility due to occlusion of Wolffian ducts, resulting in atrophy of the vas deferens (Valman and France, 1969; Wilschanski et al., Table 1.1. Organs affected in CF patients. Most complications arise fiorn CF-assriciated phenotypes of the lungs and the pancreas. Clinicai manifestations involving other organs occur in only 5% of the patient population. For an extensive review of the various disease symptoms refer to Boat et al. (1989). Definitions of disease syrnptoms were taken fiom

Stedman (1984). Biliary Cirrhosis: Progressive disease of the liver characterized by diffise damage to hepatic parenchyrnal cells, due to biiiary obstruction, with nodular regeneration, fibrosis, and disturbance of oormai architecture, resulting in disturbance of blood flow. Periportal: Surroundhg the portal vein. Hyperbilirubinemia: An abnormally large amount of bilimbin (red bile pigment, formed fiom hemogiobin during erythrocyte destruction) in the circulating blood, resulting in jaundice (yellow staining of tissues). Ascite: Accumulation of serous fluid in peritoneal cavity. Pentoneurn: Serous sac that lines abdominal cavity and coven most viscera contained therein. Edema: Accumulation of watery fluid in tissue. Squamous Metapiasia: Transformation of glandular and mucosal epithelium into stratified squamous epithelium. Microscopie Features Pathology

Microbial Acinar and Ductal Dilation infection Infiammation Mucus Block of Endobronchitis Conducting Bronchiectasis Airways Parenchymal Destruction Ventilation Pehion Irnbaiance Hypoxia Pulmonary Hypertension . . Mucus BIock of Dilation of Secretory Acini Pancreatic Atrophy of Exocrine Glands Ductules Progressive Fibrosis Malnutrition due to Inability to Release Pancreatic Enmes Mucus Block of Biliq Cirrhosis Bile Caniculi Periportal Inflammation Septal Fibrosis - Multilobular Appearance

Ascites Peripheral Edema Duodenum Mucus Block of No Complications l--- Brunner's GIands Male Mucus Block of Atrophy of Vas Deferens Reproductive Wolffian Ducts Infertility Tract Chemical Modifications of Semen Female Abnonnally Thick Reduced Fertility Reproductive Mucus Plug of Tract Cervical Os Anovulatory Cycles Salivary Glands None Observed Progressive Dilatation of Ducts Squamous Metaplasia of Lining Epitheliurn Glandular Atrophy 1 Fibrosis 1996). Fertility in fernales is also reduced because of anovdatog cycles and an abnormally thick and tenacious mucus plug of the cervical os (Kopito et al., 1973). Nonetheless, despite the multifaceted expressions of the basic genetic defect, were it not for the persistent, secondary pulrnonary infections, CF would not be a fatal disease (Quinton, 1990). Treutments - in the beginning of this century, most CF children did not live to celebrate their first birthday. Since then, various treatments were developed that greatly improved both quaIity of life and life expectancy of CF patients. Infections in the lungs initially can be controlled to some degree with high levels of antibiotics which are generally delivered intravenously (Boat et al., 1989) or in the form of aerosols. In the C latter case, careful hygiene of inhalation apparatuses is crucial (Schoen and Werner, 1991). Mucus build-up is relieved with ainvay clearance techniques (Welch and Smith, 1995) and can be made less viscous through the application of aerosolized DNase (Aitken. 1993; Bryson and Sorkin, 1994). Furthemore. various trials are underway to address / prevent lung damage with anti-inflammatory drugs or P. aeruginosa vaccines. Proper nutrition (Durie and Penchm, 1992) and supplementation of pancreatic enzymes and vitamins have eliminated pancreatic problems, and complications in al1 other organs can be treated with the same methods as used for non-CF patients with similar problems (Meams, 1993). Because of al1 these treatrnents, the average life expectancy of CF patients today is close to 30 pars (Quinton, 1990). However, this increase has presently reached a plateau and the fact is that every day three patients with CF die (Fuller and Benos, 1992). To change this, treatment strategies have to evolve fiom dealing with secondary problems to addressing the primary defect. As the following sections will show, the search for this defect was challenging but finally successful, and provided new options for therapy.

' Not relafed to or coincidental with ondation. CF - a Dlsease of ALtered Fluid Transport Since CF is inherited in an autosomally recessive fashion, the disease should result fiom alteration@) in a single gene product. However, the wide range of symptoms associated with CF made it difficult to pinpoint a single biochemical defect which would guide in the search for the mutated gene. One of the fmt clues to the nature of the cellular fault in CF came during a heat wave in New York in 1953. di Sant'Agnese and colleagues (1953) observed that CF children become dehydrated more readily than unaffected children. The dehydration is the result of NaCl depletion due to elevated sweat sodium and chloride. It was demonstrated that vimially al1 CF patients have three- to five-fold the normal concentration of sweat NaCl. Based on this observation Gibson and Cooke (1 959)

developed a sweat test that stimulates local sweat secretion utilizing pilocarpine and analyzes the chloride content. Although other tests have been developed to screen for CF candidates, the sweat chloride test is still the most reliable and most widely used diagnostic method. Physiology of the Sweat Gland - The altered sweat composition is the most consistent functional defect observed for CF patients. Eccrine sweat glands contain no mucus build-up and have no stmctural abnormality (Bonc, 1996), thus providing compelling evidence that CF is an inherited disturbance in fluid transport (Quinton, 1990). initial attempts to explain the salt elevation in CF sweat focused on the study of altered sodium transport, in accordance with the prevailing view at the time of chloride being passively distributed for charge compensation of sodium transport. Little attention was paid to the increased sweat chloride concentration until Quinton (1983) revolutionized preformed notions by demonstrating that the primary defect in CF sweat glands is a decreased chlonde conductance. In the sweat gland, an isoosmotic fluid is secreted in the secretory coi1 fiom which electrolytes are absorbed in excess of water in the reabsorptive duct to produce hypotonic sweat. Using microdissected reabsorptive ducts in isolation, Quinton (1983) observed under conditions of symrnetrical, isotonic Ehger solutions the potential difference (PD) in control ducts to be -6.8 mV relative to the serosal bath in contrast to -76.9 mV in CF ducts. The PD represents the product of ion transport rates (ionic currents} and epithelial resistance (a function of tight junctional and ce11 membrane components) (Boucher, 1994). To explore the variables that control the magnitude of the PD, the chionde in the perfusate of control tissues was replaced with impermeable sulfate. When this was done, the PD rose to -75.5 mV, which was almost identical to the PD observed in CF tissues. Therefore, the differences between control and CF ducts cm be accounted for by a decreased permeability of CF ducts to chioride. Later, more direct conductancelresistance studies utilized the sodium channel blocker amiloride and gluconate replacement to demonstrate that, in the normal duct, 85-90% of the electrical conductance is carried by chlotide and that this electrodiffusive shunt is aimost completely absent in CF ducts (Quinton, 1986; Bijman and Fromter, 1986). Based on these fmdings, it was proposed that in the normal sweat duct sodium is removed fkom the ce11 by the basolaterally located sodium/potassiurn-AïPase. Sodium follows the formed electrochemical gradient and moves into the ce11 on the apical side, with chloride passively following the entire movement first through the apical membrane and then through the basolateral membrane. In CF, the chloride movement is inhibited, which means that the apical membrane potential is depolarized, thereby reducing the electrochemical driving force for sodium entry into the cell. Overall NaCl absorption in the duct is retarded (Quinton, 1990). It was soon suggested that the lack of chloride conductance is related to an abnormal response to P-adrenergic stimulation because Sato and Sato (1984) were able to stimulate fluid secretion in normal sweat glands with isoproterenol, but not in CF glands. Cholinergie and a-adrenergic responses are unaltered. Physiology of the Ainvoys - In the ainvays, similar fmdings were obtained as in sweat ducts. When comparing lumen relative to blood, the PD of pulmonary CF epithelia is twice as electronegative as in normal epithelia (Knowles et al., 1981), and the elhination of sodium currents with amiloride demonstrated that the chionde permeability of CF tissues is one half that of normal tissues (Knowles et al., 1983). Using dual measurement on a suspended epithelium, Cotton et al. (1987) refined these findings and showed that the PD difference between CF and normal tissues occun primarily at the level of the apical membrane, with linle differences on the basolateral side. Furthemore, it was seen that in healthy tissues more than 95% of the short-circuit curent is amiloride sensitive, indicating that the major movement in ainvay tissues is sodium absorption (Boucher et al., 1988) (Fig. 1.1 gives a composite model of ainvay epithelial transport). It is intriguing and counter-intuitive that the decreased chloride permeability of CF ainvays subsequently was fomd to result in an increase in absorptive sodium fluxes and an increase in the absorption of NaCl (Boucher et ai., 1986; Cotton et al., 1987; Boucher et al., 1988), thus having the opposite effect than in the sweat duct. A model was proposed for the lung epithelium in which chloride conductance across the apical membrane is lost. However, an apparent increase in sodium permeability was then suggested (and later confirmed experimentally (Boucher, 1994)) to enhance apical sodium entry over normal. Chlonde absorption must occur passively via a paracellular path, because decreased transcellular chlotide permeability does not impede net NaCl absorption in CF (Quinton.

1990). In pulmonary tissue, again a lack of response to P-adrenergic stimulation was observed, despite an unaffected CAMP increase (Widdicombe, 1986). These findings set the stage for future CF investigations. However, the understanding is still very basic and numerous unanswered questions remain. Intuitively, the increased NaCl absorption in CF lungs suggests that the NaCl concentration in CF airway surface fluid (ASF) should be lower than in normal lungs, but the opposite was indicated to be the case1' (GiIIjam et al., 1989; Joris et al., 1993). Possibly this is a

IO Note that experiments of the previous paragaph investigated Na' and Cl- fluxes, rather than absolute NaCI concentrations of the ASF. Fig. 1 .l.Composite model of aimay epithelial ion transport. The model depicts the present understanding of ion rnovement in normal and CF proximal human airways. It has to be noted that the most si@cant loci of the disease are the more distal regions of the airways, but due to technical difficulties most preliminary investigations are conducted in the proximal areas. CF patients demonstrate an increased re-absorption of sodium 5om the airway lumen. Chloride ions follow the established electrochernical gradient via a paraceliular pathway. Ion channels are depicted as open hatches, antiporters/cotransporters as circles with arrows, and the sodiurn/potassium pump as a cide with a '-'. Adapted from Boucher (1 994)-

reflection of the fact that in the rnovement of ASF fiom alveoli to proximal areas of the ainvays, large quantities of water and electrolytes have to be absorbed and that the exact mechanisms and relative amounts are very poorly realized (Boucher, 1994); for later sîudies, it will be extremely important to clarify this basic question of 'ion status' in CF disease. Despite a lack of comprehension, the increased NaCl concentration may represent a very important finding since recent data demonstrated that bactericida1 killing of infecting organisms requires low NaCl concentrations and can be stimulated on CF

epithelia by reducing the NaCl concentration (Smith et al., 1996). The understanding of electrolyte events in CF lungs has also opened Merpossible avenues for treatrnent. To obtain normal electrolyte levels, aerosolized amiloride may be beneficial in reducing the increased sodium absorption. This could be potentiated Mer by stimulaiion of alternative apical chlonde channel activity with nucleotides such as UTP. Clinical trials of both treatments are under way (Tomkiewicz et al., 1993; Knowles et al.. 1995).

However, the basic defect still appears to be chloride imperrneability and the identification of the underlying cause for this defect was expected to provide the most effective methods of treatment.

Searcit for the Biochenrical Defect in CF The elevated sweat NaCI concentration and the presence of excess mucus in various passages were the ~o major clinical observations that guided attempts to identi& biochemical markers of CF in the search for a single defective biochemical step. AH of these investigations were performed pre-gene-discovery, involveci decades of research and produced volumes of data, but were mostly abandoned once the defective gene was identified (reviewed in great detail by Tsui and Buchwald, 1991). However, some findings rnay hold important information which is mostly ignored in the main Stream of today's CF research focuses. Therefore, for the reader's interest, several studies are summarized in the Appendix. The CF Gene and its Gene Product

Identification of the Gene Affeeted in CF After many years of conflicting data and results which seemed difficult to reconcile, major developments in the fields of genetics and molecular biology enabled CF scientists to search for the gene afTected in CF, initially without utilking any biochemical information. Linkage analysis, a method which determines the proximity of two genetic loci by measuring the fÎequency with which they cosegregate (Ott, 1985), located the CF gene together with the DOCRI-9 1 7 marker on chromosome 7 (T'sui et al., 1985). It was established that the afEected locus is flanked by the two markers D7S8 and met, each being a distance of approximately 1cM away (1 CM = 1% recombination) (Beaudet et al., 1986). To Merrestrict the area of search, saturation mapping was then applied in which clones selected randornly fiom a chromosome 7 library were localized using somatic ce11 hybrids with different segments of the chromosome deleted (Rornrnens et al., 1988). When the iimit of resolution was reached, candidate gene sequences were searched for by chromosome walking and chromosome jumping (Rornrnens et al., 1989). In chromosome jumping, linear pieces of DNA are circularized and intervening sequences are removed so that walks cm be started fiom different points sirnultaneously. This eventually Ied to the identification of a cDNA clone containing an open reading fiame, showing conservation amongst several species (conservation in 'zoo-blots' is an indication that a sequence is likely to be transcribed). and being rich in cytosine and guanine (hypomethylated 'CpG islands' occur fairly rarely and are found to consistently be associated with transcnbed sequences, generally at the 5' end of the associated gene (Bird, 1986)) - al1 indications for this clone being part of a transcribed gene. Using the clone, the entire gene could be isolated piece by piece. In RNA gel-blot hybridization experiments, the putative CF-gene was found to be expressed in tissues affected in CF, but generally not in tissues not affected by the disease (Riordan et al., 1989). Strong support that the correct gene had been identified came fiom the observation that 70% of al1 CF patients are homozygous for a mutation predicted to result in deletion of phenylalanine 508 (AF508). This defect is never observed in healthy controls (Kerem et al., 1989; Riordan et al., 1989). The Gene Product - CFTR Predicrions of Toplogy - Sequence adysis of overlapping cDNA clones predicted a polypeptide of 1480 amino acids with a molecular nass of 168,138 daltons (Riordan et al., 1989). From the primary amino acid sequence, hydropathy plots suggest the molecule

consists of two stnicturally similar halves, each accommodating six highly hydrophobic segments, of which each has the appropriate length to potentially form a transrnembrane helix (TM) of a membrane protein (Fig 1.2). In addition, each half contains a large hydrophilic domain, the nucleotide binding fold (NBF), with Walker A and Walker B motifs (Walker et al., 1982) for possible interaction with ATP. The two parts are interco~ectedby the highly charged regulatory domain or R-domain, rich in consensus sequences for phosphorylation by various kinases such as cyclic AMP-dependent protein kinase (KA) and protein kinase C (PKC). On the cytoplasrnically exposed side of the protein, the TMs are linked to each other by cytoplasrnic loops (CLs), which vary between 55 and 65 arnino acids in length. Very little of the protein is predicted to be exposed at the extenor surface, except the regions between TMs 1 & 2 and between TMs 7 & 8, with the latter containing two potential sites for Klinked glycosylation. Despite the overall syrnmetry in the two-motif structure of the protein, sequence identity between the two halves is only modest with the strongest identity occuning between the carboxyl ends of the

NBFs (Manavalan et al., 1993). Several features of this mode1 have been confimied experimentally, such as the identity of predicted extracellular and intracellular loops, the cytoplasmic location of NBFs, R-domain, as well as N- and C-termini (Denning et al., 1992a,b; Chang et al., 1994 and 1995; Chen and Zhang, 1996) and the glycosylation of the Fig. 1.2. Predicted folding of ClTR The six membrane-spanning helices in each half are depicted as cylindee, with trammembrane helix 1 king located proximal to the N-texminus

(N) of the protein and trammembrane helur 12 king located closest to the C-terminus (C). The cytoplasmically onented NBFs are show as hatched spheres with dots to indicate means of enûy by the nucleotide. The large polar R-domain, which links the two halves, is represented by a clear sphere. Charged individuai amino acids are shown as smdl circles containing the charge sign. Net charges on the intemal and extemal lwps joining the membrane cylinders and on regions of the NBFs are contained in open squares (Riordan et ai., 1989). Filled triangle, dibasic consensus site for potential interaction with PU; open inverted triangle, consensus site for potential interaction with PKC;fork, potential N-linked glycosylation site; NBF, nucleotide binding fold. Y N-linked CHO v PKC A PKA

0 K, R, H O D, E two predicted sites, Asp 894 and Asp 900 (Cheng et al., 1990). The protein was named the cystic fibrosis trammembrane conductance regulator (Cm)(Riordan et al., 1 989). A BC-superfamily of Proteins - Sequence cornparisons indicate that C FTR is strucnirally related to a family of transport proteins, collectively known as 'ATP-binding cassette (ABC)proteins' (Hïggins, 1992). To &te over 100 different ABC proteins have ken identified, in both prokaryotes and eukaryotes (Demolombe and Escande. 1996). Besides CFTR, some of the most prominent members of the super-family are periplasmic penneases such as histidine permease, a P. fakiparum protein involved in imparting chloroquine resistance to the malaria parasite, TM-1 and TAP-2 peptide transport proteins of the major histocompatibility complex associated with antigen processing, the STE6 gene product responsible for the expoa of the a-factor mating pheromone of S. cerevisiae, P- glycoprotein which exports many hydrophobie compounds fiom the ce11 including some cancer drugs (Doige and Ames. 1993) and, more recently, the sulphonylurea receptor involved in the modulation of insulin secretion (Aguilar-Bryan et al., 1995). Al1 members of the family consist of at least one unit containing six potential membrane sparming sequences and an extended NBF (Riordan et al., 1991). In most members this motif is duplicated. Interestingly, in many bacterial %insporters, such as the oligopeptide permease of S. ~phimuriurn,the four domains are encoded as separate polypeptides; in addition, bactenal uptake systems normally require a fifth periplasmic component which delivers substrate to the membrane-bomd complex. In many other members, the four core domains are fused into one single structure (e.g. P-glycoprotein), or into sub-structures as is the case for the ribose transporter of E. coli for which the two ATP-binding domains are comected, or the MHC transporter which consists of two separate peptides, each providing a trammembrane and an ATP-binding domain (Higgins, 1993). Again, most sequence homology occurs in the NBFs (Dnimm and Collins, 1993). Function ofthe Gene Product - How does the clinically observed defect in chioride permeation relate to the cloned gene product? Most of CFTR's structural relatives are transporters and because of this it was proposed that CFTR is not a chloride channel itself,

but rather the transporter of some substrate which in tum affects chloride channel activity of a second molecule. Especially in view of the expected ATPase activity of CFTR it seemed inconceivable that the ability of a channel to transport a minimum of 6x10~iondsec could be coupled to hydrolysis of ATP (Fuller and Benos, 1992) - unless the ATP is involved in gating rather than ion translocation as was previously observed for potassium channels (Petersen and Due, 1989). Firçt indications that CFTR itself might be the chloride channei came f?om reconstitution studies in which wild-type CFTR is introduced into CF cells, thereby conferring normal chioride channel regulation to the CF cells (Drumm et al., 1990; Rich et al., 1990). These fhdings were confirmecl by expression of normal CFTR cDNA in several types of non-epithelial cells, which always results in the appearance of cyclic 5'-3' adenosine monophosphate (CAMP)-sthulated anion permeability, detectable on the single-channel as well as the whole-ce11 level. The levels of stimulated anion permeability roughly correspond to the amount of CFTR protein expressed (Anderson et al.,

199 1a; Kartner et al., 1991, Rommens et al., 199 1). If the hF508 mutation is introduced into the CFTR cDNA, the anion pemeability is not produced. This gave very strong indications that CFTR itself is the channel, because othenvise a functional, but basally inactive charme1 would have to reside in al1 these cells, just waiting for CFTR to be expressed for its activation. Vice versa, if CFTR expression is decreased with antisense oligonucleotides, cells demonstrate a CF-like phcnotype (Knuss et al., 1992; Wagner et al., 1992; Kopelman et al., 1993). Further evidence for the channel theory was provided by Anderson et al. (1991b) who mutated the basic amino acids Lys 95 and Lys 335, which are predicted to reside in the TMs of CFTR, in the expectation that these basic residues might be involved in anion translocation. Indeed, introduction of acidic amino acids at either of the two positions alten the anion selectivity fiom Br-Xl-X>Fto T>BrXl~Fwhen expressed in HeLa cells and studied by analysis of whole-ce11 currents. Such a change is predicted if CFTR itself contains a pore with moderately high sites for anions. Flinher studies utilking mutagenesis in the predicted TM regions were conducted by Sheppard et al. (1993) and Tabcharani et al. (1993) and demonstrated that mutations in this section of CFTR cm influence the pore properties of the channel. In fact, Akabas et al. (1994) were able to map residues of the proposed TM helices that face the pore of the channel by cysteine replacement studies in which introduced cysteines were tested for accessibility to charged, sulfiydryl-specific methanethiosulfonate reagents added extracellularly. That CFTR itself is the channel was eventually proven unequivocaily by the purification and reconstitution of CFTR into planar lipid bilayes, which results in the appearance of regulated chloride channel activity (Bear et al., 1992a). Conductance Properties of CFTR - CFTR was studied in cells that endogenously express the protein, as well as in various heterologous expression systerns and in its reconstituted form. A summary of the observed kinetics of this anion channel is given by Hanrahan et al. (1994). CFTR cm be activated by CAMP in the whole-ce11 configuration (Rich et al., 1990) or by the catalytic subunit of the CAMP-dependent protein kinase (PKA) in the presence of ATP in the excised patch configuration (Tabcharani et al., 1990). In addition, the channel is stirnulated by other kinases such as protein kinase C (PKC), although the level of activation is much smaller and the in vivo contribution due to these kinases is not well understood. CFTR channels are voltage independent, non-rectifjmg (Cliff and Frizzell, 1990), and can be uihibited by the general chloride channel blocker

diphenylamine-2-carboxylate (DPC). Typical for CFTR are its flickery kinetics at hyperpolarizing potentials on-ce11 (but not in the excised state) and its insensitivity to extracellularly applied 4,4'-diisothiocyanatostilbene-2,2'-disulfonc acid (DIDS) that is also a general chloride channel blocker. Overall it was found that CFTR has a conductance between 6 and 10 pS with most variation between studies attributable to the different temperatures or chloride concentrations used. The calcuiated Km for CFTR is 38 mM chloride. Thus far unexplained are substates which are observed occasionally and have a conductance approximately half that of the fuily open state (Hanrahan et al., 1994; Tao et al., 1996). Estimates about the selectivity of CFTR for different anions were initialiy extremely conflicting and dependent completely on the Laboratory in which they were rneasured. The problem was evenhially solved when Tabcharani et al. (1992) demonstrated that iodide blocks the pore, so that the iodide : chloride penneability ratio is >1 before the block and €1 after the block takes place. Consistently these two anions are more penneable than bromide or fluoride. The use of various sized anions and measurement of their permeability suggested that CFTR has a pore diameter of approximately OS5 MI (Hanrahan et al., 1994). Tabcharani et al. (1993) provided elegant evidence that CFTR is a multi-ion pore. Some charnels can hold more than one ion simultaneously, in which case the destabilization which results fiom ion-ion repulsion inside multiply occupied pores is thought to enable ions to pass through rapidly despite hi& aEty binding. When rneasuring changes in conductance and pemeability ratio in solutions containing two permeant ions, conductance of a single ion pore shouid increase monotonically as the mole fraction of the more permeant species is increased. In a rnulti-ion pore, conductance may decrease in mixtures when compared to pure solutions containing either ion. This anomalous mole fiaction effect can be observed for CFTR. but is abolished if residue Arg 337 is rnutated to Asp, suggesting that this site is involved in the interaction with the permeant anion (Tabcharani et al., 1993). Lf Arg 347 is mutated to an His, the anomalous mole fiaction behaviour can be turned on and off, simply by changing the pH of the bath solutions (His is positively charged at low pH and uncharged at high pH). Interestingly, these results could not be reproduced by Kipper et al. (1 995). Very little evidence has ken presented regarding multimeric requirements for

CFTR activity, although Marshall et al. (1994) believe that the channel acts as a monomer. This would be in agreement with findings for the related P-glycoprotein (Loo and Clarke, 1996). It is conceivable that the proposed arrangement of two sirnilar halves may indicate that CFTR is a dimer within itself. In fact, when the amino-terminal half of CFTR is expressed, it forms a chloride channel with conductance properties comparable to full length CFTR. Migration on sucrose density gradients suggests that such half-proteins act as multimers (Sheppard et al., 1994). In the search for the minimal functional unit it was reported that deletion of trammembrane helices one to four fiom full length CFTR still allows the production of a finictional channel, so that the minimum requirement may be helices five and six (Carroll et al., 1995). Erpression of CF'TR - Northem as weLl as Western blot analysis demonstrated that CFTR is expressed at high levels in CF-affiected tissues such as pancreas, eccrine sweat glands, salivary glands, intestine, liver, and testis (Riordan et al., 1989; Trezise and Buchwald, 1991). In general the protein localizes to the apical surtace of the exocrine epithelia (Dennùig et al., 1992a; Kartner et al., 1992), the only exception king the sweat duct, where low levels of expression can also be detected in the basolateral membrane

(Cohn et al., 1991). This agrees well with hctional data described above that in CF a lack of chioride permeation is observed at the apical membrane of most tissues, except the sweat duct for which both apical and basolateral defects are seen (Reddy and Quinton? 1989a,b; Reddy and Quinton, 1992). Within each organ the CFïR expression is generally limited to a specific area. For example, in the pancreas moa of the protein is fomd in intercalated ducts, and in the intestine CFTR expression is largely confined to cells of the crypt (Strong, et al., 1994). Within these areas expression can be restricted to certain ce11 types, such as a sub-population of scattered villus and superficial crypt epithelial cells (Ameen et al., 1995). This is the reason that in initial studies only low levels of CFTR could be detected in the highly CF affected Iungs (Riordan et al., 1989). More detailed analysis demonstrated that in branchial tissues, CFTR is localized to cells of submucosal glands, notably ui the serous component of the secretory tubules, as well as in a sub-population of cells in ducts, especially ciliated cells (Engeihardt et al., 1992; Yang et al., 1994). Later studies also identified low levels of CFTR expression in tissues that are generally not af5ected by the disease, such as brain (Hincke et al., 1995), placenta (Faller et al., 1999, gail bladder -y- Charkr et al., 1995), and heart (Levesque et al., 1992). The isoform expressed in cardiac

tissue, although functional, misses exon 5 which corresponds to amho acids 164 - 193 (Horowitz et al., 1993; Hart et al., 1996). A similar scenario was recently discovered in kidney where, in addition to wild-type CFTR, a second isofonn is expressed which terminates C-terminal to the R-domain. Functional studies demonstrated that the conduction properties of this half protein are comparable to the wild-type molecuie (Mordes et al.. 1996). The expression of CFTR within a ce11 type is not constant, but seems to be modulated throughout the life of the cell. Continued exposure to CAMPagonists (28 hou) has been observed to allow permanent upregulation of total ce11 CFTR expression and concomitant activation of chioride secretion (Breuer et al., 1992; Kunzelmann et al., 1994). This induction occurs at the transcriptional level since blocken of both transcription and translation negate the upregulation, suggesthg the use of putative CAMPresponse elements in the CFTR gene prornoter. A possible CAMP-response element has been identified by mutagenesis at positions -48 to -41 of the CFTR promoter. Furthemore, basal expression of CFTR may require PKA activity since inhibition of PKA by expression of a dominant- negative regulatory subunit, or treatment with the PKA inhibitor N-[?-@ bromocinnamylamino)ethyl]-5-isoquinolinede, cause complete suppression of CFTR gene expression without affecting other constitutively active genes (McDonald et al.,

1995). Again, this occurs via an effect on the promoter because basal expression of a reporter gene linked to the CFTR promoter experiences the same dependence on PKA. Prolonged exposure to the PKC activator phorbol myristate acetate (PM.) down- regulates expression of CFTR by reducing CFTR mRNA levels in a dose- and the- dependent manner (Trapnell et al., 1991 ). The PMA effect rnay occur directly through PKC activation since it is blocked by PKC inhibitors. Breuer et al. (1993a) indicated that the down-replation is not only due to suppressed rates of transcription (as indicated by nni-on analysis; Bargon et al., l992a), but also due to stimulation of CFTR degradation.

CFTR mRNA levels are influenced by modulaton other than kinases. During cellular differentiation of HT-29 and Caco-2 cells, CFTR rnRNA levels were observed to increase (Montrose-Rafizadeh et al., 1991). This increase cannot be accomted for by increased transcription of the gene, which rnay indicate that CFTR mRNA stabilizing

factors are present in differentiated cells (Sood et al., 1992). Furthemore, it has been reported that increases in intmcellular divalent cation concentrations dom-regulate the expression of the CFTR gene at the transcriptional level (Bargon et al., 1992b), and that

interferon-y (Besancon et al., 1994) decreases CFTR expression post-transcriptionaIIy by destabilizing the transcript. On an even longer time scale, CFTR has ken observed to be regulated during development (Trezise et al., 1993; Gaillard et al., 1994). CFTR may especially be important in fluid rnovement during early lung development since it appears very early in the fetal lung at strategically important locations (McGrath et al., 1993; Tizzano et al., 1994; Sehgal

et al., 1996). In adult life, CFTR expression is modulated in the luminal and glandular epithelium of the uterus during the oestrous cycle and in the seminiferous tubules of the testis during spermatogenesis (Trezise et al., 1993). Ln the utem the regulation is estrogen dependent (Rochwerger et al., 1994). A curious fmding is the observation that the related multidrug resistance and CFTR genes have complementary patterns of epithelial expression (Trezise et al., 1992). In fact, induction of multidmg resistance downregulates the expression of CFTR in colon epithelial cells (Breuer et al.. 1993b).

Regulation of CFïR

Chloride charnels identified thus far are genedly ligand gated or voltage gated (Riordan, 1992) and share many structural and functional features with cation channels such as the nicotinic acetylcholine-receptor family. In contrast, CFTR does not comply with the ligand or voltage gated schemes, but rather is regdated through the concerted action of phosphorylation and ATP hydrolyçis, thus resembling transporten not only in its structure, but also in its regdation. As Riordan (1992) suggested, this is not unexpected since CFTR does not need to respond to the binding of neurotransrnitters or voltage changes. Rather, in order to participate in the controlled movement of chloride and water across epithelial barries, it might be expected to respond to rnetabolic (ATP) levels and hoxmonally stimuiated changes such as altered CAMPlevels. Indeed, Bell and Quinton (1993) described regdation of CFTR chloride conductance in secretion by cellular energy levels and Drumrn and Collins (1993) eventually summarized a cellular pathway in which chionde secretion is activated by P-adrenergic stimuli. In this cascade, stimulation cm occur by epinephrine, prostaglandin E2,vasoactive intestinal peptide, and similar agents. The invoked pathway involves P-adrenergic receptor binding, G-protein activation, stimulation of adenylate cyclase to produce CAMP, CAMP-mediated activation of PKA, and fïnally increased chloride permeability of the apical membrane. Although other modes of regdation exist, CAMP-rnediated hormonal control appears to be a predorninant pathway (Riordan et al.,

1994).

The following sections will address the phosphorylation and ATP-hydrolysis modes of regulation individually and then present the limited understanding of their interactions. Regdafion by Phosphorylation CAMP-dependent Protein Kinase (PU) - PKA is composed of two regulatory subunits and two catalytic subunits, al1 of which in the absence of CAMP bind together to form an inactive tetramer. Each regulatory subunit has two binding sites for CAMP and exhibits cooperative binding of the nucleotide. Addition of CAMP induces the catalytic subunits to dissociate fiorn the holoenzyme, making them catalytically active; hydrolysis of

CAMP by cyclic nucleotide phosphodiesterases in turn leads to reassociation and regeneration of the inactive tetramer (Walaas and Greengard, 199 1). On the protein that is to be phosphorylated, a dibasic consensus sequence (R/K - RK - X - S*/T*; X cm be any residue; astensk indicates potential site of phosphorylation) provides the optimal environment for interactions with the catdytic domain of PKA. However, peptide sequences that do not strictly adhere to this motif can also be phosphorylated, although often with slower kinetics (Kennelly and Krebs, 1991). Physical interaction of PKA with the target protein occm via the positively charged residues of the consensus sequence. The actual phosphorylation then involves a confonnational change of the target protein and transfer of the terminal phosphate group of ATP to the hydroxyl moiety of the serine or threonine in the consensus site. Several cataiytically important regions of PKA have been identified and are summarized by Taylor et al. (1 990).

CFTR contains ten of the perfect dibasic consensus sites for phosphorylation by

PKA. nine of which are clustered within the R-domain and one resides just N-teminal to

NBFl (Fig. 1.3). Therefore, in theory, CFTR appears to be an ideal substrate for phosphorylation by the kinase, so that early studies immediately focused on this potential mode of regulation of the channel. In fact later attempts to investigate other stimulatory pathways showed that phosphorylation by PKA is the most potent mechanisrn of activation identified thus far. lf recombinant CFTR is expressed in epithelial or non-epithelial expression systerns, chloride channel activity that is absent in control cells can be evoked in al1 cases by treatrnent with CAMP agonists (Dmet al., 1990; Gregory et al., 1990; Anderson et al., 199 la: Kartner et al., 1991). in general, the utilized agonists are the adenylate cyclase activator forskolin to stimulate CAMP production, the phosphodiesterase inhibitor 3- isobutyl- 1-methyl-xanthine (IBMX) to inhibit CAMP breakdown, and membrane permeant CAMPanalogues. More direct evidence that the CAMP mediated activation occurs via PKA was provided by Bear et al. (1991), who stimulated the channel by directly injecting PKA into CFTR-expressing oocytes, and Krolczyk et al. (1995), who were able to decrease chloride efflux in Caco-2 cells with a zinc-inducible mutation in PKA.Subsequent single- channel patch-clarnping demonstrated that phosphorylation and dephosphorylation of CFTR Fig. 1.3. Schematic representation of dibasic consensus sites within CFLn for interaction with PKA. The CFTR loci that have the highest likelihood to become phosphorylated by PKA possess the dibasic consensus sequence, R/K - Rn< - X - S*/T*

(astensk indicates potential site of phosphorylation). The positively charged residues provide the docking site for the kinase and phosphate attachment occurs on the hydroxyl group of the serine or threonine residue. X represents any amino acid (Kennelly and Krebs, 199 1). For each potential site, the number indicates the location of the serine or threonine in the CFTR amino acid sequence according to Riordan et al. (1 989). NBFI , nucleotide binding fold 1; R-domain, regdatory domain; S, serine (Ser); T, threonine (Thr); R, arginine

( Arg); K, Lysine (Lys). PKA consensus site: Arg/Lys - Arg/Lys - X - Ser/Thr serve as a molecular switch to gate the channel. When CFTR-transfected CHO cells are stimulated with CAMPagonists, chloride channels can be detected in the on-ce11 recording mode. However, this activity disappears within two minutes after patches are excised. If PKA is added to the cytosolic bath of the inside-out membrane patch chloride charnel activity is regained in the presence of ATP (Tabcharani et ai., 1991). This indicates that upon excision, CFTR is dephosphorylated due to the presence of membrane-associated phosphatases, but the equilibrium of phosphorylation/dephosphorylation can be moved towards the phosphorylated state by adding an excess of PKA. PKA increases the open probability (Po) of CFTR channels fiom zero to 0.407M.023. This activation can be further increased in the presence of the non-specific phosphatase inhibiton fluoride and metavanadate. Furthermore, the channel cm be deactivated more than 90% by exposure to excess alkaiine phosphatase in the continued presence of PKA, an effect that is blocked by fluoride and metavanadate. Overall, Tabcharani et al. (1991) directly demonstrated that

CFTR is regulated by both kinases and phosphatases. It was anticipated that regulation by PKA occius via phosphorylation of one or several consensus sites in the protein. In order to obtain an initial idea of the site(s) involved, Picciotto et al. (1992) phosphorylated full-length CFTR and a recombinant R- domain (residues 645-835) in vitro with PKA and y-[32~]-~~~and analyzed both by peptide mapping with TPCK-trypsin 1 V8 protease and direct amino acid sequencing. It was found that in vifru phosphorylation occurs to a stoichiometry of -5-6 moVmol of CFTR (confimed by Duihanty and Riordan, 1994a), and that serines 660, 700, 737, 8 13 and either Ser 768 or Ser 795 or both are highiy PKA radiolabeled (Table 1.2). Of the total radioactivity, 25% is incorporated at sites 768/795,20% at 700,20% at 737, 8% at 813, and

~5%at 660. Additional sites were labeled to a smaller degree, but their identity was not resolved. All phosphorylation that could be detected with this method occurs within the R- domain. When the kinetics of phosphate incorporation into sites of synthetic peptides were studied, it was detennined that serine 768 appears to be the best substrate, with the other Table 1.2. Observed phosphorylation profiles of CFTR residues by PKA and PKC in vibo and in vivo. Data is summarized fi-om Cheng et al. (1991), Picciotto et al. (1992),

Chang et al. (1 993)' and Rich et al. (1 993a) and was also tabulated by Gadsby and Naim

(1994). '+' represents that a residue was phosphorylated, '-' represents that a residue was not observed to be phosphorylated. An increasing number of '+' signs is intended to give an indication of an increased efficiency of phosphorylation. Question marks denote uncertainty in assigning radiolabeling to a specific residue. Studied Residue PKA PKC in vivo in vitro in vivo phospholabeled sites king adequate substrates as well. Picciotto et al. (1992) performed the

same procedws NI vivo, stimulating phosphorylation through forskolin. Under these

conditions the labeled sites are similar, with phosphorylation occurring on sites 660 and 700, three of 737, 768, 795, 813, and one additional, unidentified site that is mt labeled in

vim, i.e. one site is lacking and one additional site is labeled. Use of a dephospho-specific antibody indicated that at least 30% of CFTR molecules respond to the induction of phosphorylation in vivo.

In order to study the importance of individual phosphorylation sites in vivo, Cheng and coworkers (1 99 1) combined a mapping approach with expression of mutated CFTRs in

HeLa cells and measurements of functional consequences on ion halide efflux by observing changes in 6-methoxy-N-(3-sidfopropy1)-quinolinium (SPQ) fluorescence. When potential phosphorylation of each dibasic PKA consensus site was inhibited individually by changing the serine or threonine to an dzi-ine, no drastic effect on function or phosphorylation could

be observed. This indicates that no one site is the predorninant phosphate acceptor or is solely responsible for stimulation of chloride channel activity. Each mutant was subjected to

phosphopeptide mapping and it was found that serines 660,737, 795, and 8 13 are labeled in

vivo plus additional unidentified residues at very low levels. The four major sites were mutated to alanines in combination mutants; removal of any three sites had no effect on

function. but removal of al1 four sites (the 'Quad' or 4SA mutant) eliminated activation by PKA. Thus, the regdation of CFTR is degenerate, i.e., more than one site is normally involved, but no one site is essential and one site is sufficient. However, careful studies with a stable expression system and more quantitative functional methods demonstrated that the 'Quad'-mutant still retaîns 50% of its responsiveness to PKA stimulation (Chang et al., 1993; Rich et al., 1993a); slowing of the response tirne to regdatory stimuli is observed. In fact, a mutant in which ail 9 dibasic PKA consensus sites of the R-dornain are removed by changing the serine to alanine (9SA- Cm).still retains considerable activatibility by PKA. The residud activity can be Mer decreased by altering the dibasic pre-MF1 site, Ser 422, but even the resulting l OSA mutant responds at approximately 30% of wild-type levels. In the lOSA mutant ody the open probability (Po) is reduced, showing unaitered conductance and ion selectivity.

Estimation of PKA-mediated in vivo phosphate incorporation into the diEerent mutants demonstrated that the labeling of 4SA-CFTR is decreased by >95% relative to wild-type,

9SA-CFTR shows incorporation just above background and 10SA-CFTR exhibits no phosphorylation at this level of detection (Chang et ai., 1993). This indicates that the level of phosphorylation is not a direct refiection of the level of activation remaining. In related studies, Rich et al. (1 993a) investigated whether the charge of the phosphorylation event is the crucial change that evokes chloride efnux. The se~esof the dibasic sites were mutated to aspartates to shulate the introduction of charge. Ethe four sites of the 'Quad' mutant are changed, no constitutive channel activity is seen. If six or more sites are mutated simultaneously, the channei is constihitively open in the presence of ATP without addition of PKA, suggesting that negative charge is sunicient to open the channel. However, the Po is less than half that of wild-type chanoels and an 8SD mutant can still be Meractivated by addition of CAMP,hinting that additional events mut take place. A waming regarding the interpretation of al1 the mutagenic studies cornes fiom the observation that l0SA-mutagenesis not only prevents the sites fiom serving as phosphorylation substrates, but also alten the polypeptide stmcturally as estimated fkom changes in C.D. spectra (Ddhanty et al., 1995). A possible reason for this may be the fiequent localization of phosphorylation sites to predicted P-turn structures (Small et al.. 1977; Reithmeier and Deber, 1992). Replacement of serine with alanine changes a residue with hi& P-tum potential to a residue with more a-helical potential. The structural alteration was seen in a recombinant R-domain rather than the entire CFTR molecule, but it still could suggest that the fûnctionai consequences of the mutations may not be entirely due to eEects on phosphorylation. Nonetheless, mutagenesis does provide a valid fint approximation. Since the part of the R-domain that contains most phosphorylation sites maintains only 23% identity between ten species (Dulbanty and Riordan, 1994b), one couid suggest that the ten changes of the lOSA mutant rnay not af5ect structuraily-mediated function (Dulharity et al., 1995).

Protein Kinase C (PKC) - PKC is a second kinase that signincantly influences the activity of CFTR. The consensus site for PKC-mediated phosphate incorporation is very loosely defuied with phosphorylation occuming on a serine or threonine that has a positively charged arginine or lysine located in the -3 to +3 position, preferably on both sides simultaneously (Kemelly and Krebs, 1991). CFTR contains a large number of such potential sites, both within and outside of the Rdomain. Direct activation of the chloride channel by PKC can be observed with the single-channel patch-clamp method, although the increase in the Po is only one tenth of that seen for PKA-mediated stimulation (Tabcharani et al., 1991). Interestingly, the same study also demonstrated that pretreatment of CFTR channels with PKC can potentiate activation by PKA beyond a simple additive effect. It is not clear how this occurs but an increase in the level of PKA-mediated radiolabelhg of

CFTR is observed if the pretreatment is carried out. This suggests that phosphorylation by PKC rnay make additional PKA sites available (Chang et al., 1993), possibly through induction of a conformational change. The potentiation effect could be reproduced by Bajnath et al. (1993), but not by Berger et al. (1 993). Detailed phospholabeling experiments showed that PKC incorporates approxirnately 2 mol of phosphate per mol of CFTR

(Picciotto et al., 1992). If' these studies are performed in vitro, the major sites of phospholabeling are serines 686 and 790, whereas in vivo the major sites are found to be serines 686 and 700. Al1 three residues are located within the R-dornain (Picciotto et al., 1992). The fundamental role of PKC-mediated phosphorylation in ClT2 fùnction is still poorly understood. Some baseline level of PKC phosphorylation may be essential for normal regulation of the channel by CAMP, based on CFTR inactivatibility upon dom- regulation of cellular PKC levels (Dechecchi et al., 1993). Using phorbol esters to activate PKC, the same group also presented whole ce11 data indicating that the kinase itself activates CFTR in an intact ceil. In contrast, in pancreatic duct cells, the phorbol ester has no eEect on CFTR current density in unstimuiated cells, but causes a 3 1% increase in the magnitude of CFTR cunents recorded nom cells stimulated with CAMP,thus resembling the single-channel potentiation (Winpenny et al., 1995). A novel finding of the study was that prolonged exposure of stimulated duct cells to phorbol ester (which dom-regdates

PKC) significantly slows the rate at which CFTR currents nin down after establishing a whole cell recording (Winpemy et al., 1995). Similar results are observed with the PKC

mhibitor calphosth C, indicating that the usual rundown does sornehow involve PKC activity. The authors suggested that this effect might occur via phosphorylation of other molecules, possibly cytoskeletal, which regulate CFTK Addirional Kinases - Very little data is available regarding the involvement of kinases other than PKA and PKC in CFTR regulation. In a screen of various kinases, the multifunctional ~a~'lcalmodu1i.n-dependentprotein kinase failed to either phosphorylate or activate CFTR (Berger et al., 1993), whereas cGMP-dependent protein kinase (cGK) phosphorylated CFTR in viiro, but could not stimulate channel activity in excised, inside- out membrane patches. French et al. (1995) elaborated on these data and agreed that the cGK I isoform fiom bovine lung cannot activate recombinantly expressed Cmbut found that the cGK II isoform fiom pig intestinal brush borders does stimulate the channel in a similar way to PKA, but with reduced kinetics. Interestingly, both isoforms label irnrnunoprecipitated CFTR to the same extent with the same kinetics, with phosphopeptide maps revealing only subtle differences in site specificity between cGK 1 and cGK II. Because of this, it was suggested that despite CFTR king a good substrate for cGK- mediated phosphoiylation, the regulation occurs actually via a third protein which is phosphorylated by cGK and then interacts with CFTR (French et al., 1995). Expression studies in the rat intestine showed that whereas cGK Ii generally CO-localizeswith CFTR expression, cGK 1 does not (Markert et al., 1995). In addition, cGK is activated by cyclic GMP (cGMP), which has ken known to activate non-selective cation channels in a direct, phosphorylation independent manner (Kaupp, 1991). The possibility of such direct

interactions with CFTR was proposed because two residues (V397 and K420) were identified which, when changed to alanine, alter the response to cGMP independently of the response to CAMP (Sullivan et al., 1995). Furthemore, evidence has been presented that cGMP can also act via PKA (Tien et al., 1994). A well known, but poorly understood pathway of CFTR activation is the stimulation by the tyrosine kinase inhibitor genistein, which possibly may not be due to a direct kinase effect on CFTR, but rather due to a thus far uncharacterized inhibition of a phosphatase (Illek et al., 1996). Phosphatases - An ofien overlwked event that, in ternis of cellular processes, is equally important to phosphorylation of CFTR is its dephosphorylation; there are dla lot of 'muddy waters' remaining in this area of CF research (Quinton, 1996, CF Symp.).

Different laboratones obtain vexy different results, with Tabcharani et al. (1 99 1) and Hwang et al. (1993) reporthg rapid mdown of active CFTR channels upon patch excision, presumably due to endogenous phosphatases, whereas Rich et al. (1993a) observe continuation of channel activity. Thus far such differences have ken mostly attributed to differences in the ce11 types used, but no conclusive studies have been perfonned to reconcile the data. Overall, this is an important observation, since differences in various reports may ofien be attributable to usage of various ce11 types. Furthemore, in recombinant expression systerns, CFTR may be exposed to an uncharacteristic set of phosphatases and additional cellular components. The fact that phosphatases are involved in maintainhg an equilibriurn state of CFTR phosphorylation can be clearly seen fiom the observation that active channel flwes are increased by the addition of phosphatase inhibitors (Tabcharani et al., 199 1; Becq et al. 1993). Sirnilar treatments can even stimulate quiescent CFTR Pecq et al., 1994; Becq et al., 1996). Although there is uniform agreement that dephosphorylation occurs, v-g reports have been made as to which phosphatase is responsible for this event. Initial studies with severai phosphatase inhibitors indicated that phosphatase types 1, 2A, 2B, and 2C are not involved, but showed that exogenous deactivation can be stimdated by alkaline phoqhtase, thereby irnplicating this enzyme as the endogenous phosphatase associated with CFTR (Tabcharani et al., 1991). Similar findings were reported by Becq et al. (1993), but were opposite to the conclusions of Reddy and Quinton (1996a) who used phosphatase antagonists in the human sweat duct to denve that either or both of phosphatases 1 and ZA are responsible for the dephosphorylation/deactivation of CFTR chloride conductance in vivo. Berger et al. (1993) applied pwified phosphatases to excised patches and found that phosphatase 2A decreased CFTR activity, but phosphatases 1, 2B, or alkaline phosphatase did not. To complete the list of 'involved' phosphatases, studies by Hwang and coworkers

(1 993) suggested that phosphatase 2A is needed to dephosphorylate sites crucial to channel function, while 2C may dephosphorylate sites that modulate channel Po without altering the number of active channels. In addition, Fischer et al. (1995) reported that the specific phosphatase 2B blockers, cyclosporine A and deltamethrin, activate quiescent CFTR channels. Thus, the problem of ce11 types and phosphatase isoforms will require more detailed investigations. in fact, CFTR rnay possibly be regulated via a thus far unidentified phosphatase that is closely associated with the channel. Study of phosphatase activity highlighted a curious effect of phosphorylation sites.

As mentioned above, mutagenesis studies demonstrated that not al1 phosphorylation sites are equal. Some show a higher likelihood of king phosphorylated and faster kinetics of labeling than othen (Picciotto et ai., 1992). On the other han& low levels of phosphorylation of a specific site do not necessarily reflect a negligible contribution to fiction (Chang et al., 1993). Hwang et al. (1993) reported an additional distinction among phosphorylation sites. When m-down was inhibited with okadaic acid, an inhibitor of type 1 and 2A phosphatases, part of the deactivation still occurred, but part of it was inhibited, suggesting that CFTR may be phosphorylated at two different kinds of sites. The nature of this ciifference is elusive at this point. The R-domain - How does the R-domain regulate CFTR activity? The dephosphorylated R-domain must prevent gating by ATP, because channels with an intact

Rdornain, or even containing up to 9SA mutations (Chang et al., 1993), do not open in the presence of ATP until labeled by PKA (Gadsby and Nairn, 1994). The fact that the R- domain is involved in some form of an inhibitory event waç suggested fiom the observation that. upon deletion of a large part of the R-domain (A708-835), CFTR is constitutively open in the presence of ATP (Rich et al., 1991; Anderson et al., 199lc). This inhibition is ofien compared to a cork that sits in the bottle neck and blocks flux. However, in such an analogy, the observation is overlooked that CFTR with the R-domain deleted stays closed until ATP is added (Anderson et al., 1991c), indicating that this domain is not just 'blocking' the channel. That the CFTR channel can be closed by a recombinant unphosphorylated R- domain protein, but not by the phosphorylated form (Ma et al., 1996). suggests that phosphorylation is the important step. Furthemore, the activity of an 8SD mutant (Rich et al., 1993a) indicates that this event occurs due to the negative charge of the introduced phosphates. However, only charge introduction may not be sufficient since the 8SE mutant is still less active than wild-type CFTR. Both Pichiotto et al. (1992) and Dulhanty and Riordan (1994a) observed a mobility shifl upon electrophoresis of phosphorylated R- domain. The shift may be the izsult of a conformational change that can be readily detected by C.D. spectroscopy (Dulhanty and Riordan, 1994a) and that may be necessary to move the whole molecule into an open conformation. The two theories of necessity of charge introduction and conformational change are not mutually exclusive and may both prove to be important when the mechanism of R-domain function is fkally elucidated. Regulation by A TP Ndeotide BNIding FOI& - The two nucleotide binding fol& (NBFs) of CFTR are hydrophilic structures that terminate each trammembrane domain and protrude into the ce11 cytoplasm. Both NBFs contain Walker A and B motifs (Walker et al., 1982) providing potential sites for interaction with ATP. The conserved lysine of the A motif (G-X-X-G-X- G-K-T/S) may be important for maintainhg the conformation of the glycine rich loop and is thought to interact with the P or y phosphate of the bomd nucleotide. The conserved aspartate in the B motif (R/K-X7-&-D; h represents a hydrophobie residue) rnay coordinate with magnesium when MgATP is in the binding pocket. Due to the presence of these motifs it was theorized that ATP binding and potentially ATP hydrolysis may be necessary for regulation of CFTR, but since ATP is also reqwed for the phosphorylation by PKA it was initially very difficult to differentiate these two processes. Eventually this was

achieved by Anderson et al. (1991~)who were able to maintain CFTR in a phosphorylated state in excised patches in order to observe in isolation the effects of ATP on regulation. This showed that phosphorylation by PKA is necessary, but not sufficient to open the channel. Once phosphorylated, channels require cytosolic ATP to open, but addition of ATP alone does not open the channel. Phosphorylated CFTR channels close if the ATP is

removed but can be reactivated by re-adding ATP. Biochemical and Functional Findngs - Bell and Quinton (1993) hypothesized that one reason for an ATP-mediated regdation step is the need to couple transport demands to the energy level of the cell. To support this idea, cells were selectively pexmeabilized on the basolateral membrane with S. aureuî a-toxin (Fussle et al., 198 1) for depletion of endogenous ATP and to allow access of membrane impermeable ATP and CAMP to the intracellular cornpartment. In ATP-depleted cells, CAMP cmot increase transepithelial conductance. Myif ATP levels are raised close to intxacellular physiological levels (5 rnM; Williamson and Corkey, 1969), the CAMP response is restored. Similady, kinetic analysis by Venglarik et al. (1994) and Winter et al. (1994) showed that the Po of CFTR in excised inside-out patches increases with increasing leveis of ATP (0.1 -3 mM). This change in the Po is due to a decreased mean closed the, but the mean open the is not afEected. Analysis of the data indicated that CFTR channel behaviour is best described by a mode1 containhg one open and two closed states (Ci++CzttO)(Winter et al., 1994; Gunderson and Kopito, 1994). The increased ATP concentration increases only the transition rate fiom CIto C2,suggesting that ATP regulates CFTR through an interaction that increases the rate of transition fiom the closed state to a bursting state in which the channel flickers back and forth between the O and the Cz state. Al1 observed effects are most likely due to interactions of ATP with the NBFs rather than due to invoked phosphorylation for two reasons: i. ADP addition decreases the transition rate fkom CI to Cz, ii. the effects occur at ATP concentrations that are too hi& to be mediated by PKA, which has a &, for ATP of -7 pM (Sugden et al., 1976). Initial indications that the ATP-interacting domain of CFTR is an NBF came from the observation that a synthetic peptide, which corresponds to the central 67 arnino acids of NBF 1, is bound by numerow adenine nucleotides (Thomas et al., 1991). This observation was extended to recombinant NBFl proteins (Hartman et al., 1992; Ko et al., 1993) and eventually to full-length CFTR (Travis et al., 1993). Travis et al. (1993) used the photoactivatable analog, 8-azidoadenoshe S'-triphosphate (8-N3ATP), to obtain a better understanding of ATP associations with CFTR. 8-N3ATP,which substitutes for ATP in the activation of CFTR and thus interacts with the active site, shows half-maximal labeling at

10 pM in the presence of magnesium and at 100 j.M in the absence of magnesium. ATP is able to prevent photolabelhg with half-maximal inhibition at 1mM, a concentration that is comparable with the concentration of ATP required for chloride channel activity in sweat duct or T84 cells (Quinton and Reddy, 1992; Bel1 and Quinton, 1993). ADP inhibits at a half-maximal concentration of 10 m.,AMP cannot inhibit, and, importantly for later investigations, AMP-PNP is able to inhibit at 20 mM. The study of Travis et al. (1993) was not able to differentiate whether binding occurs at one or both NBFs. However, similarly to NBFl peptides, NBFZ peptides (synthetic and recombinant) can also be bound by adenine nucleotides (Ko et al., 1994; Randak et al., 1995). Is ATP binding sscient for the activation of CFTR, or is the high energy phospho- bond of the molecule utilized as an energy source? Initial functional studies suggested that ATP hydrolysis is necessary for CFTR activation since channel opening cannot be initiated by non-hydrolyzable ATP analogues such as AT'Sor magnesium-fk ATP (Anderson et al., 199lc). ATPyS is often found to serve as an adequate subçtiate for PKA-mediated phosphorylation, but not for ATPase reactions. [n contrast, Quinton and Reddy (1992) implied that ATP binding is sufncient and ATP hydrolysis is not required because, in the sweat ducf non-hydrolyzable ATP analogues can activate CFTR as long as low levels of

ATP are provided to allow for proper CFTR phosphorylation. The applied ATP levels were controlled to be below the minimal level necessary to evoke channel opening on their own.

Only recently was the controversy solved when it was demonstrated that both hydrolytic and nonhydrolytic interactions regulate CFTR in vivo. Reddy and Quinton (1 996b) showed that activation of CFTR requires low levels of hydrolyzable ATP even after PKA phosphorylation, indicating that some ATP hydrolysis is required aside from acting as a substrate in PKA phosphorylation. Subsequent to initial activation, non-hydrolyzable analogues cm then activate CFTR without the presence of ATP. Reddy and Quinton

( 1996b) suggested that ATP hydrolysis may be required to induce a conformational change in CFTR that involves the conversion of a deactivated state to a thermodynamically unfavorable activated state in which allosteric non-hydrolytic ATP binding then stabilizes activated Cm.The two steps could involve the two NBFs. The evidence presented thus far functionally shows that ATP hydrolysis is required for CFTR activation. However, it has proven very difficult to demonstrate ATPase activity of the NBFs. Many attempts failed (Ko et al., 1994) until Ko and Pedersen (1 995) were able to observe ATPase activity of a NBFl fusion protein that is stabilized by the presence of the maltose binding protein. Although significant, the observed V, of -30 nmoVmin/mg of protein is very low compared to other ATPases such as FiFrATPase or P-glycoprotein. An indication of the significance of the data is given by a negative effect due to Walker A mutations K464H and K464L. Only recently, Li et al. (1996a) rmequivocally demonstrated ATPase activity of reconstituted CFTR with an ATP turnover number of 1-UsecKFTR in the presence of 1 mM ATP. However, none of the major ATPase inhibitors thus far have been successful in the inhibition of fûll length CFTR (Schulh et al., 1996), hdicating that the mechanisms of ATP hydrolysis by CFTR may not comply with lmown pathways utilized by other ATPases.

Models of Regdation by ATP - CFTR contains two NBFs and indications that both domains may be involved in the regulation of the channel came fiom single-channe1 studies of CF-associated and novel mutations that are predicted to lie within NBFl and NBF2 (Anderson and Welsh, 1992). Mutations in the Walker A lysine of either NBFl or MF2 decrease the potency with which MgATP stimulates the channel, as do mutations of the Walker B aspartate in NBF2. Anderson and Welsh (1992) also demonstrated that in the presence of ATP, ADP can inhibit the channel; in this setting the intracellular ATP:ADP ratio is more important than the absolute concentration of ATP. The inhibitory effect of ADP is abolished by mutations in NBFZ, but is not affected by mutations in NBFl. Thus, the cornpetition of ADP with ATP must occur on NBF2 so that both domains are involved in the regulation of CFTR, but they have different roles. Whole-ce11 studies of oocytes expressing additional mutants found that substitutions for the conserved glycine and aspartate in the Walker motifs of NBFl produce a discemible reduction in the sensitivity of CFTR to activating conditions. In contrast, analogous mutations in NBR have the opposite effect, actually increasing sensitivity over that seen with wild-type CFTR (Smit et al., 1993). These observations were consistent with those of Anderson and Welsh (1992) and with the idea that modification in Walker motifs of NBF2 can increase sensitivity to activation by attenuating the inhibitory influence of ADP. Building on the above observations, Hwang et al. (1 994) delineated various functional states which led to the proposal of a model for CFTR activity that is consistent with previous data and today is cornrnonly accepted as the basic working model. Both whole-ce11 and single-channel measurements were utilized to show that a non-hydrol yza &le analog of ATP, 5'-adenosine(P,y-imin0)triphosphate (AMP-PM), cannot activate CFTR by

itself but, when added to CFTR that is already activated by ATP, does prolong this open state. The 'locking open' only is possible for WyPKA-phosphorylated channels, but not for partially phosphorylated channels. Low levels of phosphorylation can be achieved in the

whole-ceIl system by withdrawing the adenylate cyclase activator forskolin in the presence of the phosphatase inhibitor okadaic acid. This reduces the forskolin-induced chloride

movement to a low, but constant level, suggesting that phosphorylation is lost on most loci. except on some okadaic acid sensitive sites. In the single-channel system, CFTR switches f?om a high Poto a low Poupon removal of PKA,which is again thought to be the result of partial dephosphorylation by membrane associated phosphatases. In both scenarios, AMP-

PNP cannot lock open the channel (Hwang et al., 1993). This suggests that incremental

phosphorylation of CFTR at multiple sites influences the activity of the NBFs. The nature of the responses to AMP-PNP led to the conclusion that ATP hydrolysis occurs at both NBFs,

at one to initiate channel opening WDA) and at the other to initiate normal closing (NBDB).Thus, in the proposed model (Fig. 1.4) phosphorylation of the okadaic acid sensitive sites allows ATP to interact exclusively with NBDA, the nucleotide hydrolysis leading to bnef channel openings. AAer additional phosphorylation of the okadaic acid insensitive sites and ATP action at NBDA to open the channel, NBDB can then interact with ATP or AMP-PNP to stabilize the open state. This stabilization is lost upon dissociation of AMP-PNP, or of ADP plus Pi following hydrolysis of the ATP. The order of phosphorylation need not be sequential for this model. One might expect that AMP-PNP also should interact with NBDA to increase the fiequency of long closures between bursts. The fact that this is not observed is interpreted as a reflection of different afities of the two NBFs for AMP-PNP. Carson et al. (1 995c) confirmed the AMP-PNP 'locking-open' effect. Fig. 1.4. Proposed mode1 of ClTR regdation. States in the upper row represent closed CFTR channel conformations: le% dephosphorylated, D; middle, partially phosphorylated,

Pl; ri& fully phosphorylated, PiP2.Phosphorylation towards state Pi dows ATP to interact exclusively with NBDA (=NBFl), the nucleotide hydrolysis leading to brief channel openings (middle row). Mer additional phosphorylation towards the PiP2 state and ATP action at NBDA to open the channel, NBDB (=NBF2) can interact with ATP or AMP- PNP to stabilize the open state of the channel (lower row). That stabibtion is lost upon dissociation of AMP-PM, or of ADP+Pi following hydrolysis of ATP at NBDB. P. phosphate; R, regdatory domain; OA-sen, okadaic acid sensitive. Adapted fiom Gadsby et al. (1994). Brief Openings

AW+pi _l AT (AMP-PNP) (AMP-PN P)

Long Openings Evidence to Support ATP Models - Severai 'locking open' studies were performed in parallel to the AMP-PNP experiments. The inorganic phosphate (P,) analogs, V04 and

BeF,, interrupt ATP hydrolysis cycles by binding tightly in place of the released hydrolysis

product, P,, thus precluding Mercycles of ATP hydrolysis. Both P, analogs act only on CFTR channels opened by ATP and lock them open (Baukrowitz et al., 1994), again implicating that channel gating is coupled to an ATP hydrolysis cycle that is intempted if the P, analogue binds in place of the released Pi. Binding of the analog afler Pi release is possible because closing of the channel after Pi release occurs fairly slowly (-2 s-l) as estimated fiom open channel dwell times (Baukrowitz et al., 1994). The major difference between the locking open by Pl analogs and AMP-PNP is that AMP-PNP was found to work only on the Wyphosphorylated channels whereas Pi analogs lock open al1 channels regardless of their phosphorylation status. Baukrowitz et al. (1994) interpreted these differences to mean that the Pi analogs affect channel gating by interfering with ATP hyclrolysis at NBDA, whereas AMP-PNP acts only on NBDB. This gave first indications that NBDA may also have some stabilizing effect on CFTR in addition to the major stabilizing function of NBDB. Pi itself (in contrast to Pi analogues) increases the activity of CFR in a dose dependent manner, however, the effiect is more by stimulation of a rate limiting step in channel opening, rather than 'locking-open' (Carson et al., 199513). The difference between P, and P, analogue studies may be explained by the suggestion that once Pl analogs are bound they appear to mimic the transition state of ATP hydrolysis which normally precedes cleavage of the y-phosphate of ATP (Chabre, 1990). Thus, CFTR channel activity is arrested at this state in the gating cycle, while assessrnent of Pieffects on kinetics examines effects on al1 steps of channel gating (Carson et al., 1995b). Furthemore, locking open is produced by the polyphosphates PPi and PPPi (Gunderson and Kopito, 1994) in addition to increasing the rate of channel opening (Carson et al., 1995a). hterestingly, it was observed that increased concentrations of PP, increase the arnount of CFTR labeling by 8-N3ATP (Carson et al., 1995a). Modeling suggested that the effects of PP, occur via MF2 with the binding to NBF;! mimicking the effect of ATP binding. This in

turn may enhance the binding adorhydrolysis at NBFl and, because it is not hydrolyzed,

prolong the duration of the open state by preventing closure (Carson et al., 1995a). Further, carefully analyzed mutagenesis evidence also supports the NBF model.

Mutagenesis of the conserved Waker A lysine in NBFl decreases the fiequency of bursts in single channel tracings whereas parallel mutations in MF2 and mutations in both NBFs sirnultaneously prolong bursts of activity, as well as decrease the kequency of bursts (Carson et al., 1995~).This suggests that the proposed NBDA of Hwang et al. (1994) corresponds to NBFl and the proposed NBDB is NBF2. In addition, al1 mutations somewhat decrease the length of openings within the flickering activity of an open state and increase the length of the brief closures within the flickering. None of the mutations alter binding profiles of 8-N3ATP. Careful rate analysis of transition steps between the various states substantiated the conclusions, but also demonstrated that the two NBFs have some overlap in function, with NBFl mutations somewhat destabilizing the open state and NBF2 mutations somewhat decreasing the access rate of the open state (Wiikinson et al., 1996). That NBFl also stabilizes the open state in addition to the major effect of stabilization by NBF2 is confirmed by the observations that mutations in NBFl which decrease affinity for the hydrolysis product ADP destabilize the open state. It thus appears that there rnust be a significant amount of cornmunication between the two domains with reciprocal modulation. In addition, Gunderson and Kopito (1 995) proposed a somewhat different model for CFTR function based on electrophysiologic studies of mutated CFTR channels reconstituted into lipid bilayers. The bais for the investigation was the presence of two open conductance states of the CFTR channel, 0,(9.0 pS) and 9 (10.3 pS); various conductance states were also reported by Tao et al. (1996), but are not as commonly observed by other Iaboratories. The two states may, but mut not always occur in the same opening step and are not in thermodynamic equilibrium. This suggests that the movement fiom one open step to the other requires energy input, the requirement of which was cobedby the use of non-hydrolyzable ATP analogs and localized to NBR with MF- mutations. The proposed mode1 hypothesizes that ATP hydrolysis at NBFl converts the channel fiom an inactive to an active closed conformation. Binding of either M~ATP'-or ATP~to NBF2 then leads to the Oi conductance state. In the presence of rnagnesium the channel isomerizes to form a pre-hydrolysis complex in which ATP is tightly bound (the importance of magnesiun is suggested because of altered channel khetics at low rnagnesium concentrations). ATP hydrolysis at MF2 then allows the 014302transition. Subsequent dissociation of either Pi or ADP leads to channel closure. Conversion of CFTR back to the inactive state occurs when an end product of ATP hydrolysis at NBFl dissociates.

Reconciring two Modes of Regdation

Therefore, there are at least two modes of CFTR regdation, for both of which some basic understanding has been accurnulated. The ht mode, phosphorylation, is absolutely essential for channel opening to occur. In the absence of phosphorylation, channels cmot be stimulated and the level of phosphorylation appears to modulate the activity that cmbe achieved once opening occurs. Evidence fiom mutagenic phosphorylation site removal (Chang et al., 1993; Rich et al., 199?u), partial phosphorylation with okadaic acid (Hwang et al., 1993), and graded phosphorylation with fonkolin (Fischer and Machen, 1994) suggest that at lower levels of phosphorylation the Po will be lower than at higher phosphorylation levels, but the single channel conductance is not affected by this mechanism. Phosphorylation puts CFTR into a state which then can be opened through ATP binding/hydrolysis. It is not understood how this happens, but one observation is that phosphorylation of the R-domain promotes ATP binding since mutation of dibasic PKA consensus sites increases the ATP concentration required for half-maximal activity without significantly altering the final achieved open probability (Winter et ai., 1996). Also, the ATPase activity of reconstituted CFTR is elevated 2-3 fold in the presence of PKA by decreasing the Km for this activation fiom I mM to 0.3 mM without increasing the V,, (Li et al., 1996a). The most widely accepted mode1 and severai pieces of evidence suggest that

channel-opening ATP hydrolysis occurs at NBFl (Hwang et al., 1994; Carson et al., 199%). The open state subsequently is supported by ATP binding to MF2 and terminated through ATP hydrolysis/dissociation nom this domain. However, there is still very limited evidence suggesting how these levels of regulation interact. To support the idea of direct interaction between the phosphorylation and the hydrolysis levels of regulation, an experiment by

Dnimm et al. ( 1 99 1 ) is comrnonly cited. It was demonstrated that mutant versions of CFTR, expected to compromise fünction of NBFl, cm be revived by increased arnounts of the cyclic nucleotide phosphodiesterase inhibitor IBMX, thus increasing phosphorylation of CFTR. What is even less understood is how the regdatory domains then pass information to the pore fomiing domains of CFTR. One study even proposed that such passing of

uiformation is not necessary because the anion movement of CFTR occurs at least in part via NBFl itself (Arispe et al., 1992). When recombinant NBFl was reconstituted into a

planar lipid bilayer it was reported to fom a -9 pS channel that was blocked by ATP. It also has to be noted that transmernbrane helices 2 and 6 show channel forming capacities in Iipid bilayers. When reconstituted as heterooligomers they fom a 8 pS charme1 with a 95% selectivity for anions over cations (Oblatt-Monta1 et al., 1994). The functional significance of such findings remains to be detemined.

Targeting and Processing of CFTR

The AFS08 Mutation Targeting and processing of the CFTR protein have received much attention, but usually only from a single perspective. The reason for this 'narrow-minded' approach is that

70% of al1 CF patients are homozygous for the deletion of Phe 508. In fact, the AF508 mutation is found on at least one chromosome of 90% of the affected individu& (Sfena and Collins, 1993). CF is associated with irnpaired apical chloride channel activity of the exocrine tissues (Szhoumacher et al., 1987; Li et al., 1988; Hwang et al., 1 989). AAer the realuation that the mutated protein is the chloride channel itself, early speculations suggested that the AF508 mutation inhibits regdation of the channel, because it is centrally located within NBFI. However, Cheng et al. (1990) demonstrated that AFSO8-CFTR does not mature to a Mly glycosylated forrn so that normal CFTR function cannot occur in CF patients because the protein is misprocessed and never reaches its site of action. The lack of mature glycosylation was seen because of the absence of a band C, as dehed by Gregory et al. (1990). Mien CFTR is separated by SDS-PAGE, a primary translation product is observed with an apparent molecular mass of 130 kDa @and A), in addition to a core- glycosylated 135 kDa species (band B), and a diffwly rnigrating 150- 160 kDa version that represents mature, fully glycosylated CFTR (band C). This banding pattern is very typical.

However, recent studies indicated that band A is not an unglycosylated version of CFTR, but rather a second core-glycosylated fonn that is the result of utiiization of an altemate site for initiation of translation (Carroll et al., 1995; Pind et al., 1994, 1995). The absence of AF508-CFTR fiom the apical surface of affected sweat ducts was subsequently conhed by meret al. (1992) by immunolocalization studies using monoclonal anti-CFTR antibodies. Apical membrane staining, clearly evident in normal sweat duct, is entirely absent in AFSO8-homozygote biopsies, although variable amounts of apparently cytoplasmic and perinuclear granules can be observed which give positive staining reactions. In heterozygotes, staining is essentially normal although generally reduced in the apical membrane. The same defect cm be seen with nasal polyps (Puchelle et al., 1W), cultured ainvay epithelial cells (Denning et al., 1992a), branchial xenografk (Yang et al., 1994), and various recombinant expression systems (Sato et al., 1996). Since AF508-CFTR is retained within the cellular rnachinery, one way to fight disease is to promote the molecule's processing. The primary concem in such an approach is the question of whether the mutant protein will be functional once it reaches the ce11 surface. Initially, a somewhat altered structure of a synthetic 67-mer (+/- Phe 508) and less stability in 4 M urea as estimated by the loss of ATP binding ability suggested that the M508 mutation may affect CFTR function (Thomas et al., 1992; Thomas and Pedersen,

1993). However, a recombinant NBFl gave fkst indications that the total domain structure may not be severely modified by the deletion because ATP-binding capacity (Hartman et al., 1992), C.D. spectra, and the stability of the peptide (Ko et al., 1993) are similar with or without the deletion. Indeed, the function of CFTR does not appear to be affected significantly by the AF508 mutation. When measured either by reconstitution into planar lipid bilayers (Li et ai., 1993), by whole-ceii measurements in the maturationcapable baculovinis-insect ceU expression system (Li et al., 1993), or by patch-clamphg at the ER membrane (Pasyk and Foskett, 1995), the PU-stimdated AF508-CFTR currents are similar to wild-type currents. Some reports indicated that the AF508 mutation decreases the

Poof CFTR chloride channels (Dalemans et al., 199 1; Denning et al., 1W2a) but this is not consistently observed and, if present, the effect may be minor. Whatever the molecular defect is that results fiom the AF508 mutation, it cm be partially corrected with revertant mutations. Using STE6-CFTR chimeras as a mode1 system, Teem et al. (1993) were able to identi@ two mutations, NBF1-located R553M and

R5 5 3Q, which when introduced into hF508-CFTR partially correct misprocessing. This may indicate that the Phe 508 area normally interacts with the Arg 553 area, or that upon the deletion of the Phe 508, new interactions take place with the Arg 553 area (Teem et al., 1993). Interestingly, in one patient a combination of the AF508 and the R553Q mutations was found on the same chromosome and this patient showed a mked phenotype of severe lung and pancreatic disease, but normal sweat chloride (Dork et al., 199 1). Inefwnt Processing of CFTR Processing Characteristics - A curious finding about CFTR is that even the wild- type protein shows very inefficient processing; in pulse chase experiments it can be clearly seen that upon transfection of CFTR into COS-7, CHO, mouse epitheloid C127, and pig kidney epithelial LLCPKl cells ody a relatively small hction of the core-glycosylated band B matures to form the fully giycosylated band C (Cheng et al., 1990; Marshall et al., 1994). Initially, this was attributed to overpowering the cellular rnachinery in the applied heterologous expression systems. However, careful studies by Lukacs et al. (1991), Pind et al. ( 1994) and Ward and Kopito ( 1994) demonstrated that inefficient processing is inherent to CF'ïR synthesis even in endogenously expressing cek, such as the epithelial ce11 lines

T84, HT-29, Caco-2 (human colon carcinomas), and Calu-3 (human lung adenocarcinoma). Depending on the ce11 line, 50-80% of the newly synthesized wild-type CFTR is degraded by endogenous proteases, resuiting in a half-life of approxirnately 35 minutes. In the case of hF508-Cm al1 of the newly synthesized protein is degraded, but the half-life is not significantly different f?om the wild-type situation. Furthemore, mutant and wild-type CFTR both show the same rate of synthesis (Ward and Kopito, 1994). The rapid onset of degradation, 3-6 min post-synthesis, is an indication that breakdown may still occur in a pre-Golgi compartment (Ward and Kopito, 1994). The degradation is not substantially retarded by inhibition of endo-lysosomal proteolysis through dissipation of the acidic intraluminal pH with lysosomotropic agents, suggesting that the endo-lysosomal cornpartment does not participate in the degradation of CFTR (Lukacs et al., 1994). In addition, proteolysis of both wild-type and AF508-CFTR proceed after disruption of vesicuiar transport between ER and Golgi by exposure to brefeldin A. so that CFTR most likely is degraded in a pre-Golgi compartment. In fact, both AF508-CFTR and its break- down products can be detected in the ER after cellular hctionation, but not the Golgi and plasmalemmal fiactions (Lukacs et al., 1994). Thus, AF508-CFTR appears never to leave the ER and is degraded at this location. Lukacs et al. (1994) demonstrated that the hction of wild-type CFTR which is properly processed must be present in a modified state and that the transition fiom the protease-susceptible to the protease-unsusceptible forrn is energy dependent. If transport fiom the ER to the Golgi is inhibited with brefeldin A, the same hction of wild-type CFTR does not become degraded as in the control situation. The only difference is that this fiaction is not present as the Mly glycosylated protein, but as the core-glycosylated protein since it is blocked from moving beyond the ER In both cases the protease resistant fiaction of CFTR molecules has a haif-Mie of -24 hours and in both cases the fiactions are quantitatively the same. When brefeldin A is removed, normal processing can proceed. Thus, the processing of the hi&-mannose, N-linked oligosaccharides into a complex structure is not necessary for stabihtion of core-glycosylated CFTR. If cellular ATP is

depleted >96%, the transition fiom the newly synthesized, protease susceptible intermediate to the protease-resistant, transport-competent form occurs with reduced efficiency, indicating that the presurned conformational transition requires metabolic energy. Interestingly, metabolic energy is not required to preserve the stability of wild-type CFTR

after it has undergone the confoxmational transition. Chaperones - Lukacs et al. (1994) proposed that the two species of core- glycosylated CFTR (maturation-competent and maturation-incompetent) may differ in their tertiary conformation and/or in the state of association with other components of the ER. This hypothesis was Merdeveloped by Ward and Kopito (1994) who suggested that the inefficiency of maturation of wild-type CFTR reflects the kinetics of CFTR folding, that generally occurs in the context of molecular chaperones (Welch and Brown, 1996). Chaperones are a class of proteins which, during the translation process, transiently associate with nascent polypeptide chains upon initial protnision fiom the ribosome. This interaction inhibits mis-associations with other folding polypeptide chains that are present in the ce11 at high concentrations. The various classes of molecular chaperones share the property of interacting with other proteins in their non-native conformations, apparently by recognizing hydrophobie sequences or surfaces that are exposed to solvent in the unfolded,

but not in the native, state. Since chaperones do not recognize a very specific consensus

sequence, they can interact with many different proteins. Apparently chaperones only increase the yield of correctly folded protein by suppressing off-pathway reactions that compete with the productive folding pathway. They do not appear to act as folding catalysts since the major rate limiting transition state of protein folding remains unchanged in the presence of chaperones (Martin and Had, 1993). It is suggested that in addition to their active participation in the folding process, molecular chaperones also serve as a type of 'quality control system', recognizing, retainllig and targeting rnisfolded proteins for their eventual degradation (Hammond and Helenius, 1995; Welch and Brown, 1996). Three of the most widely studied chaperones are Hsp 70, -60, and calnexin, dl three of which bind ATP during the cycle of interaction and release of the af5ected protein. Thus far, newly synthesized Cmhas been observed to complex with the cytosolic chaperone Hsp70 and the ER-membrane chaperone calnexin, but not with the ER-luminal chaperones BiP and Grp94. Whether CFTR folding involves sequential or cooperative interactions of these chaperones and whether other chaperones also participate in the process remains to be seen. In both cases the association is restricteci to the immature foxm (band B) of CFTR. For Hsp70, limited evidence was provided that wild-type CFTR dissociates fiom the chaperone before its transport to the Golgi, whereas the complex with M508-CFTR is retained in the ER and AF508-CFTR is rapidly degraded in a pre-Golgi non-lysosomal cornpartment (Yang et al., 1993). Based on CO-immunoprecipitation and CO- sedirnentation through glycerol density gradients, newly synthesized wild-type and AF508- CFTR molecules also associate specifically with calnexin (Pind et al., 1994). The interaction is transient (less than 2.5 hours) but of roughly equal duration for wild-type and mutant CFTR. When a puise-chase experiment is perfomed and ce11 iysates fkom selected chase times are separated on glycerol gradients, the distributions of immature wild-type

CFTR and AF508-CFTR are initially very similar and both patterns involve association with cainexin as observed with anti-calnexin anthdies. AAer 45 and 90 minute chases, the distribution of AF508-CFTR across the gradient is unchanged, indicating that it remains included in complexes with cahexin throughout its life-tirne. In contras< the distribution of immature wild-type CFTR is shifted toward the lighter end of the gradient to a position intermediate between the initial immature-CFTR/calnexin complex and cahexin-fkee mature CFTR. However, since at these later time points immature wild-type CFIR recovered in anti-calnexin immunoprecipitates exhibits a distribution similar to that of immature wild-type CFTR in immunoprecipitates, it is suggested that complete dissociation fiom calnexin is not responsible for the shifi to the intermediate position (Pind

et ai., 1994). The shift must represent a step in the maturation of CFTR that is mattainable by hF508-CFTR, but it is unclear what it is. From the observed kinetics it is not understood what the role of the chaperones in the quality control of CFTR is and, although the end result of the interaction with the chaperone is either maturation or degradation, the cause- effect relationship is unclear. Bot. CFTR molecules that mature and those that are degraded interact with the chaperones for a similar arnount of time, so that prolonged retention is not

the mechanisrn that leads to degradation. Somehow the AF508 mutation prevents the maturation of the small fiaction of CFTR that nomally occurs, but it is not known if this is due to a slight alteration in the folding ability of CFTR or due to some other process

(Lukacs et al., 1994). An alteration in the folding ability of AF508-CFTR is only suggested, but cannot yet be verified. Degradalion of CITR - Most proteins retained in the ER are eventualiy degraded (Klausner and Sitia, 1990). In the case of CFTR this degradation is very rapid and appears at least in part to be mediated by the ubiquitin-proteasorne pathway (Jensen et al., 1995; Ward et al., 1995). In this pathway, the a-carboxyl groups of ubiquitin are reversibly joined to the

&-amino groups of acceptor lysines in the protein destined to be degraded. This step is energy dependent and generally occurs several times in repetition, forming a chah of ubiquitin molecules. Multi-ubiquitinated proteins are then broken down by the cytosolic 26s proteasome complex, which is a large supramolecular complex (Hochstrasser, 1995). The proteolytic component, the 20s proteasome, which contains multiple peptidase activities that function together in proteolysis, is inhibited by N-acetyl-L-leucinyl-L- leucinyl-L-norleucind (ALLN) (Wileman et al., 1991) and lactacystin (Fenteany et al., 1995). The ubiquitin-proteasorne pathway appears to be involved in the degradation of immature CFTR since ALLN or lactacystin treatment of HEK-293 or CHO cells expressing wild-type or AF508-CFTR leads to a several-fold increase in steady-state levels of the immature band B. However, band B increase is not accompanied by an increase in the mature band C (Jensen et al., 1995; Ward et al., 1995). Nearly all of the spared material is polyubiquitinated and insoluble in nonionic detergents. In the in vivo situation, polyubiquitination also appears to be required for CFTR degradation because coexpression of AF508-CFTR with a dominant negative ubiquitin mutant that disallows polyubiquitination results in a massive accumulation of AF508-CFTR. Again. despite this increased accumulation, most AF508-CFTR is still found in the insoluble fraction and the treatment does not promote formation of soluble, mature CFTR (Ward et al., 1995). Pulse- chase experiments suggest that the build-up of band B under al1 three conditions is the result of slowed degradation. Interestingly, a different member of the same inhibitor class, MG- 132. completely blocks the formation of the mature band C (Jensen et al., 1995). Thus, ALLN/lactacystin cause a significant inhibition of the rapid ER degradation of immature CFTR, whereas a related inhibitor, MG-132, totally blocks conversion to a maturation- competent form. Possibly, the proteolytic event that is blocked by MG432 corresponds to the ATP-dependent step described by Lukacs et al. (1994) that is needed to move core- glycosylated CFTR into the protease resistant state that eventually escapes the ER In fact, maybe one can make one more association and suggest that the core-glycosylated wild-type CFTR that shows a shift on the glycerol gradient of Pind et al. (1 994), but is still associated with calnexin, is the result of this same proteolytic step. Maybe this is the point in CFTR maturation where chaperone association and CFTR maturation/degradation converge and do so differently for wild-type CFTR and mutants such as AF508-CFTR.However, this is pure speculation and thus far no CFTR-cleavage is known to occur during its synthesis, suggesting that if the cleavage is necessary, it might be on chaperones or other interacting ER proteins. In addition to the two proteolytic pathways identified by ALLN or lactacystin and MG-132, Jensen et al. (1995) hypothesized that there mut be one more proteolytic pathway. They observed that der initial slowing of CFTR degradation with ALLN or lactacystin the protein is dl eventually degraded in the contuiued presence of large doses of the compounds. This third pathway is ATP-independent since it is very active in ATP- depleted cells. Rescue of Misprocessed Moledes - In ce11 culture systems several attempts have ken made to rescue misprocessed CFTR mutants fiom king completely degraded to a situation that is similar to wild-type CFTR in which a hction of the molecules matures fiom the ER. AF508-CFTR is nodly processed when expressed in Xenopuc oocytes (Dnimm et al., 1991) or insect cells (Bear et al., 1992b). Based on the hypothesis that in those systems processing is facilitated due to better folding at the iower temperature at which the cells are grown (26OC rather than 37OC), AFS08-CFTR expressing rnamrnalian cells were transferred to such decreased temperatures for 48 hours. The treatment does indeed allow maturation of a fiaction of the mutated molecules and hctional channels can be observed on the ce11 surface by electrophysiologicai measurements (Denning et al., 1992b). However, the processed AF508-CFTR channels have a six-fold increased tum-over rate at the ce11 surface when compared to wild-type CFTR (Lukacs et al., 1993) and evidence has ken put forward that the temperature decrease affects expression of various channel systems (Egan et al., 1995). Temperature sensitivity of the conformation of nascent proteins and their subsequent processing and transport have been reported previously (Machamer and Rose, 1988; Ljunggren et al., IWO). Like the decrease in temperature, an approach that attempts to promote proper folding of AF508-CFTR is the application of chernical chaperones. Chernical chaperones are low molecular weight compounds that are known to stabilize proteins in their native conformation. Reagents which were found to be effective in supporting AFSO8-CFTR maturation inc lude glycerol, deuterated water, dimethylsulfoxide, and trimethylamine N- oxide. To observe an effect, relatively high concentrations of the chernicals have to be applied. Unfortunately, this causes siflcant cytotoxicity with viabilities of approximately 75-90% king obsewed, depending on the cell type (Brown et al., 1996; Sato et al., 1996).

Compounds such as glycerol work because they tend to be excluded fiom the immediate vicinity of an oiigopeptide. As a result, at high concentrations, giycerol will act to increase the relative hydration around the polypeptide. In response, the polypeptide wiil tend to decrease its relative surface area by an increase in its self-association or tighter packing (Welch and Brown, 1996). It is theorized that both temperature and chernical chaperone treatrnent allow the mutant protein to adopt a native-like conformation, thereby resulting in its release fiom the ER (Brown et al., 1996). In fact, Sato et al. (1996) demonstrated that

accumulation of mature AF508 CFTR in glycerol-treated cells is not the result of an ef5ect

on mature protein that accidentally matured fkom the ER, but rather due to stabilization of the immature form and prolonging its half-life.

A third method that was found to allow some proteins to mature to the ce11 surface is the flooding of the control machinery by stirnulating over-expression. This approach was

prompted by the observation that in highly expressing Vero cells some AF508-CFTR molecules are processed (Dalemans et al., 1991). In general, in mammalian expression

systems. the same result can be achieved by stimulation of over-expression with sodium

butyrate (Cheng et al., 1995). The mahired AF508-CFTR is abundant enough to be detected

functionally as well as by the presence of band C following in vitro phosphorylation with y-

["PI-ATP. Sodium butyrate ailows upregulation of various genes, including the mdr-1 gene encoding P-glycoprotein (Mickley et al., 1989; Bates et al., 1992). It has recently ken admitted for clinicd tesîing to promote expression of the fetal globin gene to combat P- globin disorders (Perrine et al., 1993). Sodium butyrate affects several cellular processes (Knih, 1982), but its predominant mechanism of action involves inhibition of histone deacetylase with the subsequent hyperacetylation of histones. This promotes relaxation of histone-DNA interactions, thus facilitating easier access of the transcriptional machinery to the DNA (Candido et al., 1978; Sealy and Chaikiey, 1978; Vidali et al., 1978). However, the full mechanism is not completely understood and must be regulated more tightly since only some genes are upregdated by the drug treatment.

Although al1 of the methods described above process hF508-CFTR to the ce11 surface at levels which are sub-wild-type, these concentrations may be sunicient since expression as low as 10% of normal appears to restore no& chlonde currents (Johnson et al.. 1992). Synthesis of the CFTR Protein - The understanding of folding mechanisms of CFTR is limited, but knowledge regarding the initial steps of CFTR synthesis is Whially non- existent. In mammalian cells, moa membrane proteins are inserted CO-translationally into the membrane of the ER. These processes are initiated by binding of the signal sequence of the nascent polypeptide chah to a signal recognition particle (SRP). The resulting ribosome-nascent chah-SRP complex is then targeted to the ER membrane by an interaction with the membrane-bound SRP-receptor. At the membrane, the signal sequence is released fÏom SRP in a GTP-requiring step, and the nascent polypeptide is transferred into the ER translocation site, an aqueous channel through which the nascent polypeptide traverses the membrane. Membrane proteins remain anchored to the lipid bilayer by a hyclrophobic stoptransfer or signal-anchor sequence (Martoglio and Dobberstein, 1995). Only recently, a ce11 fiee translation system provided initial evidence that CFTR's membrane targeting and insertion process occurs via an SRP-mediated pathway and that CFTR utilizes an intemal uncleavable signal sequence for this process (Chen and Zhang, 1996).

Apical Processes in CFTR Expression Apical Targeting - CFTR molecules that escape &om the ER quality control machinery are targeted to the apical membrane of wet epithelia, but the precise mechanisms underlying this process have not been delineated and the determinants of targeting are not yet known (Riordan, 1993). When expressed in heterologous non-polarized cells, the wild- type CFTR protein follows the route of other integrai plasma membrane glycoproteins fkom the ER, through the Golgi apparatus, to the celi surface. However, in some epithelial ce11 lines, such as the colonic cell he HT-29, polarization appears to be an important requirement for îageting. In unpolarized and polarized cells the same amounts of CFTR rnRNA and protein are expressed, however, polarized ceUs respond to CAMP stimulation whereas unpolarized cells do not. It is found that in unpolarized ceh CFTR is retained at a perinuclear location, indicating that a peripheral targeting mechanism controls the progression of CFTR to the apical membrane and that the process does not become active mtil epithelid cells polarize (Moms et al., 1993a,b). Although non-epithelial cells are non- polarized by definition, it has been show repeatedly that exogenous expression of CFTR generates CAMP-activated chlonde conductance in these cells. Thus, mechanisms that retain CFTR within epithelial cells until pola.rization occurs are not expressed by non- epithelial cells. Recycling and Recmitwzent - Once localized to the apical membrane, CFTR undergoes fairly rapid recycling between the plasma membrane and an apically localized intracellular compartment. The presence of CFTR in an endosomal compartment has been demonstrated functionally (Lukacs et al., 1992) and irnrnunochernically (Webster et al., 1994). With both methods of estimation, the CFTR channel density per unit length of plasma membrane and subapical vesicle membrane is similar, but since the subapical compartrnent contains more total surface, the intracellular CFTR expression may exceed that of plasma membrane expression in the resting cells. To estirnate the speed of protein cycling between the plasma membrane and the endosomal compartrnent, cycling cm be stopped by cooling to 4"C, the exposed surface proteins are prepared for biotinylation with periodate, the cycling is then continued at 37*C, again stopped at 4OC, and the proteins remaining on the surface are biotinylated. This demonstrated thot 50% of ce11 surface CFTR is intemalized within minutes and mavailable for biotinylation (P~ceet al., 1994). The recycling is likely to occur via the clathrin-mediated endocytic pathway because of the rapid kinetics of intemalization. In fact, CFTR has been observed in clathrin-coated vesicles

irnmunologically and by fusion with planar lipid bilayen (Bradbury et al., 1994). interestingly, elevated CAMP levels inhibit the intemalization of CFTR (Prince et al., 1994)- demonsû-ating that CAMP utilizes another mode of regulation in addition to direct stimulation of CFTR via PKA. Similar regulation of internalization of the glucose transporter, GLUT4, has been reported (Blok et al., 1988). The inhibition requires normal chioride channe1 function since it is disailowed by mutations that disturb CFTR chloride channel activity or by low levels of cellular chloride (Prince et al., 1994). Although there was some disagreement with this observation (Santos and Reenstra, 1994) it has recently been confimied with epitope tagged CFTR (Howard et al., 1996). Cell surface CFTR was observed to elevate two- to three-fold on CAMP-mediated stimulation in a tirne course that resembles the time needed to obtain maximal stimulation in whole-ce11 experiments. The increase on the ce11 surface is not only due to an inhibition of endocytosis, but also due to recruitment fiom the subapical cornpartment (Sorscher et al., 1992; Bradbury et al., 1992a,b; Fuller et al., 1994; Howard et al., 1996). It is clear now that exocytosis is stirnulated by CAMP, but suggestions that CFTR actually regulates cellular exocytosis (Bradbury et al., 1992b) could not be confirmed @ho et al., 1993). The recruitment is dependent on intact rnicrotubules, but not on intact microfilaments (actin) (Tousson et al., 1996) and may be partially regulated by the heterotrimeric G protein Ga,-zwhich was found to inhibit exocytosis resulting in decreased chloride conductance (Schwiebert et al., 1994a). inhibition of Ga,-?has the opposite effect.

Additional Functions of the CFTR Molecule

Classes of CF-causing Mutations - In addition to the most cornrnon US08 mutation, over five hundred different gene alterations have ken identified in CF patients (CF Genetic Analysis Consortium, persona1 communication), many of which were detected only in a single individual or a very srnail group of patients. Welsh and Smith ( 1993) have classified these mutations into four groups, based on the defect that they impose on the CFTR gene product. Members of the first class of mutations introduce a premature termination signal which will either result in unstable mRNA and no detectable protein expression or the production of a trmcated or aberrant protein, which again tends to be very unstable. Class two mutations caw defective protein processing, disailowing hction at the site of action. Thus fa.no class two mutants have been identified that are processed al1 the way through the Golgi and retained at a pre-apical site other than the ER. CFTR molecules with the third class of mutations show defective regulation. Interestingly, to date mutations have not been reported that cause a constitutively open channel, possibly indicating that such a phenotype either is lethal due to manifestations such as secretory diarrhea, or causes symptoms completely different fiom regular CF-associated problems. The final class of mutations results in altered anion conduction and is generally found in the predicted TM regions of CFTR. Although there is a trend for mutations in the same areas of the protein to cause common problems, the sirnilarities are not general enough to make predictions of the functional defect due to a novel mutation. Furthermore, not al1 mutations within the same class produce the same phenotype. Often there is even variability of symptoms &om the sarne mutation (Parad, 1996), which may be due to different genetic backgrounds

(Romahel et al., 1996) andior varying environmental factors (Tsui and Buchwald, 1991 ). A Merreason that it is difficult to predict the impact of a mutation on the phenotype may be that such interpretations are generally based on the assumption that clinical problems are purely based on the altered chloride channel activity of CFTR. However, recent realizations suggest that CFTR is involved in additional activities, the modification of which could be the cause of some CF-associated symptoms. Regdation of Sodium Conductance Although the disease results fiom a molecular defect in a chlonde channel, a universai hding of the CF ainvay phenotype is increased sodium absorption of pulmonary

epithelia. This was initiaiiy amibuted to an aiteration of driving forces due to the decreased

chloride pemeability, but neither blockade of chloride channels in normal tissues with pharmacological inhibiton, nor 'blocking' in cornputer models of airway epithelial hction can mimic the observed PDs of CF lung epithelia (Boucher, 1994). In fact, it was reaiized that the increased sodium permeability was due to overactive apical, amiloride-sensitive sodium channels (ENaCs) which have an increased Po (Chinet et al., 1994), but unaltered levels of expression (Burch et al., 1995). Proof for the subsequent suggestion that the CFTR defect in CF patients has a direct impact on ENaC activity had to await cloning of the three minimal subunits of ENaC (Canessa et al., 1993; 1994). To investigate a regdatory function of CFTR on ENaC, both channels were expressed in Madin Darby canine kidney (MDCK) epithelial cells (Stutts et al., 1995). Expression of ENaC alone generates large, amiloride- sensitive sodium currents that can be stimulated by CAMP, similarly to the situation observed in CF ainvay epithelia (Boucher et al., 1986). However, coexpression of ENaC with CFTR generates srnaller basal sodium currents; these sodium currents are Mer inhibited by CM.Similar results were observed in oocytes, with the additional finding that AF508-CFT' cannot inhibit sodium currents (Mal1 et al., 1996a). Subsequent reconstitution of CFTR and ENaC into lipid bilayers demonstrated that the dom-regulation in sodium transport by CFTR occurs via several effects (Ismailov et al., 1996a). The basal Po of ENaC is decreased and the extent of Po elevation following PKA-mediated phosphorylation is decreased. In addition, the inward rectification of the gating of ENaC, nonnally induced by PKA-mediated phosphorylation, is negated thus dom-regulating inward sodium currents. The interaction of CFTR and ENaC occurs independently of whether CFTR is conducting anions, although the nonconductive GSSID-CFTR mutant cannot substitute for wild-type CFTR. Sidedness experiments indicated that a parallel orientation of CFTR and ENaC may be critical for their interaction. Furthemore, it was observed that additional associated regdatory elements may influence the interaction since the results are slightly difîerent depending on the source of ENaC (recombinant versus an

immunoprecipitated cornplex). Thus, the increased sodium conductance in CF airways appears to be the result of the absence of ENaC inhibition due to lack of CFTR on the ce11 surface. This hding provides a general mechanism of how normal pulmonary epithelia can coordinate the opposing processes of basal sait and water absorption due to sodium absorption and stimulated salt and water secretion due to chloride secretion (Boucher et al., 1994). CFTR may be the 'switch' that balances the rates of sodium absorption and chloride secretion to properly hydrate airway secretions in normal airway epithelia (Stutts et al., 1995). How communication behveen CFTR and ENaC occun is unknown, but the fact that it takes place in a reconstitution system suggests that direct physical contact is involved. Regdation of the Outward Rect~per History - Initial single-channel patch-clamp experiments on prirnary airway cultures described the presence of an outwardly rectifjhg anion channel that is activated by intracellular CAMP and has a 40 pS conductance at O mV (Frizzell et al., 1986). Ln excised patches fiom normal cells, that channel is activated by PKA or depolarization, but in CF cells it cm only by stimulated by depolarïzation, not by PKA (Schournacher et al., 1987).

Hence, this channel was presumed to be CFTR. However, Ward et al. (1991) did not observe any correlation between the presence of the outward rectifier (ORCC)in digerent ce11 types and the ability of CAMP to increase chloride conductance in those cells, or between the number of ORCCs in different ce11 types and the level of CFTR expression. Activation of the ORCC by phosphorylation is erratic, often takes minutes to work (Wine, 1995), and no traces have ever ken published showing reversible activation of ORCC by CAMP on-ce11 or deactivation by dephosphorylation in excised patches (Hantahan et al., 1994). Furthermore, the ion selectivity and response to blockers is different for the ORCC in patches relative to macroscopic transepithelid anion conductances (Hanrahan et al., 1994). As a result of these confushg findings most researchers dropped their studies 'with relief (Wine, 1995) when it was demonstrated that CFTR is an intrinsic low conductance, non-rectifying chloride channel with properties that fit both CAMP-rnediated whole-cell currents and the chloride conductance that is missing in CF ceils. Activation of Outward Recrifer - However, ORCC and CF research were eventually reunited by a report that expression of a recombinant CFTR gene in CF branchial epithelia not only introduces the typicai CFTR currents described above, but aiso restores PKA- mediated activation of the ORCC (Egan et al., 1992), suggesting that CFTR regulates more than one conductance pathway in airway tissues. One possible explanation for such a hding is that the CF gene encodes both channels (Egan et ai., 1992). This theory was disproved by Gabriel et al. (1993) because the ORCC was found to be present in CFTR knockout mice. In this setting it was again observed that ORCC is regulated by PKA in membrane patches fiorn normal, but not fiom knockout ceils, suggesting that CFTR may be involved in this regulation. Dissection of measured currents demonstrated that in a total ce11 setting both CFTR and ORCC contribute to CAMP-activated whole ce11 currents, with ORCC canying up to 40% of the CAMP stirnulated chloride conductance (Schwiebert et al.,

1994b); other laboratories estimate the in vivo contribution of ORCC at levels of -4% (Kunzelmann et al., personal communication). Although the ORCC has not been cloned, Iovov et al. (1995a,b) described a procedure for the simultaneous isolation and reconstitution of CFTR and ar, outwardly rectifyuig channel, possibly ORCC. Lf CFTR is imrnunoprecipitated fiom this preparation before reconstitution, ORCC cannot be activated by PKA. Readdition of purified CFTR rehuiis the regulatory mechanism, but only with wild-type CFTR, not with the non-functional mutant G55 ID-CFTR (Jovov et al., 1995a,b). CFTR antibodies can also inhibit activation of ORCC, but only if added before the initial

PWATP addition. Once ORCC is active, the antibody oniy inhibits CFTR, not ORCC. Recently, Schwiebert et ai. (1995) presented data to propose a mechanism for the functional relationship of CFTR and ORCC, suggesting that CFTR triggers the transport of ATP out of the cell which then stimulates ORCC through a P2U purinergic receptor- dependent signaling mechanism. Such a scenario is possible because intracellular ATP concentrations range fiom 2-5 rnM whereas nanomola. concentrations of ATP in the extracellular space can activate purinergic receptors (Schwiebert, 1996). The report demonstrated that in whole-ceii patch clamp recordings of nomal ainvay epithelial cells, CAMPactivates CFTR (Schwiebert et al., 1995). When intracellular ATP concentrations are increased fiom 1 to 10 mM, initially absent ORCC currents can also be recruited (a minimum of 2.5 mM ATP is required). It appears that the activation of ORCC depends on an efflux of ATP because the presence of the CFTR inhibitor glibenclamid prevents ORCC recruitment as does trapping of extracellular ATP by hexokinase (cleaves y-phosphate of ATP and donates it to glucose) or apyraçe (ATPase/ADPase isolated f?om potato). In a single-channel setup, hexokinase is ais0 observed to prevent activation of ORCC by PKA if present in the extracellular solution (pipette). Furthemore, 500 nM of extracellular ATP alone in the absence of CAMP activates ORCC in whole-ce11 recordings in both wild-type and CF cells. The suggestion that the receptor involved is a P2Ureceptor is prompted by the relative potency of various nucleotides. In retrospect, it had been previously reported that extracellular ATP can activate various routes of chloride secretion, including ORCC via a purinergic receptor and CFTR, possibly via an A2-adenosine receptor (Stutts et al., 1992; 1994; 1995; Cantiello et al., 1994). Thus, CFTR appean to be involved in providing the extracellular ATP that is required to activate ORCC. In fact, evidence has been presented that CFTR is a dual chloride / ATP channel, which cm conduct ATP, or at least regulate a tightly associated ATP channel. In excised patches with ATP as the only charge carrier, single-channel currents activated by PKA can be detected, which are inhibited by the CFTR inhibitor giybenclarnide; conductance and reversal potential suggest an Am* permeability approximately one half as large as for chloride. CAMP mediated release of radiolabeled ATP cm also be observed in the whole-ceIl situation for wild-type expressing cells, but not

for mutant expressing ceils (Schwiebert et al., 1995). Is ClTR an ATP Channel? - Initial evidence that CFTR can conduct ATP was provided by Reisin et al. (1994) who observed single-channe1 currents in the presence of asymmetrical chloriddATP concentrations, which to them were indicative of the same conductive pathway king responsible for both ATP and chloride movement. Single- channel patch-clamping of CFTR in the ER or in CHO membranes also measured ATP conductance (Pasyk and Foskett, 1995). Furthemore, direct ATP release assays demonstmted ATP transport in moue mammary carcinoma cells only afler expression of CFTR in those cells (Prat et al., 1996). This is in sharp contrast to an earlier study that indicated that ATP movement across T84 cells, primary cultures of ainvay epithelia, and 3T3 fibroblasts does exist, but is not influenced by CFïR expression (Takahashi et al., 1994). Additional, well controlled experiments fiom several laboratories could not detect ATP transport by CFTR in intact organs, polarized human lung ce11 lines, stably transfected rnammalian ce11 lines, or planar lipid bilayen reconstituted with CFTR potein (Grygorczyk et al., 1996; Li et al., 1996b; Reddy et al., 1996). In al1 systems the general mechanism of investigation was substitution of chionde with a chloride-fiee ATP solution which results in ceasing of the observed CFTR currents. Combined applications of ATP and chlonde also produces currents that are consistent with the sole movement of chloride, but no translocation of ATP (Grygorczyk et al., 1996). These studies suggest that the currents originally interpreted as king mediated by ATP 80w through CFTR may represent some combination of currents carried through CFTR by ions other than ATP or ATP currents through channels or permeation pathways other than CFTR. Such pathways may be related to CFTR expression, but the relationship is probably not obligatory (Reddy et al., 1996). Overall, much controversy currently exists as to whether CFTR can function as an ATP channel, but one strong argument that stands against this theory is that the size of the ATP anion (Bull et al., 1972) is much larger than the estimated diarneter of the CFTR pore (Hanrahan et al., 1994). It is, however, still possible that CFTR moves ATP via a pathway that is distinct fiom the chloride pore. Rule in Mucin Secretion Some attempts to ident* the underlying pathophysiology of CF pulmonary disease have focused on the mechanisms of abnody thick mucus production and Mpaired mucocilliary clearance (Engelhardt et al., 1994). Mucocilliary clearance fiom the airways is likely to be dependent on the quantity, composition, and viscoelastic properties of mucus and rnay be related as well to the rate of salt and water secretion near the sites of mucus production. The localization of CFTR in the mucus-producing submucosal glands of human lungs suggests a relationship between CFTR dysfunction, abnormal mucus properties, and lung disease. However, it is not clear how the presurned fiuiction of Cr- in sait and water secretion impacts on the viscoelastic properties and/or clearance of secreted mucus in CF. Several pathways have been proposed and most likely several do contribute to the overall problem. The simplest interpretation is that of a problern of flushing. In submucosal glands, mucins produced by the rnucous cells are normally flushed out by water that originates fiom serous and epithelial cells in nomal lungs, but these cells show decreased iodwater fluxes in the CF cells (Boucher, 1994; Finkbeiner et al., 1994). It is noteworthy that both serous and mucous cells show CFTR expression, but levels are higher in serouç cells. Beyond this, experimental evidence fiom mucus glands of £iog skin has indicated that saltlwater secretion and mucus secretion rnay be tightly regulated processes that influence each other (Engelhardt et al., 1994). Thus, alteration in overall composition rather than merely lack of water in epithelial secretions may be fundamental in causing the viscous mucus. ui agreement with this proposal, a defect in B-adrenergic stimulation of mucin and serous protein secretion fiom submandibular salivary tissues of CF patients was observed (McPherson et al., 1986). In healthy individuals, tracheal epithelial cells that have a serous phenotype synthesize and secrete antibacterial proteins such as lysozyme and lactoferrîn. These proteins, together with glycoconjugates, are stored in intracellular granules and their secretion is subject to P-adrenergic stimulation. CF ceb still induce CAMP upon P- adrenergic stimulation, but glycoconjugate secretion does not occur (Mergey et d., 1995). Overexpression of CFTR in gdbladder epithelial cells does upregulate the P-adrenergic stunulated mucin secretion by those cells (Kwer et al., 1994) and mucin secretion cm be inhibited by an anti-CFTR antibody (Mills et al., 1992). Furthemore, adenoviw rnediated gene transfer of CFTR to CF akay celis restores an otherwise absent PKA regdated secretion of glycoconjugates (Mergey et al., 1995). 'This evidence supports the concept that CFTR does influence glycoconjugate secretion and that a subsequently altered macromolecular composition of the airway fluid does contribute to the CF phenotype. In addition, Cheng et al. (1989) presented evidence that those glycoconjugates which can be secreted in CF epithelia via non-PKA dependent pathways do have an altered make-up, showing increased sulfation. This oversulfation appears to be the result of a perturbation in intracellular sulfate activation or tramfer of activated sulfate to glycoconjugates (Mohapatra et al., 1995). In most orgm mucus problems may arise fiom the above factors, but in the lungs an additional and possibly the major contribution to the altered viscosity of the ASF cornes fiom the bacterial infection and subsequent ce11 lysis and release of viscous DNA. It is not understood if the altered ionic (Smith et al., 1996; see section on 'Physiolog): of the

Airways ') or glycoconjugate (Engeihardt et al., 1994) ASF composition predispose the CF lung to bacterial infections, or if CFTR itself is a direct player in this process, as suggested by Pier et al. (1996): One mechanism to clear bacteria fiom the lungs is the binding and intemalkation of respiratory pathogens by epithelial cells followed by desquamation. This uptake can be observed for the most common CF infectant, P. aeruginosa. Uptake appears to occur via its lipopolysaccharide-core oligosaccharide, and is defective in CF cells (Pier et al., 1996). The defect can be reveaed when the CF cells are grown at 26OC to promote CFTR processing. Interestingly, the CFTR-mediated uptake appears to be specific for P. aeruginosa, because other bacterial pathogens are equaliy well intemalized by wild-type and CF cells. Other studies have observed increased binding of P. aemginosa to CF

respiratory epithelia (Zar et al., 1995) and an increased number of recepton for P. aeruginosa on CF epithelia (Imundo et al., 1995). Proposed mechanisrns of decreased internalization and increased binding of the bactena may be additive. The growth of P. aeruginosa in general may be promoted by a high amino acid content of the sputum from

CF patients (Barth and Pitt, 1996). The ongin of the fieamino acids is not clear. Addilionaï Proposais In addition to the proposed functions of CFTR described above, the channel has

been associated with eaux of neutral amino acids (Rotoli et al., 1994), water transport

(Hasegawa et al., 1992), and bicarbonate conductance (Poulsen et al., 1994). Furthemore, suggestions have been put fonvard that CFTR is also a regulator of amiloride-insensitive sodium channels (in addition to regulation of amiloride-sensitive sodium channels) (Zhang et al., 1996), is a regulator of a chrommol-dibitable potassium channel (Mal1 et al.,

1996b), and interacts with P-glycoprotein (Higgh, 1995) and the ROM-K' channels (McNicholas et al., 1996). Further studies have to be conducted to evaluate which of these associations are real. To allow the proposed mechanism of ORCC activation, the active complex mut contain CFTR, ORCC, a purinergic receptor and perhaps associated G- proteins since the signal cm be observed in excised membrane-patches. If endogenous phosphatases, sodium channels, and proposed other 'regulaton' and 'regulatees' are associated with the sarne complex, a super-structure of enormous dimensions would be formed. It is also not clear which of the many CF-associated defects are the clinically

relevant ones that have to be addressed in an attempt to cure the disease. A very mild fom of CF, congenital bilateral absence of the vas deferens that only produces male sterility but no other CF-syrnptoms, is associated with normal sodium conductance. it was therefore suggested that maybe only fixing the sodium defect may cure CF (Osborne, 1993).

However, the fact that CFTR-knockout mice have normal Iung function, that is attributed to the upregulation of alternative chloride conductance pathways, suggests that the chloride defect is the one îhat causes the CF problems (Clarke et al., 1994). Furthennore, biopsies fiom CF patients with the same mutations, but different disease severiw show a direct correlation between the magnitude of an altemative chloride conductance in various tissues and the resulting phenotype (Veeze et al., 1994). Also, mutations that only partially retard the CFTK chloride conductance produce a less severe phenotype (Sheppard et al., 1993). Hence, the chloide conductance defect is most likely the most sigrilficant problem in CF. This is consistent with the observation of Wine (1995) that in the three ce11 types that are most profoundly affected by CF, namely sweat duct cells, intestinal crypt cells, and lung serous cells, the CFTR channel appears to be the exclusive fom of apical chloride channel.

Thesis Problem

It is well documented that CFTR is an apically located chlonde channel, that is regulated by the concerted action of ATP binding and ATP hydrolysis at the NBFs and kinase-mediated phosphorylation and phosphatase-mediated dephosphorylation of the R- domain (Hanrahan et d., 1994). However, many unanswered questions remain regarding the precise rnechanisms and molecular events that take place during this regulation. The two unknowns addressed in this thesis are (i) whether minor PKA-phosphorylation sites exist in CFTR that can be called upon under lirniting conditions to act in the activation of the molecule and (ii) whether in addition to the R-domain and the NBFs, a third set of cytoplasmic domains, the cytoplasrnic loops (CLs), may be involved in regulation of the c hloride channel. Although PKA is not the only kinase influencing CFTR, phosphorylation by PKA is a potent stimulus for activation of the channel and is likely to mediate much of the hormonally regulated chloride secretion in CFTR-expressing epithelia (Riordan et al., 1994). CFTR contains ten dibasic consensus sites for potential interactions with PKA, nine of which are clustered within the central R-domain. Studies by Chang et al. (1993) demonstrated that, in vivo, four of the ten dibasic sites receive >95% of the PKA-induced phosphorylation, but only account for half of CFTR's activatibility. If ail ten dibasic consensus sites are removed by mutating the site of phosphoqdation (SerRhr) to Ala, in vivo phospholabeling of the resulting IOSA-CFTR variant ca~otbe detected, although this mutant retains 30% of the wild-type responsiveness to PKA. How is this possible? How can the kinase activate 1OSA-CFTR apparently without phosphorylating the channel? One possible explanation is that activation occurs via a third molecule which is phosphorylated by PKA and then in tum interacts with CFTR. Finthemore, the IOSA-CFTR variant retains al1 the positive charges that facilitate the binding of the kinase. It has been demonstrated that PKA can fonn hi&-&ty complexes with nonphosphorylatable pseudosubstrates (Taylor et al., 1990). Such complexes may induce small conformational changes, suEcient to allow some stimulation of CFTR. A third possible mechanism is that the activation occurs via additional labeling sites that do not adhere to the strict dibasic consensus for PKA interactions and, relative to other sites, are phosphorylated to such a low extent that the labeling cannot be detected by traditional methods. PKA-mediated activation via non- dibasic sites has been demonstrated previously in other proteins (Kennelly and Krebs,

199 1). Some indications favor the theory of additional low-ievel phosphorylation sites contributhg to CFTR activaticn. Viewed in a very sirnplified light, the findings by Chang et al. (1993) indicate that the four major dibasic sites which obtain >95% of the detected phosphorylation mediate 50% of the activation of CFTR by PKA. The remaining six dibasic sites which receive only <5% of the detected phosphorylation mediate another 20% of the activation of CFTR.. Thus, the level of phospholabeling of a site is not a direct reflection of the contribution it will have to function. To rationalize this, one has to consider that the observed labeling is a snapshot of many CFTR molecules that undergo ongoing phosphorylation and dephosphorylation events. If a site appears to be labeled to a lesser extent, then this wuld be a reflection of slower rates of phosphorylation or faster rates of dephosphorylation, but such a site can potentially still contribute to CFTR function. The presence of additional phosphorylation sites is also consistent with the hding of Rich et al. (1993a), that a mutant which simulates phosphorylation at eight dibasic consensus sites through the introduction of negatively charged amino acids (8SD-CFTR) can be mer activated by PKA. The same study suggested that the residual phosphorylation occurs in the

R-domain since a AR-S660A mutant does not respond to Merstimulation by the kinase. However, if carefully considered, the data only shows that the Rdomain is required to ailow PKA-mediated stimulation of CFTR. Thus, our workhg hypothesis is that the residual activation by PKA of 1OSA-CFTR results fiom Merphospholabeling of non-dibasic consensus sites. If present, this labeling mut occur at very low levels since it previously could be detected neither by in vivo approaches (Chang et al., 1993) nor by in vitro approaches (Rich et al., 1993a). To demonstrate the existence of such additional sites we propose to observe residual labeling by accumulating large amomts of in vitro phosphorylated 10SA-CFTR molecules so that the emitted radiation is raised above the limit of detection. If present, the contribution of low level phospho~ylationsites to CFTR fünction will be demonstrated by identimg a specific site which mediates some of this labeling, removing it in the 10SA-CFTR background, and observing the effect on residual CFTR activity in vivo. Such an approach will only work if the residual phosphorylation occurs on specific sites, rather than randomly on different serines and threonines in different molecules. As more knowledge accumulates about the individual modes of regulation through studies such as the investigation of PKA phosphorylation sites, it will also be important to understand how the various domains of CFTR communicate with each other. Some of the idormation processing may occur through direct links due to close proxirnity within the primary sequence of Cmbut hinctional data indicates that very distal domains are also able to influence one another. For exarnple, events at NBFl can modiQ events at NBF2 and vice versa (Wilkinson et al., 1996), and deletion of the R-domain cm suppress the uiactivating effect of a mutation in NBF2 but not of a mutation in NBFl (Rich et al., 1993a). The passing of information between domains distal in the primary sequence most likely occurs via physical contact of the functional regions, but evidence for physical interactions has been very limited and has thus far only been presented in abstract form. Preliminary experiments by Ciaccia et al. (1994) indicated that recombinant NBFl and R- domain peptides cm be CO-precipitated if cwxpressed in the baculovinis expression system. Similarly, preliminary experiments by Ostedgaard et al. (1994) showed that the N- temiinal half of CFTR CO-precipitateswith the C-terminal half if CO-expressedui HeLa cells. By deleting various regions it was seen that for interaction with the C-temiinal half, only the first four membrane spanning segments of the N-terminal haif are required, but not the R-domain, NBF 1, or amino acids 1-76, In addition to the communication between different regulatory domains, uiformation must also pass fiom the regulatory domains to the actual pore forming unit of CFTR. By analogy to bacterial relatives of CFR, it rnight be suggested that such interactions involve a third, previously uninvestigated, set of cytoplasmic domains, the cytoplasmic loops (CLs; Fig. 1.2). For several bacterial permeases the two transmembrane domains and the two regulatory dornains are supplied by four different proteins (Doige and Ames, 1993). The only parts of the trammembrane dornains that are exposed to communicate with the regulatory domains are the CLs. Thus, contact with the CLs provides a potential point of information processing in addition to the partial insertion of the regulatory segments into the transmembrane segments. More direct evidence that such interactions rnight occur cornes fiom the structurally related P-giycoprotein, in which the N-terminal transmembrane domain cm be CO-precipitated with NBFl and the C-terminal transmembrane domain cm be co-precipitated with NBF2, but not vice versa (Loo and Clarke, 1995). Besides serving as potential sites of communication, the CLs of CFTR cm be envisioned to be involved in several other functions associated with loop regions of various membrane proteins. In the voltage-gated sodium channel, removal of three cluçtered hydrophobic amino acids within the inhacellular linker between domains III and IV completely abolishes fast inactivation of that channel (Vassilev et al., 1989; West et al.,

1992). This linker region is predicted to contain 53 amino acids, creating a finictional unit quite similar in size to each of the CLs of CFTR (predicted: 55-65 amino acids). Kontis and

Goldin ( 1993), working on the same chamel, obtained evidence that various neurotoxins mediate their effects by directly interacting with a CL within domain II. Fuahermore, mutations within the CLs of P-glycoprotein alter the hgresistance profile of that transporter (Loo and Clarke, 1994a,b). Such results suggest interactions between CLs and various activators, inhibitors, or substrates, a hding that has also been demonstrated previously in bacterial transporters (Hendenon, 1990; Yamaguchi et al., 1990). In addition, the CLs may exert a functional effect through the5 mere location at the pore opening, contributhg to ion selectivity by providing the necessary charged residues. [moto et al. (1988) demonstrated that acidic residues in the mouth of the sodium conducting nicotinic acetylcholine receptor are critical for regulation of the conductance of the channel. in recent years. additional studies have documented loops to be used as the selectivity filten of ion pores (sumrnarized by MacKinnon, 1995). These are mainly extracellular loops but, in the case of the glutamate receptor ion channel, the ion selective loops are cytoplasmically located. In addition to influencing the movement of ions through the pore, it is possible that charged residues in CFTR loops play some role in ion attraction to the pore, as has been observed in other channels (Riordan, 1992). Interestingly, MacKinnon (1 995) suggested that ion channels are simply enzymes that cataiyze the selective diffusion of ions across a membrane; therefore, the sarne reasons that favor loops to be frequently found in the active sites of enzymes should also pertain to ion channel pores. MacKinnon (1995) views lwp regions of ion channels as areas with hi& potential to contribute to the function of these proteins; areas that provide architectural vesatility to allow chernical groups to be arranged optimally in space to caîalyze a specific reaction. Furthemore, to retum to the initial concept that loops may be involved in the physical contact between different domains, he also observes that immunoglobulins provide an exarnple in which loops comprise the antigen contact surface of the variable domains, thereby allowing molecuiar recognition.

Thus, it is with ease that many potential functions of the CLs of CFTR can be proposeci, but to date no evidence has been put foward that addresses the tme importance of the CLs. Because of the relatively large size of the CLs, their proximity to the pore entrance, and their potential proximity to the regdatory domains, our hypothesis is that the CLs are involved in the function and regulation of CFTR. To test this hypothesis we intend to apply a tool provided by nature. Twenty percent of al1 identified CF-associated mutations are located within the CLs. These gene alteration do cause disease, so they must somehow inhibit regular fiinction of CFTR. This may occur by disailowing proper processing of the protein, by modiwg chloride channel activity, or by afSecting any of the other proposed functions and interactions of CFTR. in this thesis we will reconstruct al1 CF-associated point mutations published for the CLs thus far and evaluate their effect on processing and chloride channel activity of CFTR in the hope that this approach will give us an initial elimpse into the importance of the CLs. Such an approach was beneficial previously in the CI study of trammembrane helices 1 and 6 (Sheppard et al., 1993; Tabcharani et al., 1993) and the NBFs (Drumm et al., 1991; Champigny et al., 1995; Sheppard et al., 1995). The starting point for this investigation wil: be CL4, solely for the reason that CF-associated mutations occur at high fiequency in this domain, which may highlight its functional importance (Mercier et al., 1994a). Data in this thesis have been published in part in: Seibert, F.S., Linsdell, P., Loo, T.W.,Hanrahan, J.W., Riordan, J.R., and Clarke, D.M. ( 1996) Cytoplasmic Loop Three of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Regdation of Chioride Channel Activity. J. Biol. Chem. 271: 27493-27499.

Seibert, F.S., Linsdell, P., Loo, T.W., Hanrahan, J.W., Clarke, D.M.,and Riordan, J.R. (1996) Disease-associated Mutations in the Fourth Cytoplasmic Loop of Cystic Fibrosis Transmembrane Conductance Regulator Compromise Biosynthetic Processing and Chloride Channel Activity. J. Biol. Chem. 271 : 15 139- 15 145.

Seibert, F.S., Tabcharani, J.A., Chang, X.-B.,Dulhanty, A.M., Mathews, C., Hanrahan, J.W., and Riordan, J.R. (1995) CAMP-dependent Protein Kinase-mediated Phosphorylation of Cystic Fibrosis Transmembrane Conductance Regulator Residue Ser- 753 and Its Role in Channel Activation. J. Biol. Chem. 270: 2 158-2 162.

Methods in this thesis have been published in part in:

Chang, X.-B., Seibert, F.S., and Riordan, J.R. (1997) Mutational analysis of phosphorylation sites in CFTR. In: Methods in Enzymology. ABC Transporters: Biochemical, Cellular and Molecular Aspects. In press.

Chang, X.-B., Kartner, N., Seibert, F.S., Kloser, A.W., Kiser, G.L.,and Riordan. 1.R. (1997) Heterologous expression systems for the study of CFTR. In: Methodr in Enzymology. ABC Transporters: Biochemical, Cellular and Molecular Aspects. In press.

Cano-Gauci, D.F., Seibert, F.S., Safa, A.R., and Riordan, J.R. (1995) Selection and Characterization of Verapamil-Resistant Multidmg Resistant Cells. Biochem. Biophys. Res. Comrn. 209: 497-505.

Chapter 1 has been published in part in:

Seibert, F.S., Clarke, D.M., and Riordan, J.R. (1997) Cystic Fibrosis: Catalytic, Channel, and Folding Properties of the CFTR Protein. J. Bioenerg. Biomembr. in press. CHAPTER 2

Materiah and Methods Co~~~tructionof Vectors and Mutants Two different vectors were used for CFTR expression. The recombinant plasmid pNUT-CFTR was assembled by Tabcharani et al. (1991) 6om an onguial constnict by Paimiter et al. (1987). Removal of the human growth hormone gene fiom the Palmiter pNUT vector lefk a SmaI site, imrnediately downstream of a mouse metailothionine" promoter, plus the human growth hormone polyadenylation sequence. The CFïR coding sequence, including nucleotides -72 to 472 1 of the complete cDNA, was inserted by blunt- end ligation into the SmaI site in the sense orientation. Since the original vector contained a mutant dihydrofolate reductase gene under the control of the SV40 early promoter, stable selection of pNLiT-CFTR transfected cells with the chemotherapeutic drug methotrexate was possible. The CFTR coding sequence, again including nucleotides -72 to 472 1 of the complete cDNA, was aiso ùiserted into the transient expression vector pcDNA3 (LnVitrogen) using the Hindm and ApaI sites of the multi-cloning-region. To facilitate rapid segment replacement, a silent mutation was created in pcDNA.3-CFTR at position 3471. The resulting BstEIi restriction site and the constitutive HpaI (nucleotide 2463) restriction site of CFTR allowed replacement of the intervening sequence by different polymerase chah reaction (PCR) hgrnents that encoded 21 nanirally occurring CF mutations in CLs 3

& 4. CL 1 & 2 mutations were created in the same constmct, utilizing either an AvaI site

(nucleotide 126) in combination with a XbaI site (nucleotide 649), or the XbaI site (nucleotide 649) in combination with a BamHI site (nucleotide 1508), depending on the location of the mutation. To insert R-domain mutations into CJTR, the HpaI site

(nucleotide 2463) was employed in combination with both DraIIi sites (nucleotides 1777, 3328) in a three-piece ligation. Mutations that were created in one vector could be shuttled to the other by directional cloning, via both DraIII sites, which have different restriction

II Metallothionines are small cysteine-rich proteins which play an important role in detoxifïcation of heavy metals and heavy metal homeostasis. There are cellular factors that, in the presence of heavy metals, interact with a heavy metal-tesponsive element in the metallothionine promoter so that gene transcription is induced by heavy bivalent metal ions such as cadmium and zinc (Levinson, 1990). sequences (CACNNNGTG, where N can be any nucleotide). PCR was performed as described by Higuchi (1990). Sequences of the PCR hgments were vedïed after insertion into the vector using the T7 Sequencing Kit (Phmcia). For general molecular biological techniques such as restriction digestions, Ligations, plasmid preparations, and transformation of E. coli, the protocols of Sambrook et al. (1 989) were applied. Expression of Mutants HEK-293,COS-1, CHO (Chinese hamster ovary), and BHK @aby hamster kidney) cells were grown at 37OC in 5% CO2 in Dulbecco's rnodified Eagle's medium containing

10% fetal calf sem. Thuty percent confluent ceiis were transfected with 2 pg/rnL of the various vector constmcts using the calcium phosphate precipitation method described by Chen and Okayama (1 987). Briefly, the DNA was suspended in water and precipitated with a final concentration of 125 rnM CaClz and lx BES (Sambrook et al. 1989) at room temperature for 10 minutes. The suspension was then diluted with ten volumes of growth medium and applied to the cells. The final volume depended on the size of the culture dish utilized (e-g.: 10 rnL per 10 cm dish, 2 rnL per well of a 6-well plate). Expression levels could not be elevated by increasing the concentration of transfected DNA. In fact, the most significant factor influencing the transfection efficiency was the confluency of the cells, with sub-confhency being an essential requirement for success. Upon transfection of COS-1 cells, increased ce11 death was observed. This was controlled by exchanging the

growth medium with DNA-fiee medium 20 hours post-transfection and by decreasing the concentration of transfected DNA. However, at concentrations below 1.5 pg of DNA per 1 mL of medium the transfection efficiency becarne greatly compromised. pcDNA- transfected HEK-293 and COS4 cells were cultured for 48 hours prior to further analysis.

In the case of pNUT-transfected CHO and BHK cells, 50 pM and 500 pM methotrexate respectively was added to the medium 72 hours post-transfection (depending on the initial conflwncy the cells had ken split to various dilutions 24 hours post-transfection). Cells continued to grow in the selective medium for twelve days. Sunriving individual colonies were picked and arnpmed in the selective medium. CFTR expression in cells destined for phosphorylation studies was induced with 0-30 rnM sodium butyrate as indicated, 20 hours pnor to the experiment. The wild-type CFTR (BQ2) and 10SA-CFTR expressing CHO ce11 lines of chapter 3 were prepared by Chang et al. (1993). Temperature Shzp adGlycerol Exposure To explore the temperahue/glycerol-semitivity of processing mutants, celis expressing these mutants were shifted to 26°C for 48 hours or left at 37T for 48 hours with the addition of 10% glycerol to the growth medium. A control sample remained at 37OC without glycerol. Protein Detection Confluent cells were lysed in 1% sodium dodecyl sulfate (SDS) containing several protease inhibitors (10 pM E-64, 12 pg/rnL leupeptin, 100 U/mL aprotïnin 0,50 p@mL

AEBSF, 25 pg/mL bentamidine; the sarne composition was utilized whenever protease inhibitors were applied in later rnethods; applied volumes depended on the sample size, for example, 500 pL of SDS were sufficient per confluent well of a 6-well plate). Total ce11 protein content was determined with the BCA Protein Assay kit (Pierce), which is based on a color reaction due to reduced copper ions interacting with bicinchoninic acid. AAer diluting the lysate 1:l with 2x sample buffer (3% SDS, 5% P-mercaptoethanol, 10% glycerol, 62.5 mM Tris-HCl, pH 6.8), it was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analysed by Western bloaing (Towbin et al., 1979) using

M3A7 as the primary antibody. M3A7 is a monoclonal antibody, generated against a fusion protein containing residues 1 197- 1480 of CFTR (Kartner et al., 1992). The secondary antibody was a goat anti-mouse antibody labeled with horse-radish peroxidase (Gibco), that is detected by cherniluminescence with the ECL kit (Amersham Corp.). Optimal CFTR detection with minimal background was achieved by first blocking the nitrocellulose with 'milk' [5% milk powder in TBS (10 mM Tris-HCl, 150 mM NaCl, pH 8) plus 0.5% Tween 201 for 15 minutes, followed by a 2 hour incubation with the primary antibody suspendeà in 'rnilk' at a concentration of 1 pg/mL. Afler 3 washes with TBS plus 0.5% Tween 20 for 5 minutes each, the secondary antibody was added in 'milk' for 45 minutes. The blot was washed 6 x 5 minutes with TBS plus 0.5% Tween 20, 2 x 5 minutes with TBS, and then incubated for 1 minute in the chemiluminescence b&er (Amersham). For less stringent conditions to allow detection of low levels of 1 ISA-4SA-CFTR processing, Tween 20 was omitted fiom the 'rniik' and decreased to 0.1% in the washes. The entire Western blotting procedure was performed at room temperature.

Endoglycosidase H Digestion

Forty-eight hours post-transfection, confluent cells fiom a 6-well dish were washed with PBS (150 mM NaCl, 3 mM KCI, 10 mM Na2HP04x7H20, 1.5 mM

KH2P04)and lysed in 1 ml of a denahiration solution (0.5% SDS, 1% P-mercaptoethanoi, 10 rnM EDTA, protease inhibitors). DNA was removed by centrifugation. Supernatant

(45 pL) was incubated with endo H buffer (5 pL of 10x stock; NEB Biolabs) plus or minus 1 jL endoglycosidase H (NEB Biolabs; 1000 U/a) for 10 minutes at room temperature. After dilution in 2x sample buffer the samples were analysed by Western biotting (Towbin et al., 1979) as described above. Phosphorylafion of CFTR Moderately confluent CHO or BKcells, untransfected or stably expressing various CFTR constructs, were grown at 37OC in 5% CO2 for 20 hours in a-rnodified minimal essential medium (Life Technologies, Inc.) plus 0-30 rnM sodium butyrate (as indicated). After ce11 iysis with 1 mL RIPA buffer (1 % Triton X- 100, 1% deoxycholic acid, 0.1% SDS, 150 mM NaCl, 20 mM Tris-HCl, pH 8, 0.25 rnM phenylmethylsulfonyl fluoride, 2 pg/mL leupeptin, 2 pg/mL aprotinin) per 10 cm dish, the total protein content was determined for each sample. Equal arnounts of total protein were utilized for the subsequent step (corresponding to the amount of the most dilute sample) and adjusted with RIPA buffer to a fmal volume of 1 mL. CFTR was irnmunoprecipitated fkom the lysate by incubation with 2 pg/d monoclonal antibody (M3A7; Kartner et al., 1992) for 2 hours and subsequent addition of 10 PL of packed protein G-Sepharose 4B beads (Sigma) for 1 hou. This 3 hou incubation was performed at 4OC with shaking. The beads were separated fiom the lysate by centrifugation, and washed three times with RIPA b&er to remove background proteins and three times with PKA buffer (140 mM NaCl, 4 mM KCl, 2 mM MgCI2, 0.5 rnM CaC12, 10 mM Tris, pH 7.4) to remove detergents.

Washes were performed as cycles of resuspension (4OC) and centrifugation (room temperature) for 25 seconds at 15,000 rpm. CFTR was then phosphorylated by incubating the beads in 100 pL of phosphorylation buffer (180 nM catalytic subunit of PKA

(Prornega), 20 FM ATP, 10 pCi y-[32~]-~~~(Amersham), 10 pg BSA, 140 mM NaCI, 4 rnM KCI, 2 rnM MgCI2, 0.5 mM CaC12, 10 mM Tris, pH 7.4) for 15 minutes at room temperature. The sample was centrifbged (room temperature), the supernatant was removed, and the beads were resuspended in 1 mL of RIPA-buf5er (4OC). This step was repeated at lest five times to wash out contarninating background phosphopeptides. AAer removing the last wash, the phospholabeled protein was eluted by incubating beads in 40 pL of 2x sarnple buffer for a minimum of 5 minutes at room temperature. Elutions could be improved if two separate 20 pL elution steps were performed. Efficient separation of the sample buffer fkom the beads was achieved by piercing the bottom of the Eppendorf tube, placing the pierced Eppendorf tube into a second Eppendorf tube, and subjecting both to a brief, low speed spin. The sarnple was then analyzed by SDS-PAGE and autoradiography or subjected to Meranalysis. It was observed that non-specific background in the autoradiognph could be decreased by increasing the number of RIPA-buffer washes; for some experiments up to ten post-phosphorylation washes were beneficial. In addition, non-specific adherence of proteins to the protein G beads may be avoided by briefly preclearing the lysate with beads pnor to the addition of antibody. The efficiency of the actual phosphorylation reaction could be slightly increased by lowering the amount of unlabeled ATP, which in some cases did also decrease the amount of background phosphorylation. For our

experiments we could go as low as 15 pM ATP, but below this level, phosphorylation of

CFTR was greatly compromised. In our hands, increasing the amount of PKA or y-[32~]- ATP somewhat elevated the level of CFTR phosphorylation, but was of no practical value since background phosphorylation also increased. Furthemore, performing the

expenment at 30°C, as suggested by the manufacturer, or elongating the incubation time with PKA did not improve phospholabeling efficiencies. Puified R-domain was prepared as described (Dulhanty and Riordan, 1994a) and labeled in solution under the same conditions as whole CFTR protein. The entire phosphorylation mixture was applied directly to the polyacrylamide gel after addition of

1/ 10 volume of 1 0x sarnple buffer.

Cyunogen Bromide (CNBr) Cleavage ofPhosphorylated CFTR FoIIowing protein separation by SDS-PAGE, the phospholabeled protein was transferred to a polyvinylidene difluonde (PVDF) membrane using the buffer system described by Towbin et al. (1979). The PVDF membrane was utilized because it allows eflicient protein elution. Different methods have been described to transfer protein ont0 a PVDF membrane (Mozdzanowski and Speicher, 1990), but in our case the transfer efficiency was not improved by the use of more complicated buffer systems. Afier detection by autoradiography, membrane segments containing CFTR were excised and incubated in the dark for 48 houn in 200 pL of 20 mg/mL CNBr (Sigma) in 70% formic acid at room temperature. Observed variables to optimize the cleavage efficiency: avoid use of aged CNBr (as indicated by yellow colored crystals), increase the concentration of CNBr, elongate the CNBr cleavage time, and occasionally vortex the cleavage solution.

Cleaved protein was eluted fiom the membrane with two 100 minute incubations in 200 pL of 2 pL/rnL trifluoroacetic acid in 70% isopropanol. Occasional vortexing of the elution solution greatly promoted elution efficiency. The cleavage and elution solutions were pooled and evaporated to dryness under a stream of nitrogen. The resulting pellet was washed with double distilled water to remove interfering excess acid, redried, and solubilized in either 2x sample buffer for SDS-PAGE or in RIPA buffer, pH 8, for a second immunoprecipitation with different antibodies. When analyzing the observed banding patterns afier the final SDS-PAGE, it is important to remernber that cleavage at some sites may be ody moderately efficient and partially cleaved entities are commonly observed. The purified R-domain, which was phosphorylated in solution, was also cleaved in solution. The phosphopeptide which was still in the phosphorylation buffer was suspended in approxirnately 1 mL of 50% formic acid, concentrated to 80-100 pL by spinning in Centricon 10 units (Amicon), and mixed 1: 1 with a solution of 40 mg/rnL CNBr in 90% formic acid. Cleavage also occurred for 48 hours in the dark before drykg and washing as above.

An tibody Recognition of CNBr Clemage Products

Radiolabeled, cleaved and eluted proteins were solubilized in RIPA buffer, pH 8, and subjected to a second immunoprecipitation (as above), the difference being that

M3A7 was replaced by either 2 pg/mL P-Ab 6 @olyclonal antibody generated against a synthetic peptide corresponding to CFTR amino acids 724-746) or 2 pg/mL Ll lE8 (monoclonal antibody generated against a CFTR fusion protein containing CFTR residues

341-702; Kartner et al., 1992). The precipitated proteins were solubilized in 2x sarnple buffer and subjected to SDS-PAGE.

Cell Surface Labeling

A confluent 10 cm tissue culture dish of transiently transfected HEK-293 cells was washed with PBS containing 0.1 mM &Cl2 and 1 mM &Clz (+ CM) before a 5 minute incubation with 4 mL of 10 mM sodium periodate in PBS + CM in the dark. The penodate solution was aspirated, the cells were washed with 0.1 M sodium acetate + CM, and overlayed with 2 mL of 1 mM biotin-LC-hydrazide (Pierce) dissolved in 0.1 M sodium acetate + CM. To promote solubility of biotin-LC-hydrazide, it was initially suspended in 100 pL dimethylsulfoxide @MS O). The biotin-LC-hydraude incubation was perfomed for 5 minutes in the dark before stopping the labeling reaction with a 5 minute incubation in 10 mL of 0.1 M Tris-HCl, pH 7.5. To this point al1 steps were performed at room temperature. The cells were washed hvice with PBS and solubilized in

1 mL of buffer 1 (25 rnM Tris-HCl, pH 7.5, 150 mM NaCI, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM EDTA, protease inhibitors) at 4" for 30 minutes with agitation. DNA was removed by centrifugation and CFTR was irnmunoprecipitated as described above. After elution fiom the protein G-Sepharose 4B beads (Sigma) with 2x sample buffer, the sample was split into two parts, both of which were separated by SDS- PAGE and transferred to a nitrocellulose membrane (Towbin et al., 1979). One sample was immunoblotted with the primary anti-CFTR antibody M3A7 (1 pg/mL; Kartner et al., 1992) and the secondary goat anti-mouse antibody labeled with horseradish peroxidase (Gibco) to detect total CFTR in the cells. The nitroceI1ulose with the second half of the sample was blocked for 15 minutes with 2% mik powder in TBS and then for 15 minutes with 2% BSA in TBS plus 0.05% Tween 20. BSA was utilized as a rnilk powder substitute to avoid background reactions. AAer subsequent incubation with streptavidin-conjugated horseradish peroxidase ( 1 :1006; Amersham) in TBS plus 0.05% Tween 20 and 2% BSA, the blot was washed 6 times with TBS plus 0.5% Tween 20, each wash lasting 10 minutes. Both reporter molecules were detected by cherniluminescence.

CFTR Proteolysis in Mem brme Vesicles

Forty-eight hours post-transfection, ten confluent 10 cm dishes of HEK-393 cells were washed twice with PBS before collection by centrifugation. The cells were resuspended in a total of 3 mL of lysis buffer (10 mM Tris-HCl, pH 7.5, 0.5 mM MgCl*, protease inhibitors) and homogenized with 40 strokes in a Dounce homogenizer. After

addition of 2x solution A (2x: 0.5 M sucrose, 0.3 M KCI, 10 mM Tris-HCI, pH 7.5) the

mixture was centnfùged at 6.5 krpm for 10 minutes at 4OC. The supernatant was brought

to a total of 20 mL with lx solution A and the membranes were pelleted by high speed

centrifugation (100,00Og, 60 min, 4OC). The membranes were resuspended in 500 pL of 50 mM Tris-HCl, pH 7.5 to give a total protein concentration of approximately 1-5 mg/mL. Proteins were cleaved by incubation on ice with various concentrations of TPCK-trypsin for various time penods (as indicated) before detection by Western bloaing.

iodide Eflw Studies

The same method was used as descnbed previously (Chang et al., 1993). Briefly, stably transfected CHO cells or transiently transfected COS- 1 cells, grown to confluency in a 6-well tissue culture plate, were washed three times with iodide loading buffer (136 mM Nd, 3 rnM MO3,2 rnM Ca(N03)2, 11 mM glucose, 20 mM HEPES, pH 7.4) and

incubated in 2 mL of this buffer for one hou at room temperature (the loading buffer should be fieshly prepared due to the light-susceptibility of iodide; volumes are per well). The iodide loading buffer was aspirated. For subsequent steps, cycles were performed of overlaying cells with 0.5 mL of iodide efflux buffer (136 mM NaN03, 3 mM KNO3, 2 mM Ca(NO3)2, 11 mM glucose, 20 mM HEPES, pH 7.4), leaving the iodide efflux buffer on the cells for 60 seconds, then aspirating the buffer. The aspirated buffer was disposed for the first ten cycles. Buffer fiorn the following three cycles was collected as individual aliquots in a 24-well tissue culture plate; these samples represented the baseline before CFTR stimulation. The iodide efflux buffer for subsequent cycles was supplemented with 10 pM forskolin (final concentration; lOOOx stock was prepared in dimethylsulfoxide) and these samples were again collected in the 24-well tissue culture plate; generally twelve forskolin-containhg cycles were sufficient to obtain a full activation curve. The amount of iodide in each sample was evaluated with an iodide- specific electrode relative to a set of reference solutions of iodide efflux buffer containing

lod M to 10'~M Nd. Iodide-specific and reference electrodes are generally supplied as separate electrodes. To allow measurement of 0.5 mL of beer in a 24-well dish, we decreased the surface area of the reference electrode by immersing it into a 10 mL syringe, filled with 1 M KNO3, sealed with 1% agarose / 1 M KNO3. Thus, the point of contact with the sample was the narrow tip of the syringe, rather than the wide surface of the electrode. Recently, a single-body iodideheference electrode has been developed (Analytical Systems) which was utilized for some of the measurements. Highest efflux rates were obsewed fiom cultures of moderately confluent cells relative to lower efflux rates fiom sub-confluent or over-confluent cultures. During the efflux procedure, care had to be taken not to scrape cells into the collected aliquot since these cells will continue to release iodide and skew the results. To avoid such a problem, each ce11 line was analyzed in triplicate. It has to be noted that the iodide efflux technique has several limitations. The natural substrate of CFTR is chloride. In the present assay a different anion, iodide, is used because of the ease of detection. An advantage of iodide over chloride is the fact that besides CFTR very few channels are capable of iodide transport, in contrast to the many chlonde transporting proteins on the ce11 surface. Thus, the potential for interfering processes is smaller with an iodide-based method (Rich et al., 199 1). The disadvantage is that CFTR transports iodide with less eficiency than chloride due to a pore block that occurs in the presence of iodide (Tabcharani et al., 1992). Furthemore, some aspects of the anion efflux are poorly understood. Fine et al. (1995) showed that the chloride content of the ce11 influences CFTR activity, because in HT28- 184, cells chloride depletion ïnhibited CAMP activated chloride fluxes to a greater extent than predicted by modified driving forces. The authors suggested that chloride may interact with a non-CFTR protein to regulate fiction of CFTR. The evidence for this is very limited, but if it is the case, it is conceivable that CFTR mechanisrns are somewhat altered in the contrary situation of the iodide emux experiment where cells are loaded with very high levels of anion. We are confident that the currents evaluated in the iodide efflux are CFTR-

rnediated currents because they are absent in mock-transfected cells. However, the iodide

efflux approach varies from the single-channel evaluation of CFTR in the aspect that it is a whole-ce11 technique. In iodide enlw the cellular machinery is intact whereas single- channel patch-clamping utilizes a patch of channel-containing membrane that is excised from the cell. Thus, in iodide efflux experiments such aspects as phosphorylationl dephosphorylation due to non-CFTR-associated endogenous kinases/phosphatases

(Berger et al., 1993; Picciotto et al., 1992; Becq et al., 1994; Reddy and Quinton, 1996a), cellular energy levels (Bell and Quinton, 1993), cytoskeletal interactions (Fischer et al., 1995; Prat et al., 1995), CFTR recruitment (Bradbury et al., 1994; Prince et al., 1994;

Schwiebert et al., 1994a; Webster et al., 1994), and channel cooperativity (Krouse and Wine, 1995) influence the overall measurements. Although rnany of these cellular mechanisms are only poorly understood, they have the potential to affect CFTR measurements in whole cells, which may not apply to the excised patch situation. Both

approaches, the whole-ce11 iodide efflux and the cell-removed single-channel patch-

clamping, are therefore valid approaches, but they are expected to give slightly different results because they evaluate CFTR channels in different environments.

Patch-chmp Studies of CFTR-expressing CHO CeIls

Electrophysiological studies were perforrned in the laboratory of Dr. J. W. Hmahan, McGill University, Montreal, Quebec. Data for the ISSA-CFTR variant was collected by Dr. Cen Mathews and data for CL 3 & 4 mutants was collected by Dr. Paul Linsdell. CHO cells stably expressing wild-type or mutant CFTRs were plated at low density on glass coverslips and cultured under stmdard conditions for 2-5 days before use. Single channel currents were measured in excised inside-out membrane patches (Hamill et al., 198 1 ; Pemer and Neher, 1989). The pipette and bath solutions contained 145 mM NaCI, 4 mM KCI, 2 mM MgCh, 10 mM TES, pH 7.4. Excised channels were activated by the addition of 1 mM MgATP and either 180 nM of the catalytic subunit of PKA (prepared in the laboratory of M. P. Walsh for 11SA-S753A-CFTR studies; Tabcharani et al., 1991) or 75 nM of the catalytic subunit of PKA (prepared in the laboratory of M. P. Walsh for 1SSA-CFTR studies and by Promega Corp., Madison, WI for CL studies). Currents were filtered at 50 Hz using an 8-pole Bessel filter and digitized at 250 Hz. Recordings were made at room temperature (Tabcharani et al., 199 1, 1993).

Measurements of channel open probability (Po) were made fiom recordings that lasted fiom 5-13 minutes at a membrane potential of -30 mV. To ensure a reliable estimate of the nurnber of channels in the patch, 1 mM 5-adenylylimidodiphosphate (AMP-PNP; Calbiochem) was added to the bath solution at the end of each experiment. This nonhydrolysable ATP analogue causes CFTR channels to be locked in the open date in the presence of ATP (Hwang et al., 1993). Calculations were perfonned according to

Tabcharani et al. (199 1 ; 1993).

Openings of CFTR channels occur in bursts, with very short closures within bursts (a few milliseconds) and much longer closures separating bursts (Gunderson and Kopito, 1994; Venglarik et al., 1994; Winter et al., 1994). The mean durations of bursts of openings and of closings betiveen bursts were calculatèii for patches containhg more than one CFTR channel using the following equations:

where to and t, are the mean bunt duration and the mean closed duration between bursts of openings (interburst duration), N is the number of channels in the patch (estimated as described above), t is the total length of the recording, n is the number of identifiable bursts of openings, and x represents the multiplication syrnbol. Bursts were defined as groups of openings separated by closings briefer than 10 ms. Although this method assumes only a single open state and a single hterburst -te, it has ken used successfully to analyze CFTR kinetics fkom multi-channel patches and is of use in identimg gross kinetic differences between different chamel variants. Data were presented as the mean +/- S.E. (Gray et al., 1988). CAMP-dependentProtein Kinase-mediated Phosphorylation of CFTR Residue Ser 753 and its Role in Channel Activation Chapter Summary Hormonally induced activation of CFTR is largely mediated via phosphorylation by the CAMP-dependent protein kinase (PKA). Previous studies showed that

approximately 30% of the wild-type CFTR response to PKA remains upon inhibition of detectable in vivo phosphorylation by mutagenesis of al1 ten dibasic consensus sites

(1 OSA-CFTR; Chang et al., 1993). To identify potential additional sites that do not adhere to the dibasic consensus but are responsible for the remaining activity, an in vitro approach was taken in which large amounts of IOSA-CFTR were phosphorylated with

PKA using hi& specific activity y-[32~]-~~~.Cyanogen brornide cleavage indicated that a large portion of the observed PKA phosphorylation occurred within a 5.8 kDa fragment of the R-domain between residues 722-773. Mutagenic removal of serines at potential

PKA sites in this hgment showed that Ser 753 accounted for al1 of the y-[32~]-labeling of the 5.8 kDa peptide. Replacement of Ser 753 with alanine reduced the level of residual chloride channel activity by a Mer 40%, indicating that phosphorylation at this previously unidentified site indeed contributes to the activation of 1OSA-CFTR. Introduction The chloride channel activity of CFTR is regulated by the concerted action of ATP binding/hydrolysis at the NBFs and phosphorylation/dephosphorylation of the R-domain

(Hanrahan et al., 1994; Hwang et al., 1994). In this mechanism the phosphorylation step is essential, although not sufEcient, to allow opening of the channel and the degree of

phosphorylation appears to serve as a regulator of the extent of activity that cm be achieved

by CFTR (Fischer and Machen, 1994). Hence, the balance between phosphorylation and dephosphorylation events determines the set-point of a metered response. While PKA is not the only kinase iduencing CFTR, phosphorylation by PKA is a potent stimulus for activation of the channel (Tabcharani et ai., 1991) and is likely to mediate much of the homonaily regulated chloride secretion in CFTR-expressing epithelia

(Riordan et al., 1994). In the CFTR molecule there are ten dibasic consensus sites for recognition by PKA (R/K - R/K - X - S*/T*; astensk indicates the site of potential phosphorylation; X represents any amino acid; Ke~ellyand Krebs, 1991), nine of which are clustered within the central R-domain (Riordan et al., 1989). In vivo, four consensus sites were found to cany 95% of the PKA-mediated phosphorylation (Cheng et al., 199 1 ), yet their functional blockade only retards CFTR activation to 50% of the wild-type level. In fact, Chang et al. (1993) demonstrated that a mutant, that had al1 ten dibasic sites eliminated by changing the serine or threonine to an alanine (10SA-CFTR), couid still be activated by PKA. Activation was approxirnately 30% effective compared to the wild-type rnolecule, however, in vivo phosphorylation was no longer readily detectable. The aim of the work presented in this chapter was to address the mechanisrn of the residual sensitivity of IOSA- channels to PKA. Is there any evidence that this sensitivity is mediated by phosphorylation of additional sites within CFTR at levels that are below the level of sensitivity of standard techniques, or does the residual activity solely involve pathways other than the direct phosphorylation of CFTR by the kinase? Resdts and Discussion Expression SIudies - Our strategy to observe low levels of residual phosphorylation involved an attempt to accumulate large amounts of the potential phosphoprotein and to thereby raise the total ernitted radiation, if present, above the limit of detection. CFTR production in the Chinese hamster ovary (CHO)expression system utilized in this study is under the control of a metallothionine promoter. To test whether exposure to heavy metals cm stimulate the metallothionine promoter and thereby elevate CFTR expression, various cations were applied to the CHO ce11 Iine 50-1 that stably expresses AF508-CFTR. This ce11 line was utilized as a mode1 because it expresses CFTR at relatively low levels, thus facilitating easy differentiation of any irnposed effects. The final cation concentrations were optimized to allow maximal protein production without ce11 poisoning. To summarize, it was observed that after 20 hour treatments both 5 pM

Cd" and 100 pM ~n*elevated CFTR expression 3- to 5-fold, whereas 100 pM CU+had no effect (Fig. 3.1 ). Although significant elevations were achieved, even higher CFTR expression levels could be hduced with the drug sodium butyrate (Fig. 3.1). This reagent has various reported effects on cultured cells (Knih, 1982), but most importantly it was found to increase the expression of several genes through inhibition of histone deacetylase (see

Chapter 1; Candido et al., 1978; Sealy and Chalkiey, 1978). In addition, sodium butyrate was observed to specifically upregulate the activity of the metallothionine promoter (Birren and Herschman, 1986; Andrews and Adamson, 1987). For al1 CHO experiments in this report a final concentration of 2 mM sodium butyrate was used because higher concentrations did not Merincrease CFTR expression (Fig. 3.1) and at 8 mM of the dmg, ce11 death started to occur. Interestingly, the sodium butyrate treatment of 50- 1 cells produced such high amounts of hF508-CFTR that the ER quality control mechanism apparently became flooded. A sufficient number of AFSO8-molecules rnatured to allow functional detection of CAMP-induced chloride channel activity using the fluorimetric Fig. 3.1. Promotion of CFïR expression with heavy metals and sodium butyrate. 50-1 CHO celis, which stably express A508-CFTR, were incubated for 20 hours in growth media, supplemented with various concentrations of sodium butyrate (But), &Cl2 (Zn"), CdClt (Cd"), or CuCl (CU?. CFTR expression was detected by Western blotting with the anti-CFTR antibody M3A7 (5% SDS-PAGE). For the 50-1 ceus, both core-giycosylated bands A and B can be seen, whereas in the wild-type expressing ceiis (BQ2) the core- glycosylated band B and the Myglycosylated band C are the predominant species. 50-1 CHO membrane potentiai measurement of Lukacs et al. (1993) (data not shown). Anion conductance could not be stimulated in untreated 50-1 cells. Subsequently, the same observation was reported and quantified by Cheng et al. (1995) and has recently been applied to enhance production of retroviral vectors expressing CFTR cDNA (Olsen and Sechelski, 1995). The effects of heavy metals and sodium butyrate were seen to be

additive (Fig. 3.1 ), as described previously (Andrews and Adamson, 1987)

10SA-CFTR is still Phosphorylated by PKA - Since a 20 hour, 2 mM sodium butyrate treatment induced higher protein expression than exposure to heavy metals, the sodium butyrate approach was chosen to accumulate maximal levels of 1OSA-CFTR protein in stably transfected CHO cells. Sequential dilution of lysate from sodium butyrate treated 1OSA-CFTR expressing cells in Western blottùig demonstrated a? least a

30-fold increase in CFTR levels relative to untreated cells (Fig. 3.2, panel A). Wild-type CFTR fiom uninduced cells and 10SA-CFTR fiom sodium butyrate induced cells were irnrnunoprecipitated with the monoclonal antibody, M3A7 (Kartner et al., 1992). Upon subsequent in vitro phosphorylation with y-[32~]-~~~and PU, phospholabeling of

1OSA-CFTR was indeed detected (Fig. 3.2, panel B). Notably, sodium butyrate did not induce expression of endogenous CFTR in untransfected KI-CHO cells (Fig. 3.2, panel B) and omission of the kinase inhibited al1 in viwo labeling (data not shown). Because of the larger amounts of 10SA-CFTR protein fiom induced cells relative to the lower amounts of wild-type CFlR protein frorn uninduced cells, the level of overall phosphorylation was comparable between the two samples. However, when a different source of CFTR protein was utiIized that did not require sodium butyrate induction, it was clearly seen that 1OSA-CFTR is phospholabeled to a lower extent than wild-type CFTR. For this, large but equal quantities of 10SA-CFTR protein and wild-type CFTR protein were produced in baculovim-transfected SF9 insect cells and PKA-labeled by the same in vitro technique (Fig. 3.3). Thus, the remaining activation of IOSA-CFTR by the kinase could potentially be mediated by the observed residual phosphorylation, but to test Fig. 3.2. Detection of phosphorylation of IOSA-CFïR by PKA. CHO K1 cells were stably transfected with wiid-type or 1OSA-CFTR. (A) Expression of 1OSA-CFTR protein was induced with 2 mM sodium butyrate for 20 hours and deteded by Western blotting (M3A7; 5% SDS-PAGE). Amounts (pg) of total cell protein applied are indicated above each lane. Migration of Mr (~10-3)markers is show on the nght. This cornparison indicated that sodium butyrate induction elevated 1OSA-CFTR expression approxirnately 30-fold. (B) CHO K1 untransfected control cells and IOSA-CFTR expressing CHO cells were induced with 2 rnM sodium butyrate for 20 hours; wild-type (WT) CFTR expressing

CHO cells were not induced. Immunoprecipitated CFTR was incubated in phosphorylation buffer including 180 nM catalytic subunit of PKA and 10 pCi / 100 pL y-[32~]-~~~,and visualized by autoradiography following SDS-PAGE (5%). In al1 lanes CFTR is seen as the diffuse fully glycosylated -170 kDa species and the distinct core glycosylated 150 kDa species. + Sodium Butyrate t 1 30 30 15 75 5 25 1 Fig. 3.3. In viiro PKA phosphorylation of CETR harvested from SF9 insect cells.

CFTR variants were immunoprecipitated fiom SF9 insect cells without prior sodium butyrate induction, in vitro PKA phosphorylated, and detected by autoradiography.

Although weaker than for wild-type CFTR, phospholabeling was clearly visible on IOSA- CFTR as well as a 9SA-CFTR variant in which only the dibasic sites of the R-domain are mutated, not Ser 422. Note that insect cells are incapable of complex glycosylation of proteins. The core-glycosylated CFTR band B is marked (CFTR). this hypothesis it was necessary to identiQ one specific labeled site and demonstrate its contribution to CFTR hinction. For subsequent experiments, the CHO-expression system was utilized because of its conveaience. Loculization of PKA Phosphorylotion in IOSA-CFTR - In order to map the location of the remaining PKA phosphorylation site(s) to a specific area within the protein, immunoprecipitated y-[32~]-phosphorylated 1OSA-CFTR was subjected to peptide mapping. Cleavages with various enzymes and chernicals were utilized including thrombin, hydroxylamine, factor Xa, and 3-bromo-3-methyl-2-(2'-nitrophenylsulphenyl) indolenine. However, the best separation was obtained with cyanogen bromide (CNBr) cleavage, a procedure that cuts the phosphopeptide C-temiinally to methionine residues

(Stryer. 1988). Gel eiectrophoresis of the CNBr cleavage products and subsequent autoradiography produced the same three major labeled bands for both IOSA-CFTR and wild-type CFTR (Fig 3.4, panel A). The segments generated &om wild-type CFTR showed slightly less mobility, which rnay result fiom an increase in the apparent molecular weight due to a largzr nurnber of phosphoryl groups attached. Such effects of phosphorylation on R-domain mobility have been reported previously (Picciotto et al ., 1992; Dulhanty and Riordan, 1994a). AI1 three observed bands most likely originated from within the R-dornain since wild-type R-domain (produced as a recombinant protein in E-coli and purified; Dulhanty and Riordan, 1994a), showed a comparable banding pattern as wild-type CFTR upon in vitro phosphorylation by PKA and CNBr cleavage (Fig. 3.4, panel B). The lower molecular weight species, bands 2 and 3, CO-migrated, suggesting that these bands are denved fiom the R-domain. Band 1 had a lower mobility in the CFTR sarnple which may in part be attributed to the fact that in CFTR the fmal CNBr cleavage site involving an R-domain segment occurs at residue 837 which lies C- terminal to the C-terminus of the isolated R-domain (residues 595-83 l), therefore generating a slightly larger fiagrnent. The additional 13 kDa band seen in the R-domain Fig. 3.4. Localuation of residual 10SA-CFTR phosphorylation by cyanogen bromide cleavage. (A) Protein expression was induced with 2 rnM sodium butyrate in untransfected K 1 control cells and in 1OSA-Cm cells (CHO). Immunoprecipitated CFTR protein was phosphorylated in vitro with PKA, cleaved with cyanogen bromide (CNBr), and detected by autoradiography following 16% SDS-PAGE (WT, wild-type).

(B) Upon ifi vitro phosphorylation wiîh PKA and CNBr cleavage of wild-type full-length

CFTR (denoted as 'C') and isolated R-domain (denoted as 'R'), bands 2 and 3 CO- migrated. (C) Predicted CNBr cleavage map of the CFTR R-domain. CNBr cleaves C- terminal to methionine residues. Numbers above the schematic indicate the position of each methionine and numbers below the schematic show the predicted Mr of the resdting segments. The 5.8 kDa segment (residues 722-773) is enlarged to show serines which were aiready replaced by alanines in IOSA-CFTR (open circles) and remaining serines

(closed circles). S, serine. (D) Immunoprecipitated wild-type CFTR was labeled in viîm with PKA and cleaved with CNBr. A controI sample was imrnediately subjected to SDS- PAGE whereas the other two samples were reprecipitated with either P-Ab 6 (epitope:

724-746) or Ll lE8 (epitope: 341-702) before separation by SDS-PAGE (1 6%) and visualization by autoradiography. - - cas 1. < 143

3 preparation rnay correspond to the 13 kDa natural cleavage product previously observed by Duhanty and Riordan (1994a) in the presence of SDS. Based on this idormation it was possible to use antibodies to assign each band to a specific segment of the predicted CNBr cleavage map of the R-domain (Fig. 3.4, panel C). Fig. 3.4, panel D, illustrates re-precipitation of the phosphorylated CNBr cleavage products pnor to gel electrophoresis and autoradiography with antibody P-Ab 6 (raised against a synthetic peptide containhg residues 724 to 746). P-Ab 6 recognized band 3, suggesting that band 3 corresponded to the predicted 5.8 kDa segment (residues 722 to 773). The intermediate band 2 was recognized by the monoclonal antibody L11E8 (raised against a GST-fusion protein contauiing CFTR residues 34 1-702; Kartner et al., 1PZ), suggesting that band 2 contahed the predicted 8.7 kDa segment (residues 646-721). Both antibodies interacted with band 1, indicating that it rnay represent a partial cleavage product containing the 5.8 kDa segment, the 8.7 kDa segment, and possibly other segments. The Mersearch for a remaining phosphorylation site focused on the predicted 5.8 kDa segment (band 3). That this region may play an important role in CFTR regulation through PKA phosphorylation was suggested by the observation that most labeling of wild-type CFTR occurred within band 3 (Fig. 3.4, panel A). Band 3 contains two serines (Ser 737, Ser 768) which were removed in the IOSA-CFTR mutant and mut therefore account for the large difference in the arnount of phosphorylation between the 5.8 kDa segments of wild-type and IOSA-CFTR. This is in agreement with the results of

Picciotto et al. (1992), who demonstrated that Ser 737 is phosphorylated in vitro by PKA. Furthemore, these authors showed that either Ser 768 or Ser 795 is the most strongly phosphorylated site in the R-domain but could not distinguish between the two residues. Our studies indicate that Ser 768 is this strongly phosphorylated residue since it lies within the heavily labeled 5.8 kDa segment of the R-domain, whereas Ser 795 does not. In addition, the data coincides with the report that, relative to other PKA consensus sites, the phosphorylation kinetics are the fastest for a synthetic peptide containing the PKA consensus site around Ser 768 (Picciotto et al., 1992).

Mut~genesisof PossibZe Phosphoylution Sites within the 5.8 kDa Segment - PKA- catalyzed phosphate incorporation in CFTR has only been detected on serine residues (Cheng et al., 1991; Picciotto et ai., 1992). The 5.8 kDa segment of the R-domain contains 6 serines at positions 728, 737, 742, 753, 756, and 768, two of which (Ser 737, Ser 768) have already been changed to alanines in LOSA-CFTR (Fig. 3.4, panel C). To investigate whether any one of the remaining four serines is phosphorylated by PKA, each one was individually converted to an alanine in a IOSA-CFTR construct (Chang et al.,

1993) by in vitro mutagenesis, thus creating four different 11 SA-CFTR variants ( 1 1SA- S728A-CFTR, 1 1SA-S742A-CFTR, 1 ISA-S753A-CFTR, 1 1SA-S756A-CFTR). The mutants were stably expressed in CHO cells. When CFTR fiom each 1 1SA variant was in vipo phosphorylated with PKA and cleaved with CNBr, it was observed that in 11SA- S728A-CFTR,1 1 SA-S742A-CFTRand 1 1SA-S756A-CFTRthe 5.8 kDa segment (band

3) was still phospholabeled (Fig. 3.5). The 5.8 kDa segment in 1 1SA-S753A-CFTR, however, was not phosphorylated, indicating that the only remaining PKA phosphorylation site within this fiagment was Ser 753. In 1 1 SA-S753A-CFTR the phospholabeling of the partially cleaved band 1 also was greatly diminished, suggesting that Ser 753 made a major contribution to its phosphorylation. Yet, phosphorylation of band 2 was unaffected, illustrating that there is Merresidual phosphorylation outside the 5.8 kDa segment of the R-domain. Evaluurion of the FunctionaZ Efect of Ser 753 Mutagenesis - Thus far, we had demonstrated that Ser 753 is a phosphate acceptor in vitro, but to illustrate the residue's fûnctional contribution in vivo the effect of its removal on PU-mediated stimulation had to be demonstrated. To address the functional role of Ser 753, IOSA-CFTR, 1 ISA- S753A-CFTR and wild-type CFTR were stably transfected into BHK cells which Fig. 3.5. Identification of the site phosphoryiated in the 5.8 kDa segment. Four different 1 1 SA-CFTR mutants were created, each based on the IOSA-CFTR construct (Chang et al., 1993) and each changing a different serine residue of the 5.8 kDa segment to an alanine (1 1SA-S728A-Cm 1 1SA-S742A-CFTR, 11 SA-S753A-CFTR, 1 1SA- S756A-CFTR). CFTR comtructs were stably transfected into CHO KI cells or BHK cells and protein expression was induced by 2 mM sodium butyrate. hunoprecipitated CFTR protein was labeled in vitro with PKA, cleaved with CNBr, and visualized by autoradiography following SDS-PAGE ( 16%).

consistently express similar amounts of CFTR protein for different mutants (Fig. 3.6, panel A, insert), thereby facilitating a rigorous quantitative functional cornparison of cell lines expressing different CFTR variants. Fig. 3.5 confïxms that in BHK cells Ser 753 also accounted for dl the labeling within the 5.8 kDa segment of the R-domain (band 3), so that this phosphorylation is not cell-type specific. Iodide efflw assays demonstrated that forskolin-stimulated channel activity was reduced in the cell line expressing 1 1SA-

S753A-CFTR relative to the ce11 line expressing IOSA-CFTR (Fig 3.6, panel A). In addition, the chloride channel activity of 1ISA-S753A-CFTR was evaluated in cornparison to IOSA-CFTR by single-channel patch-clamping. Fig 3.6, panel B, illustrates that in inside-out membrane patches excised fiom BHK cells expressing wild- type CF'TR, IOSA-CFTR, and 11SA-S753A-CFTR, quiescent channels can be activated by addition of 180 nM PKA and 1 mM ATP to the bath solution. The opening of an individual channel is seen as a distinct downward step in the recorded trace. Both lOSA and 1 lSA-S753A channels demonstrated typical CFTR charactenstics of low conductance and non-rectification (data not shown). Significantly, the open probability (po)12 in the presence of ATP and PKA was 40% lower for 1 ISA-S753A-CFTR channels when compared to IOSA-CFTR channels (Pos were 0.203 I 0.012 and 0.346 t 0.023 respectively; mean k S.E.; n=5; Fig. 3.6, panel C). This result was consistent with the iodide efflux data and showed that Ser 753 does significantly contribute to the residual activity of 1OSA-CFTR. To hctionally confirm the phosphorylation data that Ser 753 is the only residual phosphorylation site within the 5.8 kDa segment, a 16SA-CFTR variant was created in which al1 rernaining serines (S728, S742, S753, S756) and theonines (T757, T760) within the 5.8 kDa segment were simultaneously mutated to alanines in the IOSA-CFTR

- - " Note that conductance is a measure of the rate at which ionic current flows through an open channel in the presence of a specific voltage dnving force. The Podescribes the fraction of thethat a single channet remains in an open or conducting state. Fig. 3.6. Funetional evaluation of llSAS753A-Cm (A) Iodide efflux fiom BKK cells expressing mutant CFTRs. (+) indicates that fiom tirne O onward cells were stimulated with 10 @lforskolin dissolved in Me2SO. The same amount of Me2SO without forskolin was used in the control sarnples (-). The profiles of control sarnples were similar, so îhat only the efflux curve for 11SA-S753A-CFTR(-) is presented in this figure. The insert shows a Western blot of the various CFTR mutants in BHK cells. Total protein applied per lane: 50 pg. (B) Activation of wild-type (WT), lOSA and 1 ISA- S753A (1 ISA) CFTR channels in excised inside-out membrane-patches fiom BHK cells by addition of 180 nM PKA and ImM ATP. Vm = -30 mV. Openings are downward. (C)

Open probabilities (Po) of I OSA-CFTR and 1 1SA-S753A-CFTR channels upon exposure to 180 nM PKA and 1 mM ATP. IOSA-CFTR channels expressed in BHK cells showed a

Po of 0.346+/-0.023 whereas 1 1SA-S753A channels had a Po of 0.203+/-O.O 1 2 (mean +/-

S.E., n=5). The Po for wild-type CFTR in BHK cells could not be accurateiy estimated because gigaOhrn seals were lost during the vigorous response to PKA before the channel number could be detemined.

background. When this mutant was expressed in BHK cells and the cells were subjected

to iodide efflux, they could be stimulated by forskolin to the same level as 1 1SA-S753A- CFTR expressing cells, confimiing that none of the other potential sites were subject to PKA interactions of functional importance (Fig. 3.7).

Overall, this study indicated that the remaining responsiveness of 1OSA-CFTR to PKA at least in part is the result of low levels of phosphorylation at additional sites which do not adhere to the strict dibasic consensus for PKA-mediated phosphorylation. One such phospholabeled site is Ser 753 which accounts for approximately 40% of the residual activity. In retrospect, among the four remaining serines in the 5.8 kDa segment of IOSA-CFTR., Ser 753 was the most likely candidate for PKA-catalyzed phosphorylation since it is the only serine having a basic residue in close proximity (P - R

- 1 - S753*). Rich et al. (1993ayb).as a result of experiments conducted on an R-domain deletion mutant, had already speculated that Ser 753 may be a PKA substrate. Our results support this speculation, although it hzs to be noted that Ser 753 was found to contribute to fimction in a IOSA-CFTR mutant, devoid of al1 other major phosphoryiation sites. This does not prove that Ser 753 phosphorylation contributes to activation of the wild-type protein. It has, however, been difficult to evaluate the functional importance of individual phosphate acceptors in vivo since CFTR phosphorylation sites appear to act in a redundant manner so that removal of a single site has Iittle effect on function.

Since a fiaction of the residual in vitro phospholabeling of IOSA-CFTR occurs on a monobasic PKA consensus site, the rernaining monobasic consensus sites seem valid candidates to carry the remahhg phosphorylation by the kinase. Monobasic consensus sites for potential interactions with PKA reside in CFTR's N-terminus, cytoplasrnic loops,

NBFs 1 & 2, R-domain, as well as C-terminus. The following chapter will address whether any of these loci are in vitro labeled by PKA and contribute to CFTR fùnction. Fig. 3.7. Anion conductsnce of 16SA-CFTR expressing cells. The 16SA-CFTRvariant, which changes ail residual serines and threonines of the 5.8 kDa segment simultaneously to alanines, was stably expressed in BHK cells. Iodide eaux was stimdated with 10 pM fonkolin as described in Fig. 3.7. The induced activation profile was similar to fluxes fiom 11 SA-S753A-CFTR expressing cells. 2 4 6 8 10 12

Time (min) CHAPTER 4

Monobasic Consensus Sites for CAMP-dependentProtein Kinase- mediated Phosphorylation of CmR Chapter Summary Residual in vitro phosphorylation of 1 1SA-S753A-CFTR by PKA was observed to occur within a distinct -9 kDa band following CNBr cleavage, separation on SDS- PAGE and visualization by autoradiography. In this chapter, we utilized several R- domain deletion mutants to demonstrate that the -9 kDa band contains both the predicted 8.7 kDa segment (residues 646-721) and the predicted 7.3 kDa segment (residues 774- 83 7) of the R-domain, apparently migrating with equal mobility on SDS-PAGE. The serines and threonines of dl four monobasic consensus sites (RIK - X - SV*) within these sequences were simultaneously changed to alanine in the IlSA-S753A-CFTR

background. The resulting ISSA-CFTR variant was stably expressed in BHK cells. Although the residual in vitro phosphorylation of 1 HA-S753A-CFTR decreased upon removal of the four additional sites, the ISSA-CFTR expressing cells still showed the same anion efflux profile as the 1 ISA-S753A-CFTR expressing cells. In agreement, single-channel patch-clamping demonstrated that the mean open tirne, the mean closed time, and the overall open probability al1 did not significantly differ between 1 ISA- S753A-CFTR and ISSA-CFTR channels. Therefore, monobasic consensus sites of the R- domain other than Ser 753 do not significantly contribute to the molecule's function. Cryptic sites, that utilize distally located positive charges for the interaction with the kinase, must carry the residual phosphorylation and may be involved in the activation of the 1 1 SA-S753A-CFTR charael by PKA. In addition, we attempted to use a mutagenic approach to investigate the importance of monobasic consensus sites for PKA interactions in NBFI. However, upon stable expression of the NBFl-mutants in CHO cells, the only observation that couid be made was that alteration of such residues results in severe misprocessing of CFTR, thereby precluding functional analysis. Introduction Ln chapter 3 it was demonsîrated that the residual responsiveness of IOSA-CFTR to PKA at least in part is the result of low levels of phosphorylation at sites that do not adhere to the strict dibasic consensus for interactions with the kinase. One such site was identified as Ser 753. It became apparent that Ser 753 in fact resides within a monobasic consensus motif that has a single positively charged residue at the -2 position in the primary amino acid sequence (RM - X - S*/T*; asterisk indicates site of phospholabeling; Kemeily and Krebs, 199 1). In the investigation, an I 1SA-S753A-CFTR variant was created that was based on the IOSA-CFTR construct (Chang et al., 1993) and also

changed Ser 753 to Ala. When expressed in CHO cells, 11 SA-S753A-CFTR still was activated by PKA to some degree and could be in vitro phosphorylated by the kinase. Using CNBr-based phosphopeptide mapphg, the phosphorylation was traced to a single band with an apparent molecular weight of -9 kDa, called band 2. The aim of the present investigation is to unequivocally detemiine the ongin of this band 2 within the amino acid sequence of CFTR. If this is achieved, any of the Ser or Thr residues within the sequence could in theory serve as a phosphate acceptor. To narrow down the search for additional sites, the hypothesis will be made that, of the many potential sites, the one(s) that idare phosphorylated idare the one(s) which possess a positively charged residue in the -2 position as seen for Ser 753. Whether this is the case will be tested with a similar combination of site-directed rniitagenesis and functional assays as described in chapter 3. This last attempt to remove al1 phosphoryIation/activation by PKA is undertaken because such an unresponsive mutant would be beneficial to the study of CFTR regulatory mechanisrns. Al1 phosphorylation sites cm be re-introduced individudly and thus investigated as to their contribution to channel stimulation. Furthemore, in such a mutant, regulation of CFTR by ATP hydrolysis, targeting, and degradation events can potentially be studied in isolation fiom phosphorylation effects. Results and Discussion Origin of the -9 kDa CNBr Cleavuge Producl (Band 2) - CNBr cleavage and antibody re-precipitation experiments had demonstrated that PKA-labeled wild-type CFTR produced three R-domain derived phosphorylated bands, of which band 3 was identified to contain the predicted 5.8 kDa segment (residues 722-773), band 2 to contain the predicted 8.7 kDa segment (residues 646-721), and band 1 to contain at least these two segments plus potentially additional segments. However, there was no extra band detected that could correspond to the predicted 7.3 kDa segment (residues 774-837). This was unexpected because this segment contains the two sites Ser 795 and Ser 81 3 which were clearly demonstrated to contribute to phosphorylation and function of wild-type CFTR (Cheng et al., 1991). It was thus conceivable that the 7.3 kDa segment had equal mobility to either the 5.8 kDa segment or the 8.7 kDa segment, but was hidden from our detection because of the lack of a specific antibody to this area of the protein. To address this speculation, two deletion mutants were utilized which were generously provided by

Dr. J.M. Rommens. The A43 mutant is lacking residues 734-777 and the A 174 mutant has the 603-777 sequence deleted. Both CFTR constnicts were transiently expressed in HEK- 293 cells, in vitro PKA labeled, and subjected to CNBr cleavage. As seen in Fig. 4.1, both mutants produced banding patterns consistent with the interpretation that the 7.3 kDa segment was PKA labeled biit could not be detected previously because it had equal mobility to the phospholabeled 8.7 kDa segment. The schematic illustrates that the A43 deletion removes most of the 5.8 kDa segment (including al1 phosphorylation sites: S737, S753, S768) and attaches the N-texminal twelve amino acids (a.a.) of the 5.8 kDa segment to the 7.3 kDa segment, which itself loses four Noterminal amino acids ('7.3 kDa + 8 a.a.' segment, labeled as -8.0 kDa). In the autoradiogram this resulted in the disappearance of band 3 and in the splitting of band 2 into the unaffected 8.7 kDa segment and an upwards shifted '7.3 kDa + 8 a.a.' segment. Band 1 shifted down upon the deletion, but could only Fig. 4.1. CNBr cleavage of R-domain deletion mutants. The R-domain deletion mutants A43 and A1 74 respectively are rnissing residues 734-777 and residues 603-777 in the wild-type CFTR background. Both constnicts and the wild-type (WT)form of CFTR were transiently expressed in HEK-293 cens, Mmunoprecipitated with M3A7, in viîro phosphorylated by PU,and CNBr cleaved pnor to analysis by SDS-PAGE (16%) and autoradiography. CNBr cleaves C-terminal to methionine residues; in the R-domain methionines are located at positions 595, 607, 645, 721, 773, and 837 (Riordan et al., 1989). In the schematic, the numbers below the bars represent the predicted molecula. weight of each CNBr cleavage product. Cyanogen Brornide Digest of the R-Domain be seen at longer exposures. Furthemore, the A1 74 deletion removes al1 of the 4.4 kDa,

5.8 kDa, and 8.7 kDa segments and attaches the N-temiinal 7 a.a. of the 1.4 kDa segment to a 7.3 Dasegment which itself is shortened by four a-a. ('7.3 kDa + 3 a.a.' segment, labeled as -7.7 kDa). The fact that in the autoradiogram the slightly upwards shifted '7.3 kDa + 3 a.a.' segment was still phosphorylated confimied that (i) the 7.3 kDa and 8.7 kDa segments CO-migrateand that (ii) the 7.3 kDa segment is in vitro phosphorylated in wild- type CFTR. Thus, band 2 corresponds to two regions of CFTR, residues 646-721 and residues 774-837. Muragenesis of Monobasic Consensus Sites for PKA Interactions in the R-domain

- Within these two regions of the R-domain there are 20 additional serines and threonines located, each one of which or a combination thereof could potentially represent cryptic site@) for PKA-mediated phosphorylation and act as phosphate acceptors in the 1 1SA- S753A-CFTR variant. To simultaneously mutate ail 20 sites to alanine was unrealistic because the tme effect of such a large number of mutations cannot be evaluated. Thus, to limit the search for residual site(s), four candidate residues were selected that were judged to have a high likelihood of being phosphorylated. Of the 20 possible sites, three are monobasic consensus sites for PKA-rnediated phosphorylation that, similarly to Ser 753, have a positively charged residue in the -2 position: Ser 670, Thr 690, and Thr 787. In fact, Ser 670 is part of a dibasic consensus site in CFTR of several species other than man (Gadsby and Naim, 1994). One more site, Ser 789, was also selected as a possible candidate because it is swounded by several positively charged residues and is located in an area which is highly phosphorylated in wild-type CFTR (Fig. 4.2). To obtain an initial estimation of the importance of these sites in PKA-mediated activation, al1 four residues were simultaneously changed to alanine in the 1 ISA-S753A-CFTRbac!cground. The resulting ISSA-CFTRvariant was stably expressed in BKK cells. Processing Characteristics of ISSA-CFTR - Since CFTR is ver- susceptible to recognition by the ER quality control machinery, a primary concem was whether Fig. 4.2. Schematic of monobasic consensus sites in the R-domain. The schematic shows the three labeled CNBr cleavage products of the R-domain. Numbers beside the schematic indicate the predicted M, of each segment. Depicted are serines and threonines that were already replaced by alanines in 11SA-S753A-CFTR (open circles), three monobasic consensus sites for PKA interactions (closed circles), and a single serine located in an area containhg several positively charged residues (shaded circle). The serines and threonines of these four additional sites were simultaneously changed to alanines, thus creating a ISSA-CFTR mutant. S, serine; T, threonine; NBF, nucleotide binding fold.

alteration of 15 residues in the R-domain modifies the protein's structure sufficiently to cause misprocessing of the channel. However, as show by Western blotting, the steady- state amounts of fully glycosylated band C relative to core-glycosylated band B were similar in both wild-type CFTR and ISSA-CFTR expressing cells, indicating that

processing was not af3ected by the mutations (Fig. 4.3, panel A). The presence of both CFTR variants on the cell surface was confirrned by surface labeling (data not shown). As suggested by Dulhanty and Riordan (1994b), this flexibility of the R-domain may also be reflected in the fact that it is the least conserved region of the molecule with only 23% identity between 10 species. To observe srnall levels of phosphorylation in the ISSA-CFTR mutant, its expression in BHK cells was stimulated with sodium butyrate. Optimization demonstrated that BHK cells could tolerate much higher levels of the dmg than CHO cells, with maximal expression occurring afier a 20 hour treatment with 10-50 mM sodium butyrate (Fig. 4.3, panel B). However, densitometry readings and serial dilution showed that 30 mM sodium butyrate stimulated CFTR expression only 7- to 8-fold in BKcells, rather than the 30-fold increase in CHO cells. Similarly lower levels of stimulation were reported by Cheng et al. (1 995) in C 127 cells. Nonetheless, BHK cells were chosen over CHO cells for the investigation of monobasic PKA consensus sites since in the study of 1 ISA-S753A-CFTRthe electrophysiological characterization of BHK cells proved to be easier than that of CHO cells. Labeling Characteristics of USA-CFTR - To observe whether the novel mutations influenced in vitro phosphorylation by PKA, the high levels of ISSA-CFTR protein were immunoprecipitated with monoclonal M3A7, labeled with y-[32~]-~~~and PKA,and cleaved with CNBr. This showed that 1SSA-CFTR could still be PKA labeled, indicating that in the CFTR molecule cryptic sites mut act as phosphate acceptors (Fig. 4.4, panel A). To quantitate the decrease in the in vitro phosphorylation, the intensity of band 2 was quantified with densitometnc scanning and normalized relative to a non- Fig. 4.3. Expression of ISSA-CFIn. (A) Wild-type CFm and various R-domain mutants were stably expressed in BHK cells and analyzed by Western blotting (5% SDS-

PAGE, M3A7). 1 1 SA, 1 1 SA-S753A-CFTR; ISSA, residues S670, T690, T787, S789 are modified to Ala in the 1lSA-S753A-CFTR background . (B) BHK cells stably expressing

ISSA-CFTR were incubated for 20 hours in medium supplemented with O mM to 100 mM sodium butyrate (concentrations are shown above each lane). Equal arnounts of total cell protein were applied (6 pg) and again detected by Western blotting (S%SDS-PAGE,

M3A7). The core glycosylated band B and the Myglycosylated band C of CFTR are indicated. Occurrence of core glycosylated band A, a product of the utilization of an alternative site for initiation of translation (Pind et al., 1995), is ce11 type dependent and appears to be low in BHK cells.

Fig. 4.4. CNBr cleavage of CFTR phosphoproteins. (A) Untransfected and CFTR mutant expressing BHK ceils were induced with 30 mM sodium blrtyrate.

immunoprecipitated CFTR was phosphorylated in vitro with PKA (using 1/10 the arnount

of y-[32~]-~~~for wild-type CFTR), cleaved with 20 mg/mL CNBr, and visualized by

autoradiography (only 1/10 of the wild-type sample was applied). Positions of CNBr cleavage product bands are indicated on the left. A non-specific band, also present in the

control sample, was seen to nin at a molecular weight lower than band 3. Residual

phosphorylation of band 2 for each mutant was evaluated by densitometry and is

indicated in panel @), nomalized in relation to the non-specific band (data are the mean

IS.E., n=2). Control 11 SA-S753A 15SA R-domain Mutants specific band which was aiso present in untransfected control cells. Thereby it was seen

that the removed monobasic sites contributed to approximately 30% of the residual PKA- mediated phospholabeling remaining in the 11SA-S753A-CFTR mutant (Fig. 4.4, panel

BI. Anion Permeation of 15SA-CFTR Expressing BHK Cells - In the mutagenic study of phosphorylation sites the most critical characterization is the functional evaluation since two misleadhg possibilities exist: (i) loss of in vitro labeling may not represent loss of functionally significant phosphorylation or (ii) a site that cannot accept labeling in

vina does still serve in the in vivo function, possibly by only becorning available for

phosphorylation when the molecule is in an active or serni-active state. When ISSA-

CFTR expressing ceIls were cornpared to 1 1SA-S753A-CFTR expressing cells by iodide efflux, no difference could be detected in their pemeability to the anion (Fig. 4.5). Thus, the four sites studied in this chapter do not appear to contribute significantly to Cm function. To ensure that the residual activation of I SSA-CFTR expressing cells by forskolin is not a forskolin-induced artifact, the analogue 1,9-dideoxy-forskolin (Sigma), which is

unable to stimulate adenylate cyclase and therefore does not elevate intracellular CAMP concentrations (Bhat et al., 1983; Seamon et al., 1983), was applied to the cells. The

inactive analogue could not stimulate iodide emux, demonstrating that the activity is indeed CAMP-mediated(Fig. 4.6). Evaluation of l5SA-CFTR by Single-channel Patch-clamping - Electrophysiological data obtained from excised single CFTR charnels confinned the whole-ce11 iodide efflux observations, in that the residual Po was not decreased significantly when the fow potential phosphorylation sites were mutated in the 1 ISA- S753A-CFTR background (Fig. 4.7, panel B). We note that in the present study the absolute Po value for 1 ISA-S753A-CFTR was lower than reported in the previous Fig. 4.5. Functional analysis of ISSA-CFTR by iodide efnux. BHK cells, expressing various R-domain mutants at similar levels, were loaded with 136 mM Nd. Using an iodide sensitive electrode, iodide efnw was then measured before and afler addition of

10 pM forskolin at time O. The residual activity of ISSA-CFTR was not significantly different fiom 1 ISA-S753A-CFTR.Control mtransfected BHK cells were treated with

1O pM forskolin. Control 15SA llSA lOSA

2 4 6 8 1 O 12

Time (min) Fig. 4.6. Application of an inactive forskoh analogue to the iodide enlux. The iodide efflux was conducted as described in Fig. 4.5, replacing forskolin with its analogue 1,9- dideoxy-fonkolin, which is incapable of activating adenylate cyclase. Cont., untram fected control cells; For., forskolin; DDF.,dideoxy- forskolin. -4 -2 O 2 4 6 8 10 12 Time (min) Fig. 4.7. Funetional analysis of ISSA-CFTR by single-channe1 patch-clamping. (A)

Traces of single-charme1 activity of ISSA-CFTR channels in a patch excised fiom BHK cells (i) in the presence of 1 rnM MgATP and 95 nM PKA and (ii) following addition of 1 mM AMP-PNP, in the continued presence of MgATP and PKA with the subsequent locking open of channels. Channel openings are shown as an upwards shift in the recorded trace. Data were acquired at 500 Hz following filtering at 100 Hz. Total duration of the record shown is 14 minutes. (B) Plot showing single channel Po's for 1 ISA- S753A-CFTR (1 1SA) and 1SSA-CFTR (1 5SA) channels. The mean Po's of the channels were not significantly different (PO.11; unpaired t-test). (C) Plots showing (i) the open burst durations and (ii) interburst durations for 1 I SA-S 753A-CFTR and 15SA-CFTR channels. Neither was significantly different between the mutants e0.95 in each case; unpaired t-tests). n=9 for I 1SA-S753A-CF?R and n=6 for 15-SA-Cm.

chapter. This difference may be attributable to the use of different batches of kinase and

buffen in the two sets of experiments. While evaluating the single-channel characteristics of ISSA-CFTR, two facts became apparent that did not agree with predictions fiom the current mode1 for CFTR

fimction (Hwang et al., 1994): (i) ISSA-CFTR could be locked open by AMP-PNP (Fig. 4.7, panel A) and (ii) the estimation of burst and interburst durations indicated that these parameters did not differ between 1 I SA-S753A-CFTRand ISSA-CFTR (Fig. 4.7, panel C), but that ISSA-CFTR relative to reported wild-type values (Carson, et al., 1995; Gunderson and Kopito, 1995) showed an increase only in the interburst duration with little changes in the burst duration. According to the mode1 proposed by Hwang et al. (1994; Fig. 1.4) a low level phosphorylated CFTR molecule sucn as ISSA-CFTR is expected to not be locked open by AMP-PNP and should be unable to obtain regular full- Iength openings.

Monobasic Consensus Sitesfor Interaction with PKA Ni NBFI - With our level of sensitivity we could not detect PKA-mediated phosphorylation outside the R-domain.

However, studies by Ko and Pedersen (1994) suggested that NBFl, in the form of a fusion protein, was in vivo phosphorylated by endogenous kinases of the expressing E. coli without extemal stimulation of phosphorylation. Ko and Pedersen (1994)also were able to phosphorylate NBFl in vitro with PKA when present as a purified peptide. Furthemore, the dibasic pre-MF1 consensus site Ser 422 was found to contribute to PKA-mediated stimulation of CFTR (Chang et al., 1993) although the study could not see phosphorylation of that residue. We were thus considering the possibility that phosphorylation of NBF 1 occurs at sub-sensitivity levels, but still contributes to the activation of the channel. In MF1 there are no dibasic, but four monobasic, consensus sites for PKA interactions with a positively charged residue in the -2 position. Al1 four potential phosphate acceptors of these sites (Ser 466, Ser 485, Ser 489, Ser 557) were simultaneously changed to alanine in either the wild-type CFTR background (WT-4SA- CFTR) or in the 11SA-S753A-CFTR background (11SA-4SA-CFTR) and stably expressed in CHO cells. Unfomuüitely, introduction of these four mutations resulted in misprocessing of CFTR which was so severe that in Western blonuig extremely low

levels of band C could only be detected if very mild conditions were applied (Fig. 4.8, panel A). Although the low levels of band C that were processed were similar for WT- 4SA-CFTR and I 1SA-4SA-CFTR., activity could only be detected fiom WT-4SA-CFTR

expressing cells, but not fiom I MA-4SA-CFTR expressing cells by iodide efflux (Fig. 4.8, panel B). Thus, removal of the four potential sites in NBFl could have disallowed the residual responsiveness of 1 1SA-S753A-CFTR to PKA.From the present studies, this can ody be speculated, not concluded, since the mutations modified the overall protein structure so significantly that the bulk of the CFTR molecules was not allowed to process to the ce11 surface. Therefore, it cannot be differentiated whether the functional effect was (i) due to removal of a phosphorylation site or (ii) the result of other structural effects such as inability of NBFl to communicate with other regdatory domains or (iii) that

simply the number of molecules at the ce11 surface has dropped below detection threshold when combined with the decreased activity of 1 I SA4753A-CFTR. It is noteworthy that in the R-domain 16 residues could be modified without affecting processing (Fig. 3.7) whereas a small number of NBFl mutations had a devastating effect on overall CFTR conformation. Interestingly, the introduction of alanines into NBF 1 (SA) allowed slightly more processing than the introduction of glutamates (SE), whereas the replacement of dibasic serines with glutamates in the R-domain (8SE) allowed maturation of CFTR (Fig. 4.8, panel A). Summary The present studies demonstrated that Ser 753 is the only monobasic consensus site for PKA-mediated phosphoxylation in the R-domain that is of functional significance. Since the Cmmolecule can still be labeled in the R-dornain in vitro by PKA upon removal of al1 monobasic and dibasic consensus sites, the phosphate acceptance must Fig. 4.8. Study of monobasic PKA consensus sites in NBFl. The serine of four monobasic consensus sites was replaced with alanine in the Md-type (WT-4SA-CFTR) or I 1SA-S753A-CFTR background (1 1SA-4SA-CFTR). The constnicts were stably expressed in CHO cells and evaluated by (A) Western blotting (5% SDS-PAGE,M3A7) and (B) iodide efflux as described in Fig. 4.5. In the Western blot it is seen that the Ser to Ala mutations (SA) in NBFl severely retard maturation of CFTR, but allow more processing than the corresponding Ser to Glu mutations (SE). Control, unbansfected CHO ceIis. 4 6

Time (min) occur at cryptic sites that utilize distally Iocated charged amino acids for their interaction with the kinase. Ii remains to be determined whether the residual activation of 1 1SA-

S 753A-CFTR by PKA occurs via the phosphorylation observed by h vitro techniques. CHAPTER 5

Disease-associated Mutations in the Fourth Cytoplasmic Loop of CFTR Compromise Biosynthetic Processing and Chlotide Channel Activity Chapter Summary

A cluster of eighteen point mutations in exon 17b of the CFTR gene has been detected in patients with CF. These mutations cause single amino acid substitutions in the most C-terminal cytoplasmic loop (CU,residues 1035-1102) of the CFTR chioride channel. Heterologous expression of the mutants showed that twelve produced only core- glycosylated CFTR, which apparently did not reach the plasma membrane; the other six mutants matured and reached the ceU surface. In some cases substitution of one residue resulted in misprocessing, whereas substitution of its neighbor had no effect on the biosynthetic maturation of the protein. Thus, the topology of CUmay contribute crucially to the proper folding of the entire CFTR molecule. CAMP stimulated iodide efflux was not detected fiom cells expressing the misprocessed variants but was from the other six, indicating that their mutations cause relatively subtle channel defects. Consistent with this, these latter mutations generally are present in patients who are pancreatic suflïcient whereas the processing mutants are rnostly fiom patients who are pancreatic insufficient. Single- channel patch-clamp analysis demonstrated that the processed mutants had the sarne ohrnic conductance as wild-type CFTR, but a lower open probability, generally due to an increase in the channel mean closed the and a reduction in the channel mean open the. This suggests that mutations in CL4 do not affect pore properties of CFTR, but disrupt the mechanism of channel gating. Introduction In the previous chapten we have bdt on the understanding that phosphoxylation of CFTR is essential for its activation and fider developed the concept that various degrees of phosphate binding aliow a graded response to an activation signal; a decrease in the molecule's labeling capacity was seen to reduce its probability of residing in the open state. However, one aspect of CFTR regulation that has not been ddineated is the mechanism of communication between the regulatory domains themselves and between the regulatory dornains and the pore foming unit of the channel. How, for example, does the R-domain impose the graded response on the ion pathway? We propose that potential sites of communication between the regulatory subunits and the pore-fo&g domain are the cytoplasrnic loops (CLs, Fig. 5. l), which are the only parts of the pore suggested to protrude fÏom the membrane. The idea that inter-domain communication may occur by dornain contacts other than direct links in the primary sequence is based on an analogy to bacterial memben of the ABC-superfamily of proteins for which membrane and cytosolic regions of the assembled oligomer are provided by entirely different proteins (Doige and Ames, 1993). The CLs link the TMs to each other on the cytoplasrnically exposed side of the protein and are predicted to range between 55 and 65 amho acids in length (Riordan et al., 1989). They are highly conserved between CFT% expressed in different species (Diarnond et al., 199 1) and moderateiy conserved in cornparison to the CLs of other ABC transporten (Manavalan et al., 1993). However, little information is available regarding their importance in CFTR function. Only recently, a report by Xie et al. (1995), that studied the effect of a nineteen amino acid deletion in CL2 (residues 242-307), suggested that this region is involved in stabilizing the fidl conductance state of the CFTR chloride channel. The CLs' possible significance is further highlighted by the fact that 20% of al1 identified CF- associated mutations lie within the CLs. Exon 17b in particular, which approxirnately corresponds to CM (amino acids 1O3 5- 11 02)' appears to be a 'hot-spot' for such mutations (Mercier et ai., 1994a). Thus far, eighteen different point mutations have been described in this exon, in cornparison to seven, two, and three point mutations described respectively in

CLs 1, 2, and 3 (CF genetic analysis consortium, personal communication). nie gme alterations within CL4 produce various degrees of CF, but there is no account as to the underlying biochemical defect that in each case results in the observed disease symptorns. Interestingly, of the 500+ mutations hown to be associated with CF, only two have been found to arise de novo, Le. were not iniierited bm either parent; one of these de novo mutations is R1066H in CU,which rnay fiirther indicate that this area of CFTR is highly susceptible to nucieotide alterations (Cremonesi et al., 19%). The mutations in the CLs do result in CF, so that they must have some effect on proper CFTR activity. This tool provided by nature was utilized in the presented study. The point mutations were reconstructed in the expectation that evaluation of changes in hction will allow speculation about the nomial task of the CLs. Previously, the detailed analysis of amino acid alterations in other CFTR domains had shown that the CF phenotype usually is attributable to either a lack of the channei at the ceIl surface or the production of a channel with impaired function (Welsh and Smith, 1993). The aim of this chapter was to observe whether point mutations in CUaffect processing and intraceilular transport of CFTR or channel activity. CL4 was used as the starting point of the loop investigations solely because the large number of disease-associated mutations in this single area rnay indicate functional significance of that arnino acid stretch (Mercier et al., 1994a). Results and Discussion Identlpcation of Processing Mutants in CL4 of CFTR - To date, eighteen different point mutations in CUhave ken reported in the DNA fiom CF patients (Fig. SA), but no information is available regardhg their general eEects on the protein which give rise to the associated disease symptoms. Because the CFïR molecuie is known to be extremely susceptible to the ER quality control system (Cheng et al., 1990; Lukacs et al., 1994; Pind et al., 1994), we fint examined the capacity of each of the mutants to exit fiom that compartment and to acquire complex N-linked oligosaccharide chains in the Golgi apparatus. This process can be readily assessed fiom mobility shifts on SDS-PAGE (Pind et

al., 1994). In order to allow a rapid screen of ail 18 mutations, a transient expression system was developed that utilized the expression vector @NA3 (InVitrogen) to carry the CFTR gene. pcDNA3 contains an SV40 ongin of replication and the human cytomegaloWus promoterlenhancer region which has proven to drive high levels of expression in eukaryotic systems. Immunoblot analyses of transiently transfected HEK-293 ceiîs demonstrated that in this expression system CFTR could not be detected in cells transfected with vector alone (Fig. 5.2, panel A). However, if the vector camîed the wild-type CFTR gene, the core- glycosylated band B was produced as well as the fully glycosylated, diffuse band C. For twelve CL4 mutants the major products had apparent masses of 150 kDa corresponding to the core-glycosylated band B, indicating that these proteins were irnproperly processed. The mature fom of the other six mutants (F1052V, KlO6OT, A1067T, G1069R, R1070W, R 10704) were produced in relatively normal amounts (band C), although for A 1O67T- and R1070W-CFTR the ratio of the complex-glycosylated to core-glycosylated bands was significantly lower than for wild-type CFTR. The same results were obtained when the mutants were transiently expressed in COS-1 cells and screened by Western blotting (data not shown). To Merconfirm that the misprocessed CFTR mutants were core-glycosylated molecules, the protein samples were digested with endoglycosidase H prior to detection by Fig. 5.1. Simpiïfied representation of CF'-associated point mutations identified in CL4 CFTR was drawn according to the mode1 proposed by Riordan et al. (1 989). The eniarged CL4 corresponds to amino acids 1035-1 102. Mutated residues are depicted as open circles.

Each mutation is described as the on@ residue (£htletter), its location (number), and the amino acid it was changed to (second letter). NBF, nucleotide binding fold; R regdatory domain; TM, transmembrane helix; CL, cytoplasmic loop.

Fig. 5.2. Evaiuation of mutations in CL4 regarding their effect on processing. Using the expression vector pcDNA -3, wild-type, control (vector only), and CL4 mutated CFTRs were trançiently expressed in HEK-293 celis for 48 hours. (A) The cells were Iysed in 1% SDS plus protease inhibitors and directiy analysed by Western blotting, using 5% SDS- PAGE and M3A7 as the primary antibody. It codd be clearly distinguished that six mutations allowed the formation of Myglycosylated Cm(band C), whereas the remainiog twelve mutants produced only core-glycosyiated protein (band B). Below band B, traces of a lower rnolecular weight species can be seen, which most likely originate fiom the use of an altemate site of initiation for translation (Carroll et al., 1995; Pind et al., 1994,

1995). (B) Cells expressing wild-type or CLA mutated mis-processed CFTRs were lysed in lysis bfler and incubated for 10 min at room temperature with (+) or without (-) endoglycosidase H (endo H). Subsequently, the sarnples were analysed by Western blotting, using M3A7 as the prirnary antibody (5% SDS-PAGE). Al1 twelve mutants were endoglycosidase H sensitive.

Western blotting. Endogiycosidase H is an enzyme secreted by several S~eptomycesspecies that hydrolyzes the glycosidic bond between the core Kacetylglucosamine residues of eukaryotic asparagine-linked oligosaccharides. This bond is only sensitive to digestion in core-giycosylated proteins of the ER and early Golgi. In the intermediate Golgi cisternae, several mannoses are removed and an N-acetylglucosamine is added to the oligosaccharide chahs. This renders the core-glycosidic bond resistant to cleavage by endoglycosidase H (Alberts et al., 1989). Hence, sensitivity to endoglycosidase H of an oligosaccharide generally indicates that the carier protein has not ben processed through the Golgi. Al1 twelve processing mutants were endoglycosidase H sensitive (Fig. 5.2, panel B), suggesting that they indeed were incapable of leaving the ER Thus, the heterologous transient expression analysis indicated that for twelve of the eighteen CF-associated point mutations in CUthe underlying basic defect of the disease symptoms is the physical lack of the CFTR channel at the ce11 surface due irnproper protein processing. In the case of previously studied CFTR mutations known to result in ER capture, including AF508, it is believed that local misfolding prevents the aitainment of the global conformation of CFTR that is required for maturation and ER to Golgi transport. CL4 wodd be expected to contribute to the cytoplasmic aspect of the overall tertiary structure of the molecule in the ER membrane.

It appears that disturbance of the local folding may prevent it fiom adequately making this contribution. This result was not unexpected in analogy to the stnicturally related P- glycoprotein for which mutations in the CL regions also frequently prohibit proper processing (Loo and Clarke, 1994a,b). It is not obvious why amino acid substitutions at 9 of the 14 different positions compromised CFTR maturation, whereas those at the remaining 5 positions appeared not to (Table 5.1). Interestingly, when the obsewed processing characteristics of each CFTR- variant were correlated to the pancreatic status of the CF patients, it was seen that for 8 of 11 mutations resulting in misprocessing of CFTR, the patients are pancreatic insufficient (PI) and for 2 of 3 mutations allowing the processing of CFTR, the patients are pancreatic Table 5.1. Summary of processing characteristics of CL4 mutants versus the patient phenotype '+' indicates presence of band C in Fig. 5.2 and '-' its absence. Tbere is a general trend that pacreatic insufficient (PI) patients carry mis-processed mutants (8 PI / 1 1 mis-processed) and pancreatic sufncient (PS) patients cary processed mutations (3 PS / 4 processed). R1070Q is designated PSPI because, of five patients analyzed, one was PS, one PI and three were uflspecified. The original paper describing each mutation is indicated. not reported. Mutation

Mercier et al., 1993 Ferec et al., 1993 Casais et al., 1995 Mercier et al., 1993 Ghanem et al., 1994 Fanen et al., 1992 Ferec et al., 1992 Mercier et al., 1993 Ferec et al., 1992 Savov et al., 1994 CasaIs et ai., 1995 Mercier et al., 1993 Ghanem et al., 1994 Bozon et al., 1994 Mercier et al., 1993 Zielenski et al., l995a Zielenski et al., 1993 Mercier et al., 1993 sufficient (PS) (Table 5.1). Hence, on this basis, exon 1% mutations are corsistent with the general notion that biosynthetic amst of CFTR in the ER causes severe disease whereas some point mutations, compatible with transport of the protein to the ce11 surface, may result in less severe disease (Kerem et al., 1989; Welsh and Smith, 1993).

For the most common processing mutant, the S508 deletion, exposure of CFTR expressing cells to a low temperature environment forces the transport of some molecules to

the plasma membrane (Denning et al., 1992b). In order to investigate whether CL4 processing mutants show a similar temperature sensitivity, both HEK-293 and COS4 cells were transientiy transfected with pcDNA3 carrying wild-type and CL4 rnutated CFT&.

Fifteen hours &er transfection the cells were moved to an 26°C environment for 48 hours

while a control set remained at 37OC. Subsequently, equal amounts of total protein were analyzed fiom each batch by Western blotting. For none of the mutants was the increase in processing significant enough to be detected by Westem blotting. The HlO54D-CFTR variant is illustrated as a representative example (Fig. 5.3). However, a cornparison of the results obtained for the expression in HEK-293 and COS4 cells highlighted some noteworthy differences: (i) glycosylation patterns for Wly glycosylated wild-type CFTR diEered in the two ce11 lines, as band C migrated at a higher molecular weight if expressed in COS-1 cells than if expressed in HEK-293 cells. Such an effect of glycosylation-pattem- dependence on the ce11 type has been previously descnbed (Bear et al., 1992a). (ii) when COS-1 cells were cultured at the reduced temperature, increased amounts of the immature band B accumulated, an effect that was not obvious in HEK-293 cells. (iii) for CFTR expressed in HEK-293 cells a lower molecular weight band A was prominent, that was only a minor species if expressed in COS-1 cells. (iv) upon switching the cells to a lower temperature, band A production was inhibited in HEK-293 celis. Recent studies implied that band A results fiom initiation of translation at an alternative site (Carroll et al., 1995; Pind et al., 1994, 1995). Thus, growing the cells at a lower temperature appears to reinforce the use of the initiation site that leads to the synthesis of the core-glycosylated band B. Fig. 5.3. Effect of temperature shifk on processing of CL4 mutants. HEK-293 and COS-

1 ceils were switched to an 26OC environment 15 hours pst-transfection for 48 hours. A control sample remained at 37°C for the same time-period Subsequently, the protein expression was analyzed by Western blotting (M3A7,5% SDS-PAGE). The same negative results were obtained for ali mis-processed mutants. Mutant H1054D is show as a representative example. WT, witd-type; A, core glycosylated band A; B, core giycosylated band B; C, hlly glycosylated band C.

Functional Analysis of CL4 Mutants by Iodide Eflux - Although the above observations suggested strongly that biosynthetic arrest and mislocalization is the primary cause of dysfûnction in twelve of the CL4 mutants, it was possible that very low amounts of these reach the ce11 surface and provide some anion conductance. Similady, it might be suspected that the other six that mature and reach the cell surfâce may be defective in their chloride channel activity. To assess these possibilities, iodide efnux assays were used as a measure of the CAMP activated anion channel activity of CFTR. COS-1 cells were employed because of their better adherence to culture wells than HEK-293 cells. Ail six processed CFTR mutants could be activated by forskolin to various degrees (Fig. 5.4, top panels). To estimate the amount of CFTR protein present in each ce11 batch used for the iodide-enlux, the ceils were lysed with equal volumes of 1% SDS and subsequently equal amounts of ce11 lysate were anaiyzed by Western blotting (Fig. 5.4, bottom panel). This showed that the level of activity for cells expressing each CFTR mutant did approxirnately correspond to the amount of manire protein in the sample, i.e. in COS-1 cells F1052V-

CFTR, K1060T-Cm and G1069R-CFTR produced efflux levels similar to wild-type CFTR in accordance with a protein expression similar to wild-type. A 1067T-CFTR, RI 070W-CFTR, and R1070Q-CFTR had significantly lower efflux levels and significantly lower protein expression than wild-type CFTR in COS4 cells. Hence, none of the processed mutants were grossly defective in their ability to form CAMP activated chlonde channels. The sarne flux assay was perfonned with the twelve CL4 mutants that caused misprocessing of the channel. COS- 1 cells expressing these mutants experienced no or very rn-inute (e.g. Q 107 1P-CFTR) activation with forskolin (Fig. 5.4, middle panels), consistent with low levels of mature CFTR on the ce11 surface of these variants. Patch-clamp Analysis of CL4 Mutants - Since the six processed mutants do result in disease, some alteration in the normal function of CFTR was expected. In order to identiQ more subtle changes in their activity, the mutant channels were stably expressed in CHO cells and analyzed by the single-channel patch-clamp technique. Other disease- Fig. 5.4. Evaluation of mutations in CL4 by iodide efflux. Wild-type, control (vector only), and CU mutated CFTRs were transiently expressed in COS-1 cells for 48 hours. Cells were treated with loading baer containing 136 mM sodium iodide. Subsequently, the cells were washed with efflux beer fke of sodium iodide. From time O onward, the efflux buffer contained 10 pM forskolin. The stimulateci iodide e8nuxes were deterrnined with an iodide sensitive electrode. Top panels: Iodide effluxes of ceIis expressing mutants that allowed processing of CFTR. Middle panels: Iodide effluxes of cells expressing mutants that inhibited processing of CFTR. Bottom panel: Immediately after the flux experiment, cells expressing processed mutants were lysed with an equal volume of 1% SDS plus protease inhibitors and an equal amount of celi lysate was analysed by Western blotthg (5% SDS-PAGE,M3A7). This indicated that for these variants the amount of efflux roughly corresponded to the amount of protein expression. The data are fiom representative experiments, each of which was repeated at Ieast three times. Tie (min) The (min) causing CFTR mutants, which are appropriately processed and tmfiïcked to the plasma membrane, show defective ion conduction properties (e.g. R3 34 W, R347H, and R347P; Sheppard et al., !993; Tabcharani et al., 1993) or defective regulation of channel activity (e.g. GSSIS, G1244E, S I255P, and G1349D; Anderson and Welsh, 1992). Stable expression of each of the CL4-CFTR variants in CHO cells led to the appearance of PKA and ATP dependent channel activity in excised membrane patches (Fig. 5.5, panel A). The unitary conductance for dl channels, measured with symmetrical chloride concentrations of 154 rnM, was identical to that of wild-type CFTR (-7.6 pS; Fig 5.5, panels B and C), suggesting that once the channel was open, chloride permeation was not altered by CL4 mutations. This indicated that despite their close proximity to the putative pore entrance, these residues do not contribute to interactions between the channel and the permeant anions. However, as is suggested by Fig. 5.5, panel A, different CL4 mutations had distinct effects on channel gating. The mean Po for each mutant was reduced compared to wild-type (Fig. 5.6, panel A). In some mutants this reduction in Po was associated with a significant reduction in mean burst duration (FI O52V, G1 O69R) or an increase in mean interburst duration (R1070W). In other cases, there were smaller changes in both of these parameters (Fig. 5.6, panel B and C). The fact that partial activity remained correlated well with a less severe phenotype in that two out of three of the fùlly processed variants are found in PS patients (Table 5.1). Despite the relatively small aiterations in the Pos, their conceptual significance is highlighted by the fact that each of the single amino acid substitutions in CL4 that allowed processing reduced Po of the channel, whereas deletion of 19 residues in CL2 had no effect on the Po (Xie et al., 1995). Overall, these functional data suggest that residues in CL4 affect both the rate of channel opening and the stability of the open state, and that mutations in this region may lead to CF by causing a reduction in channel activity. In summary, it was found that twelve of the mutations identified in CUresult in the CF phenotype due to misprocessing of CFTR, thus showing that point mutations in CU Fig. 5.5. Single channel activity associatecl with processed CL4 mutants. (A) Examples of wild-type CFTR and CUmutant CFTR single channel currents recorded fiom inside-out membrane patches at a membrane potential of -30 mV. In each case, the closed state is indicated by a horizontal he on the lefi. (B) Mean single-charnel curent-voltage relationships for wild-type CFTR (open circles) and an example of a CL4 mutant channel (filled circles, G1069R). Each point represents the mean of data fiom 3 to 12 patches. No error bars are show since in eacb case the S.E. was sderthan the size of the symbol. (C) Mean single-chanoel conductaxice of those CU mutants studied, showing mean data fkom 1 1 patches for wdd-type 0and 4- 10 patches for different mutants. In each case, error bars represed one S.E.. SINGLE CHANNEL PROPERTIES OF CYTOPLASMIC LOOP IV MUTANTS

Wild Type Fig. 5.6. Mean kinetic properties of single CL4 mutant channels. Open probability, burst duration and interburst duration are descnbed. In each case, data are the mea.+ S.E. fiom 12 patches for wild-type and 4-5 patches for each mutant. * represents a significant difference fiom wild-type @ c 0.05, two-tailed r test). SINGLE CHANNEL KINETIC PROPERTIES OF CYTOPLASMIC LOOP IV MUTANTS often perturb the overall stmcttm of the molecde sdliciently to mark it for recognition by the ER quality control syçtem. Six mutations ailow the protein to mature, and in these, the CF phenotype is likely a consequence of the decreased Po detected. The decrease in Po could generally be contributed to an increase in the channel mean closed time and a decrease in the channel mean open tirne. This result together with the unaffecteci conductance suggests that mutations in CUdo not affect pore properties of CFTR, but rather the mechanism of channel gating. Finally, these data provide the first functional evidence that this set of mutations does cause disease. Cytopiasrnic Loop Three of CFTR Contributes to Regulation of Chloride Channel Activity Chapter Summary To examine the contxibution of cytoplasmïc loop three (CL3, residues 933-990) of CFTR to channel activity, all three point mutations that have ken detected in CL3 of patients with CF (S945L, H949Y, G970R) were characterised CHO cell Iines stably expressing wild- type CFïR or mutant G970R-CFTR yielded polypeptides with apparent masses of 170 kDa as the major products, whereas the major products of mutants S945L-CFTR and H949Y-CFTR had apparent masses of 150 kDa. The 150 kDa fom of CFTR were sensitive to endoglycosidase H digestion, indicating that these mutations interfered with maturation of the proteh Increased levels of mature CFTR (170 kDa) could be obtained for mutant H949Y when cells were grown at a lower temperature (26OC) or incubated in the presence of 10% glycerol. For al1 mutants, the open probability (Po)of the Cmchannels was significantly altered. S945L-CFTR and G970R-CFTR showed a severe reduction in the Po,whereas the H949Y mutation doubled the Po relative to wild-type. nie changes in Po predominantly resulted fiom an alteration of the rnean burst durations which suggests that CL3 is involveci in obtaining and/or maintaining stability of the open state. In addition, mutants S945L and G970R had 1-V relationships that were not completely linear over the range i80 mV, but showed slight outward rectification. The fact that CL3 mutations can have subtle effects on channel conductance suggests that this region rnay be physically close to the inner mouth of the pore. Introduction To investigate the contribution of the CLs to CFTR activity, CUwas chosen as the starthg point becaw of the high fiequency of CF-associated mutations in this domain. However, reconsûuction of CL4 alterations indicated that the reason for the susceptibility of this peptide segment to deletenous point mutations appears to be its significant contribution to the correct overall folding of the CFTR molecule rather than its fûnctional importance. Mutations in CUrnust disrupt this contribution since a majority of the CF-associated point mutations cause the channel to be retained intracellulady and to not reach its major site of action, the plasma membrane. The following two chapters will address the question whether mutations in other CLs have more severe effects on CFTR activity than the relatively small effects of mutations in CIA. The airn of the curent study is to apply the same approach of reconstnicting CF- associated point mutations to the previously iminvestigated loop CL3 (predicted residues:

933-990; Riordan et al., 1989; Fig 6.1). CL3 comects TMç 8 and 9 of CFTR. Thus far, only three CF-associated point mutations have been published for this loop: S945L (Claustres et al.. 1993), H949Y (Ghanem et al., 1994), and G970R (Cuppens et ai., 1993). The fact that relatively few amino acid substitutions have been identified in CL3 of CF patients could indicate that gene alterations in this domain often evade detection because of their negligible functional consequence; altematively, CL3 may tolerate very litîle change, so that many -O acid substitutiors are lethai. Indeed, characterïzation of the identified mutations will dernonstrate that mutations in CL3 have much more dramatic effects on CFTR activity than mutations in CU, and will suggest that CL3 is significantly involved in regulation of the chloride channel. Results and Discussion Processing Mutanis in CL3 - Three different point mutations have been identifieci

within CL3 (Fig. 6.1) of Cmfiom patients suffiring fiom CF. To hvestigate theu effects

on protein pmxssing, the mutations were recomtrwted in a CFTR-containing pNUT vector (Chang et al., 1993) and stably expressed in CHO celis. Western blotting with the CFTR-

çpecific monoclonal antibody M3A7 (Kartna et al., 1992) demomtmted that wild-type and

G!97OR-mibant CFTRs yielded Mymature protein (1 70 kDa, band C) as the major product whereas mutants S945L and H949Y yielded Meof the mature fonn Fig. 6.2). In these two cases the predominant producf had a molecular rnass of 150 kDa (band B). Band B was fdly

sensitive to digestion with endogiycosidase H (data not shown), indicating that it was a core- glycosylated biosynthetic intemiediate, incapable of le-g the ER The same results and

relative protein maturation levels were observed den the mutations were reconstructed in pcDNA3-Cm tnmsiently expressed in either HEK-293 or COS-1 cells, and screened by

Western blotting (data not shown). These findings indicated that the S945L and H949Y mutations cause a defed in the biosynthetic processing pathways of CFTR maturation. The most common mutation of CFTR (M508) associated with CF has ken found to disnipt processing of the protein in a similar marner (Cheng et al., 1990, Sheppard et al.,

1995). However, maturation of AF508 is sensitive to the celi culture conditions; when cells

were grown at reduced temperatures @enning et al., 1992b) or exposed to elevated leveis of

glycerol (Brown et al., 1996; Sato et al., 1996) an increase of mature AF508-CITR at the ce11 surface has been observed. To explore the temperature/glycerol-semitivity of the processing

mutants in CL3, CHO cells stably expressing these mutants were shifted to 26°C for 48 hours (Fig. 6.3, panel A) or lefi at 37°C for 48 hours with the addition of 10% glycerol to the growth-media (Fig. 6.3, panel B). In cornparison to a control sarnple that remained at 37OC without glycerol, it was observed that for the severely affecteci mutant S945L these procedures did not irnprove maturation of CFTR sigdicantly. In the case of H949Y, however, the amount of protein in band C was Merincreased under both conditions, with a more strikùig Fig. 6.1. Schematic npresentation of CFLa CFTR is depicted according to the predictions of Riordan et al. (1989). The numbers 1, 2, 3, and 4 indicate cytoplasmic loops CL1, CU,

CL3, and CURspectively. CU,which is predicted to conespond to amino acids 933-990, is enlargecl to depid the CF-associated point muîations identifieci within this region of the protein. For each mutation the fkt letter corresponds to the original residue, the number gives the location within the primar). sequence of Cmand the second 1- describes the residue which results from the mutation. CL, cytoplasmic loop; MF, nucleotide binding fold; R, regdatory domain; TM, transmembrane helix.

Fig. 6.2. Immunoblots of CU mutants. CHO cells were stably transfected with pNUT

vector ody (contml), or pNUT vector @g the wild-type CFTR gene or the CFTR gene

with CF-associateci point mutations within CL3. (A) Afier ce1 lysis, equal amounts of total cellular protein were separated by SDS-PAGE (5% acrylamide gel) and CFTR was detected by Western blotting using the monoclonal antibody M3A7. The positions of core-glycosylated

band B and Myglycosylated band C fomof CFTR are indicated. (B) 'The relative amolmts of bands B and C were measured by densitometry. The values were first normalised according to the amount of band B relative to wild-type 0.The amount of band C was then calculated as a percentage relative to wild type (n=3). S94SL H949Y C970R

CL3 Mutan ts Fig. 6.3. Temperature- and glyceroi-sensitivity of CL3 mutants. Stably expressing CHO cells were incubateci for 48 hours (A) at 26°C or 37°C or (B)in media in the presence (+) or absence (-) of 10% glycerol. The ceils were then lysed with SDS and dysed by Western bloning (5% SDS-PAGE;M3A7). Equal amoimts of total ceil protein were applied to each lane. Position of the fUy giycosylated form C of CFTR is indicated.

effect observeci with 10Y0 glycerol. It has to be noted that protein degradation was an apparent

side effect of the glycerol treatment and rnany ceik died upon exposure to glycerol. In hct, when a concentration gradient was evaluated, pracessing could only be promoted at glycerol levels which also induced ceil death ('th effi commencecl at 5% giycerol; data not show). This increase in ce11 death rnay explain altentions in the maturation/degradation profile of wild-type CFRThe extreme culture conditions did not induce the expression of endogenous CFïR in mock transfected CHO celis (Fig. 6.3, panels A and B). Improper tra£ücking of gene products is the cause of a growing number of diseases,

including Tay-Sachs disease or ai-antitrypsin deficiency, and is often attributed to misfolding of the protein (Thomas et al., 1995). In the case of CF, misfolding is also cornmody stated as the reason for retention in the ER of mutants such as AF508-CFTR.

This suggestion was prompted by observations like the reduced stability of AF508-CFTR at the plasma membrane (Lukacs et ai., 1993), slightly altered circular dichroism spectra and reduced stabiiity of a synthetic peptide representing a portion of NBFl that comprises the

AF508 mutation (Thomas et al., 1992 a,b), and finally by the alleviation of the processing defect due to reduced temperatures @enning et al., 1992b) and chernical chaperones (Sato et al., 1996; Brown et al., 1996). However, al1 of this evidence is indirect and the actual misfolding of CFTR mutants has never been demonstrated. Ody a recent study by Qu and

Thomas (1996) with recombinant NBFl indicated that the AF508 mutation significantly reduces the folding yield at a variety of temperatures as well as the rate of folding, suggesting that Phe 508 may make crucial contacts during folding. Since a large nurnber of

CL 3 & 4 mutations cause misprocessing of CFTR, an attempt was made to use these mutant proteins to provide more direct evidence for induced misfolding.

One approach to indicate misfolding was based on the hypothesis that the mutant proteins rnay show different accessibility to exogenous proteases while still inserted into the membrane. To test this, HEK-293 cells were transiently transfected with the pcDNA3 vector carrying wild-type or CL3 mutant CFTRs; forty-eight hours pst-transfection, cmde membranes were prepared and subjected to tryptic cleavage at various concentrations for

various time periods. This procedure demonstrated different banding patterns for the rnisprocessed S945L and MY-CFTRs relative to the properly processed G970R- and wild-type CFTRs (Fig. 6.4, panel A). However, the additional bands in the later two cases were diffuse, indicating that they may result fiom the presence of mature glycosylation. That this indeed was the case was demo-ted in Fig. 6.4, panel B, which showed that (i)

AFS08-CFTR produced the same banding pattern as S945L and W49Y-CFTR which was diEerent fiom wild-type CFTR, but that (ii) this pattern was dso obtained fiom wild-type CFTR if the proteins were cornpletely deglycosylated with N-glycanase F prior to trypsin cleavage. Thus, misfolding of CFTR variants could not be demonstrated with this approach. A second investigation was based on a report that in the related P-giycoprotein, CL3 can be found on the extracellular side of the membrane if the protein is produced by cell-ke synthesis (Skach et al., 1993qb). Since this area appears to be susceptible to altered folding, we speculated that CL3 mutations may perturb the conformation of CFTR sufficiently to place the loop on the extracellular side. To demonstrate such an event, in al1 three CL3 mutants the native glycosylation sites were eliminated by changing N894 and N900 to Ala.

Subsequently, a P960N point mutation was introduced into CL3, thereby creating a potential glycosylation site with the sequence 959-A-N*-M-S-T-963 (Fig. 6.5, panel A, * indicates potential site of glycosylation). That the introduction of the potential glycosylation site did not inhibit processing of wild-type CFTR was demonstrated by Western blotting (Fig. 6.5, panel B) and that none of the mutations af5ected the charnel fùnction of CFTR was seen in iodide eaux experirnents (Fig. 6.5, panel C). The observation that glycosylation of the protein is not essential for function has ken described previously (Moms et al., 1993~).If CL3 mutants, fiom which native glycosylation has been removed and into which a novel intemal glycosylation site has been introduced, can become core- glycosylated when expressed in HEK-293 cells, then this wouid demonstrate that the mutations cause the molecde to missfold to such an extent that the loop becomes onented Fig. 6.4. Tryptic cleavage of membrane embedded CU mutants. Forty-eight hom pst- tmsfecton cdmembranes were prepared hmHM-293 ceils expressing wild-type CFTR and various mutant CFTRs and subjected to ûyptic cleavage. Cleavage was performed for 15 min on ice with the ûypsin concentrations çhown above each lane (cig/mL). The reaction was temiinated by the addition of 3 pg trypsin inhibitor per 1 pg trypsin. Cleavage products were separated by SDS-PAGE (9%) and analysexi by Western blotting (M3A7). Positioning of the 200 kDa molecular weight rnarker is indicated. (A) Cleavage of wild-type and CL3 mutant CFT& demomtmted that the processed variants (wiid-type and G970R-Cm) produced one extra, dinuse band that was absent in the mis-processed variants (S945LCFTR and H949Y-

CFTR). (B) Prior to exposure to the same cleavage procedures, wild-type and A508-CFTR were incubated for 1 hou in the presence (+) or absence (-) of N-glycanase F. This showed that processed wild-type and mis-processed A508-Cm showed the same differences in banding patterns as the processed and mis-processed CL3 variants of (A); however, the differences were eliminated if glycosylation was removed with N-glycanase F and were therefore due to the presence of complex giycosylation. O 10 30 nnn Fig. 6.5. Deglycosyiation/regiycosytation of CU variants. (A) The schematic shows the

location of endogenous glywsylation sites N894 and N900 that were removed by mutagenesis to Ala and the location of the potentiai glycosylation site that was introduced with the

mutation P960N, creating a N*-M-S consensus sequence (* hdicates the potential site of glycosylation). (B) Western blotting with M3A7 (5% SDS-PAGE) demonstrateci that

introduction of an Asn at position 960 into wild-type CFIR did not affect processing of the

protein. This is in contrast to the introduction of an Asn at position 953, which eliminated processing (various potential giywsylation sites were tested for their feasibility). Note that L953N-CFTR and P960N-CFTR stiu containeci the endogenous glycosylation sites N894 and

N900, so that the L953N mutant is still core-glycosylated. (C) None of the mutations affecteci

the efnux profle of CFTa Iodide efflux was performed as descri'bed in Fig. 6.6. N2, N894/900A; N3, P960N+N894/900A; Control, cels transfected with pcDNA3 vector only. (D) The S945L-, H949Y-, G970R-mutations were introduced into the N3 background, i.e. into

a mutant which had the natural glycosylation sites removed and the potential htracellular site

introduced. Mer cell lysis, the deglycosylation/reglycosylationmutants were hcubated in the presence (+) or absence (-) of endoglycosik H and analyseci by Western blotting (5% SDS-

PAGE, M3A7). Since none of the novel Asn 960 residues became glycosylated, this locus was not utilised by the glycosylation rnachinery.

into an extracellular location, now accessible to the glycosylation machinery. However, endoglycosidase H cleavage showed that none of the novel comtructs becarne core-

glycosylated (Fig. 6.5, panel D). The same observation was made when this mutagenic

strategy was applied to all eighteen CF-associated point mutations of CL4 (data not shown). Thus, neither the tryptic nor the mutagenic approach could demonstrate misfolding of the misprocessed mutants. This does not prove that the CFTR mutants are not misfolded, however misfolding still was not demonstrated directly.

EvaZuation of CL3 Mmomby iodide E~ZLT- Iodide efflux (Chang et al., 1993) was used to estimate whether the macroscopic anion permeability of cells expressing CL3 mutant CFTRs deviated fiom wild-type Ievels (Fig. 6.6). Upon exposure of iodide loaded CHO ceils to the agonist forskolin, there was CAMP-mediatedenlw observed for cells transfected with wild-type CFTR, but no activation for mock transfected cells. Very little stimulation occurred in cells expressing S945GCFTR in agreement with the severe inhibition of protein maturation described above (Fig. 6.2). H949Y-CFTR containhg ells exhibiteci iodide efflilxes which were sirnilar to wild-type (Fig 6.6), although there was much less mature protein than in wild- type expressing cells as judged by Western blotting (Fig. 6.2). This suggested that the H949Y mutation may actuaily produce a hyperactive fom of CFTR. For G970R-CFTR a foskolin induced efflux was almost absent (Fig. 6.6), despite the large amounts of complex glycosylated protein observed in Western blottuig (Fig. 6.2). The same results of relative total ce11 activities were obtained when iodide efflwes were rneasured fiom COS-1 cells tmnsiently expressing these mutants (data not shown).

It was apparent that G970R allowed the production of Myglycosylated CFT'R, but that there was little anion charnel activity in the cells expressing this mutant. To ensure that the glycosylated form of the protein did in fact reach the plasma membrane, surfàce labeiing was cauied out with the membrane irnpermeant ragent biotin-LC-hydrazide (Lisanti et al., 1989; Prince et al., 1994). The chemical bhds to the sugar entity of glycoproteins exposed on the plasma membrane and thereby attaches a biotin molecuie which cm then be detected Fig. 6.6. Fundionai evaluation of CL3 mutants by iodide eQux. Stably expressing CHO ceh were incubated in 136 mM Nd for one hour. After removal of extracellular Nd, the ce& were stimdated with 10 @id forskolin stamng at the '0'. The resdting anion effluxes were detected with an iodide sensitive electrode. These data are hmrepresentatîve experirnents, each of wtùch was repeated at leasî three times. WT, wild-type CFTR expressing cek; Control, ceils which stably express pNUT without CFTR. 7/ Control WT S945L - H949Y + G970R

Time (min) through association with a streptavidin-carrying reporter molecule. With this approach, it was observed that sdararnoimts of mature forms of G970R-CFT'R and wild-type protein were biotinylated (Fig. 6.7). The lack of biotinylation of band B serveci as an intemal control that biotin-LC-hydrazide did not permeate the bilayer. This showed that G970R-CFTR did reach the cell surface and therefore must be severely impaired in fùnction to explain the very low level of iodide efflux observed. The absence of fimction is Likely to be the cause of the CF syrnptorns in patients affectecl by the G970R mutation. Mutational A~k'yskof Residue Gly 970 - The mutation of a Gly to an Arg at position 970 is a siflcant alteration since a smd, uncharged amino acid is replaced by a buUcy, positively charged amino acid. To investigate whether the fùnctional effect observed was due to the introduction of a charge or due to the introduction of sk, several 0thmutations were generated: Gly 970 was changed to either Ala (srnail residue), Met (bulky residue), Glu (negatively charged residue), or Lys (positively charged residue). The pcDNA3-CFTR constnicts, containing the desired mutations, were transiently expressed in COS-1 cells, and the cells were subjected to iodide etnw experirnents (Fig 6.8, panel A). The G970A and

G970M alterations dowed attainment of efflux levels similar to wild-type, suggesting that amino acid size is not the major factor deteminhg disturbance of CFTR hction. Introducing a negative charge (G970E) decreased iodide efflux while the introduction of a different positive charge (G970K)again abolished CFTR fundon. Therefore, the loss of CFTR activity observed with the mutation G970R appears to be due to the presence of a positive charge at this site. To mure that none of the observed fiinctional effects of the different mutants was caused by misprocessing, Western blotting was caxried out with monoclonal anti-CFTR antibody M3A7. As show in Fig. 6.8, panel B, sirnilar levels of mature CFTR were observed for al1 mutants. Patch-clamp Ana[ysis of CL3 Mutants - Iodide efflux measurements suggested that mutations within CL3 have signifiant influences on overail CFTR fûnction. To obtain a more detailed understanding of these effectç, single-channel patch-clamp analysis was applied. AI1 Fig. 6.7. Surface labelling of CFTR expressing ce& Forty-eight hours post-tmmfection, biotin-LC-hydrande was covdently hked to the oxidized sugars of all glycoproteins presented on the siirface of HEK-293 ceh, including the various CFTR variants that these ceik exprd Subsequent to cell lysis, CFTR was imrnunoprecipitated with M3A7. separated by SDS-PAGE (5% acrylamîde) and detected by Western blotting using (A) peroxidase labelled sûeptavidin (Kirkegaard and Perry Laboratones) to detect biotinylation or

(B) M'A7to detect CFTR-

Fig. 6.8. Ioàide etnu measuremenh of cells expressing CFIn Gly a70 mutants. Gly 970

was changed to various amino acids in the transient expression vector pcDNA3-CFTR (A)

COS-1 cek were tninsiently transfd with these comtmcts and 48 hom pst-transfection

were subjected to the idde efaw methods described in Fig. 6.6. (B) Ce& were lysed immediately after the iodide efflux experiment with equai amormts of lysis biIffer and equal amoimts of ceil lysate were analysed by Western blotting (5% SDS-PAGE; M3A7). These data are fiorn representative experiments, each of which was repeated at least three times.

Control, celis mock-transfected with pcDNA3 vector. -4 -2 O 2 4 6 8 10 12 Time (min) three CL3 mutants formed PKA- and ATP-dependent cbannek when stably expressed in

CHO tells. For each mirtant, however, the level of channel aaivity was clearly difTerent fiom wild-type CFTR; channel mean open probability (Po) measurements demonstrateci that

S945GCFiX and G970R-CFïR had signincantly lower mean POSthan wild-type channels.

and the mean Po of W49Y channels was signifïcantIy greater than obsened for wild-type

(Fig. 6.9, panel A). Analysis of channel burst kinetics indicated that for each mutant the

alteration in Po was mainiy the result of a change in mean open burst duration (Fig. 6.9, panel

B), with a smaller change in mean interbufit duration also contributing to the reduced Po seen in S945L-CFTR (Fig. 6.9, panel C). The fict that CFTR channels with CL3 mutations could still be opened through PKA/ATP-mediated stimulation, but that the duration of their open states was sigrilficantly altered suggested that CL3 may be involved in obtaining ancilor maintainhg çtability of the open state. Within the hework of curent models (Gundenon and Kopito, 1994; Hwang et al., 1994; Carson et al., 199%; Carson and Welsh, 1995:

Wilkinson et al., 1996; Fig 1.5) the dtered duration of the open state observed in the present study may indicate that mutations in CL3 can affect events at MF2 or ai3ect communication fiom NBF2 to the pore. The magnitude of the differences in activity levels of each mutant relative to wild-type CFTR was more pronounced using iodide efflw assays compared to the Po measurements of singleshannel patch-clamping. This may in part resdt fiom differences in CFTR activity in whole cells that have an intact cellular machinery versus excised patches, but more likely is due to limitations of the techniques themselves. Specifically, the wild-type-like iodide efflw level together with the low expression of H949Y-ClTt suggested a very hi& activity for that mutant, which could not be fully accounted for by the doubling of the Po observed in single- channel patch-clarnping. However, iodide efnux may overestimate H949Y-CFTR activity if both wild-type CFTR expressing and H949Y-CFTR expressing cells reach an efflux plateau which masks a merence in the activity of their respective CFTR channels. This was observed by Rosenfeld et al. (1994) who showed that increasing expression of normai human CFTR Fig. 6.9. Mean kinetic properües of single CL3 mutant channeis. (A) Open probability, (B) open burst duration, and (C) interbunt closed duration were calculateci as describeci. In each case, the data are the mean * SEM hm 12 patches for wild-type and 5-6 patches for each midant. Paramm that are significantly different hmwild-type are indicated (*P<0.05;

**P

cDNA in CF epithelial celis results in a progressive increase in the level of CFTR protein expression, but a lunit in the level of CAMP-stimulateci chloride secretion. Similarly, Trapneil

et al. ( 1991 ) used PMA-mediated dom-regdation to show that when the mRNA level in T84

cells is 267% of control, the rate constant for CAMPstimulated chloride secretion is normal.

At mRNA levels of Q2% of conml the stimulation is negligible and at values in between the stimulation is progressively depressed with decreasing mRNA levels. Furthemore, Tabcharani et al. (1991) observed similar efflux levels for wild-type CFTR tmsfected CHO

cells and endogenously CFTR expressing T84 cells, aithough the protein levels are fairly

different. If such an efflux plateau cornes into play, the overall iodide efflux can only be viewed as a qualitative or semiquantitative esthate. To obtain true quantitative data, the

initial slopes of the flux curves have to be determineci, which has thus far not been possible due to technical difliculties. Even if these dBiculties are overcome, iodide efnux will always be an artificial system that uses iodide as a substitlbe for chloride; the two anions are handled somewhat differently by CFTR (Tabcharani et al., 1992). Thus, iodide efflux provides an efficient tool to identlfy gross alterations in CFTR channel activity, but the quantitative details are bea evaluated by more subtle techniques such as single-channel patch-clarnping.

However, for low activity channels such as G970R-CFTR, which give little iodide efflux, the Po may somewhat overestimate residual channel activity shce only open channels can be observed electrophysiologically whereas channels which never open during the course of the experiment will be missed. Two of the CL3 mutations studied by patch-clamping also had subtle effects on chlonde permeation through the Cmchannel. Under the applied recording conditions, wild- type channels had a linear singlechamel curent-voltage (1-V) relationship over the range h80 mV (Fig. 6.10), with a mean dope conductance of 7.8I0.1 pS (n=ll). Both S945L and G970R mutants, however, had 1-V relationships that were not completely linear over this voltage range, but instead showed slight outward rectification (Figs. 6.10, panels A and C). In S945LCFTR, this outward rectification was due to a significant reduction in current at Fig. 6.10. Chionde conductance properties of single CL3 mutant charnels. (A)-(C) show mean single channel 1-V relationships for each mutant ((filled chles) wmpared to wild-type (open circles) CFTR. Each point represents the mean * SEM (wtzere this is larger than the size of the symbol) of data fiom 3-12 patches. (D) shows the mean single-charnel conductance for each mutant measured at both negative potentials (inward curent; open bars) and positive potentials (outward curent; hatched bars). * qresents a significant difference hmwild-type

(PcO.0 1, two-tailed t-test), indicating that S945L shows decreased inward (but not outward) cumnts and G970R has in& oldward (but not inward) currents. Ail data are the mean k

S.E. fiom 6- 15 patches. -100 -80 -60 -40 20 40 60 80 100

Wild Type O Wild Type S945L

O Wild Type negative membrane potentials compared to the wild-type channel (Figs. 6.10, panels A and D), wMe in G970R-CFTR ordward rectification resulted hmincreased current at positive potentials (Figs. 6.10, panels C and D). In contrast, H949Y channels had a hear 1-V relationship (Fig. 6.10, panel B) with a conductance similar to wild-type (7.%0.1 pS; ~7). The rasons for the outward rectification seen in both S945L-CFTR and G970R-CFTR are unclear; however, the fact that CL3 mutations can have subtie effects on channel conductance suggests that this region may be physically located close to the inner mouth of the CFTR chlonde channel pore. Does the R-domain Play a Role in Observed CL3 Effects? - The functional effects observed as the result of CL3 alterations suggested that this dornain is involved in the regdation of CFTR chloride channel activity through its interactioa with the regdatory domains, particularly NBF2. However, one could also envision the Rdomain irnplementing its regdatory hction on the channel through contact with the parts of the pore that promide fi-om the membrane, the CLs. If'some of the effects of the CL3 mutations occur because of alterations of such an interaction with the R-domain, it is possible that these effects would be different in a CFTR-molecule thai cannot be phosphorylated to the full extent, such as the 1 1 SA-CFTR variant produced in Chapter 3. To test this hypothesis, the three CL3 mutations were introduced into 1 1SA-CFTR. The constructs (1 1SA-S945LCm 1 1SA-H949Y- CFTR, 1 1SA-G970R-CFTR) were ûansiently expressed in COS-1 cells and evaiuated by iodide efflux, which showed that the effects due to the CL3 mutations on the residual 11SA- CFTR activity were similar to their effects on wild-type CFTR activity: 1 1 SA-H949Y-CFTR activity was comparable to 1ISA-CFTR, despite decreased processing, 11 SA-S945L-CFTR had reduced activity and reduced processing, whereas I ISA-G970R-CFTR could not be activated by forskolin, although processing was normal (Fig. 6.1 1, panels A and B). ïhis observation would be consistent with (but not exclusive for) the notion that CL3 mutations affect CFTR function not because of altered interactions with the R-domain, but because of aitered interactions with NBF2. Fig. 6.11. CL3 mutations in the LISA-CFTR background The CL3 mutations were ineoduced into a 1ISA-pcDNA3-CFTR construct that Cames the S753A gene variation and transiedy expressed in COS4 ce&. Both, (A) Western blotting with M3A7 (5% SDS-

PAGE) and (B)iodide enlw perfonned as in Fig. 6.6 indicated that the CL3 mutations had the same impact on the residual 1 ISA-CFTR activity as on wild-type activity and produced the same processhg profXe for I 1SA-CFTR and wild-type CFTR Positions of immature band

B and mature band C are indicated. Control, ceiis rnock-transfected with pcDNA3 vector. -4 -2 O 2 4 6 8 10 12 Time (min) CHAPTER 7

N-terminai Cytoplasmic Loops of CFTR What Do They Do? Chapter Summary Since Mie is known about the contribution to finiction of CFTR's N-terminal cytoplasmic lwps (CL 1, residues 139-194; CU, residues 242-307), ail nine point mutations identified in CLs 1 & 2 fiom patients with CF were reconsûucted in the expression vector pcDNA.3-CFTR and ean~ientiyexpressecl in COS4 and HEK-293 cek. Four amino acid substitutions retarded production of mature, fully glycosylated CFTR, suggesting that misprocessing of the chamel causes the disease symptoms in the &ted patients. Protein maturation could not be promoted by celi culture conditions of reduced temperature (26°C).

When properly pmessed mutants were evaluated for fimctional defm by the iodide efflux method, the G178R- and E193K-CFTR expressing ce11 lines showed irnpaired anion translocation activities. Singlechanne1 patch-clarnping elucidated that for the E ;93K-variants the significantly decreased open probability resulted fiom an increase in the mean closed time of the channels; this was opposite to disease-associated point mutations in CL3 which were seen to mainly aEit the mean open the of CFT'R. None of the maturation competent mutants demonstrated modifïed channel conductances. Thus, the Ktemiinal CLs appear not to contribute to the anion translocation pathway of CFTR; rather, mutations in CL1 impede the molecuie's ability to achieve the open state. Interestingly, the I148T and G178R amino acid substitutions negated locking open by the non-hydrolyzable ATP-analogue AMP-PM. Introduction

Reconstruction of CF-associated point midations in CLs 3 & 4 aliowed an evaluation of the cause-effect relationship of protein alterations versus the resulting disease symptorns.

From the data, two major concepts evolved regardhg the importance of the CLs so that an initiai idea could be obtained of the contributio~iof each loop to CFTR fiaidon. CL4 appears to be involved in the achievement of the proper overall protein codguration since a majority of the ClA mutations disallowed protein maturation to the cell surface. Sirnilarly, hvo of the three mutations reported in CL3 cawd misprocessing of CFTR; more strilüngly, however, al1 three CL3 variants dernonstrated major alterations in chloride channel activîty relative to wild- type, much more so than CU variants. Hence, the functional data suggested CL3 to be involved in the regulation of CFTR.

Because the two C-tedCh were found to be distinct in îheir contributions to

CFTR activity, we will complete this initial investigation of the CLs by applying the same muîagenic approach to CL1 (predicted residues 139-194; Riordan et al., 1989; Fig. 7.1) and CL2 (predicted residues: 242-307; Riordan et al., 1989; Fig. 7.1). Thus far, seven CF- associated point mutations have ken published for CLI, but only two for CL2 Interestingly, in a cornparison of CFTR sequences fiom various species, al1 nine of the affected arnino acids are 100% conserved (Manavalan etal., 1993). The N-terminal half of CFTR may be an important component of the channel fomiing imit since it was reported to constitute a regulated chlonde channel by itself as observed through introduction of a preliminq stop codon at residue Asp 836 and expression in HeLa cells (Sheppard et al., 1994), or through the investigation of a shortened rend isoform of CFTR (Morales et al., 1996). It is conceivable that the N-terminal CLs are necessary constituents for this half of the rnolecule. Thus far only two studies have addressed CLs 1 & 2 of CFTR, both of which were based on sequence deletions. Delaney et al. (1993) deleted exon 5 (residues 163- 193). This splice variant is present as an active isoform in human cardiac cells, however, when the mutant was introduced into HeLa ceils, it was misprocessed, so that the functional effects couId not be evaiuated. A similar deletion of residues 267-285 in CL2 dso caused misprocessing of CFTR, yet when the variant was reconstituted into lipid bilayers it formed a fiuictiond channel which was indistinguishable fiom wild-type CFTR in ail aspects of conductance and regdation, except for an altered probability of mition between a high and a low subconductance state (Xie et al., 1995). Thus, very drastic approaches have elucidated little of the importance of CLs I & 2 and more information rnay be obtained by investigating specific residues which should be key sites since their aiteration results in disease. Results and Discussion Biochernical Analysk - AU nine CF-associated point mutations (Fig. 7.1) of CLs 1 & 2 were reconstructed in the vector pcDNA3-CFTR and transiently expressed in either HEK-293 or COS-1 cells. Western blotting demonstrated that five of the protein variants (I148T. II 75V, G178R E193K, R297Q) were unaltered in their processing (Fig. 7.2). producing banding patterns comparable to wild-type CFTR. Four mutations did decrease the yield of the fully glycosylated band C with relative amounts of bF508-CFTR 5 G149R- CFTR < Hl 39R-CFTR < R258G-CFTR c D192G-CFTR wild-type CFTR, so that lack of CFTR at its site of action seems to produce the disease symptoms in the affected patients. Thus, alrnost half of the published mutations in CLs 1 & 2 retarded processing of the channel. However, this sornewhat overestimates the importance of these loops for protein maturation. In total, sixteen mutations have ken identified in CLs 1 & 2 (CF Genetic Analysis Consortium, persona1 communication). When al1 sixteen variants were transiently expressed in HEK-293 cells, twelve (Le., 75%) were processed to a significant degree (data not shown). The mutants that did not achieve the fully glycosylated fonn appeared to be defective in their ability to mature fiom the ER since the core-glycosylated band B was 100% sensitive to cleavage by endoglycosidase H in every case (data not shown). Surface labei hgwith biotin-LC-hydrazide demonstrated that the molecules which matured fiom the ER quality control rnachinery were properly transported to the ce11 surface since, for every mutant, the amount of CFTR protein that could be labeled with biotin-LC-hydrazide (Fig.

7.3, panel A) was proportional to the total amount of fully glycosylated band C in Westem blotting (Fig. 7.3, panel B).

In order to investigate the temperature sensitivity of the CL1 & 2 variants, the four misprocessed proteins were stably expressed in CHO cells and incubated for 48 hours at

26°C. This treatment did not promote processing to a degree detectable by Western blotting (Fig. 7.4). Yet, two points are noteworthy fiom Fig. 7.4: (i) the relative processing characteristics (band C versus band B) of al1 four misprocessed mutants were the same Fig. 7.1. Schematic represenbition of CL 1 & 2 mutations. CFTR is shown according to the predictions of Riordan et al. (1989). CL 1, predicted to correspond to aoiino acids 139- 194 and

CL2, predicted to correspond to amino acids 242-307, are edarged to depict the CF-associateci point mutations identifid within these regions of the protein. For each mutation the first letter corresponds to the original residue, the number gives the location within the prirnaxy sequence of CFTR, and the second letter describes the residue that resdts hm the mutation. CL, cytoplasmic loop; NBF, nucleotide binding fold; R Rdomain; transmembrane helix.

Fig. 7.2. Processing chsracteristics of CFTR carrying CL 1 & 2 mutations. The mutations were întroduced into pcDNA3-Cm transiently expressed in HEK-293 cel Is, and analyzed by Western blotting (5% SDS-PAGE, M3A7). Above the figure the loop of origin is shown for each mutation. Positions of wre giycosylated band B and fûily glycosylated band C are indicated. A long over-exposure tirne was chosen to illustrate the relative amounts of band C for the maturation-defective CFTR-variants.

Fig. 7.3. Surface labelhg of CL 1 & 2 CFTR varïants. The surface sugars of transiently üxnsfected HEK-293 cells were oxidized and covdentiy Linked to biotin-LC-hydrazide. AAer ceU lysis, immunoprecipitation with M3A7, and separation by SDS-PAGE (5%), Western blotting was performed with either (A) peroxidase labelled streptavidin (Kirkegaard and Peny Laboratones) to detect biotiny1ation or (B)M3A7 to detect CFTR.

Fig. 7.4. Temperature sensitMty of CL 1 & 2 CETR variaots. The misprocessed mutants were stably expressed in CHO ce&. For each cell line one batch was grown at 26OC for 48 hours, Mea second batch remained at 37OC for the same time period. The ceiis were iysed with 1% SDS plus protease inhibitors and equal amounts of total protein for each sarnple were separated by SDS-PAGE (5%) and atliilysed by Western blottlig (M3A7).

whether expressed stably or transiently and (ii) in stable expression, the levels of CFTR production were less evenly matched between the various mutant ce11 lines than in transient expression (compare to Fig. 7.2; in ail cases equal amounts of total protein were applied to each lane). In stable expression, the amount of protein production is dependent on the fiequency and locus of gene insertion into the genome, which will be different for each transfected cell; if functional cornparisons are desired, many colonies have to be screened for each ce11 line to enable picking of those which demonstrate simila. leveis of CFTR synthesis. In transient expression, only the transfection efficiency influences the amount of protein production, which generally is sirnilar between the various mutants in a single experiment, but may Vary between difEerent experiments.

Funciional Analys& - When the CL 1 & 2 variants were transiently expressed in COS-1 cells and analyzed by iodide efflux, the observed anion translocation efficiency (Fig. 7.5) of each mutant-expressing ce11 line roughly corresponded to the arnount of processing of that CFTR variant (Fig. 7.2); amino acid substitutions which decreased maturation to the ce11 surface allowed Iess cellular anion conduction and mutations with little effect on processing enabled higher iodide movements. The oniy exceptions to this generalization were the G178R- and E 193K-variants which both produced activities that were lower than predicted from theu wild-type like maturation profile; in al1 five effluxes analyzed, the decrease was more severe for Gl78R-CFTR expressing cells. Western blotting confirmed that expression was similar for al1 mutants in the applied transient system (data not show). Thus, by analogy to observations for CLs 3 & 4, the G178R and El 93K mutations must significantly decrease the anion translocation capability of CFTR since changes could be detected by the rough iodide efflux estimation, whereas for the other CL 1 & 2 variants alterations in the channel characteristics, if present, are relatively small. To fbrther quanti@ these observations the more sensitive single-channel patch-clamping technique was applied. For this purpose, the properly processed mutants were stably expressed in CHO cells, which in al1 cases led to the appearance of PKA-and ATP-dependent channel activity in excised Fig. 7.5. Funetional evaluation of CL I & 2 CFïR variants by iodide efflul Each CFTR variant was transiently expressed in COS4 ceUs. 48 hours pst-transfecton the celis were

Ioaded with 136 mM Nd and then stimulated with 10 ph4 forskolin starthg at time '0'.The resulting iodide efflux was determined with an iodide sensitive electrode. The bottom right panel depicts effluxes fiom CL2 mutant expressing ceil lines; ail other panels show results obtained hm CL1 variant expressing ceiI lines. These data are fiom representative experiments, each of which was repeated at least five thes. WT, wild-type CFTR expressing cells. Control, cells mock-transfected with pcDNA3 vector. membrane patches (Fig. 7.6, panel A). Al1 CFTR variants showed the same linearity and level of conductance as the wild-type channel (Fig. 7.6, panels B and C) and generally had unaltered gating properties (Fig. 7.7). Only the E 193K mutation, in agreement wiîh iodide efnw data, produced a significant decrease in the Po of CFTR, with the magnitude of the decrease correspondhg to changes seen for CL3 variants. Interestingly, the reduction in Po was due to an increase in the mean closed tirne of the channels (Fig. 7.7) that is opposite to the effect of CL3 mutations which were seen to modify predominantly the mean open tirne of the molecuies. According to curent models of CFTR hction (Gunderson and Kopito,

1994; Hwang et al., 1994; Carson et al., 19952; Carson and Welsh, 1995; Wilkinson et al., 1996; Fig. 1.4) a modification of the mean closed tirne is consistent with the E 193K mutation affecting events at NBF1 or af5ecting communication fiom NBFl to the pore. In the case of G178R-CFTR, which had very reduced anion transIocation capabilities in the iodide efnux, dtered channel khetics could not be observed by single-channel patch- clamping. Possibly the reason for this is the additional observation that G178R-CFTR(as well as I148T-Cm) could not be locked open by AMP-PNP. Since in the acquisition of the electrophysiologicai data, our standard procedure involves locking open of CFTR with AMP- PNP at the end of each experiment to obtain a more accurate estimation of the number of channels in the patch, the number of channels per patch may have ken underestimated for

I148T-CFTR and G178R-CFTR expressing cells, thereby skewing the data of Fig. 7.7. and overestimathg the Po of thes2 inutants. It is intrîguing that arnino acid substitutions in CL1 within the fkst half of the molecule can disturb locking open, which has been ascribed to a stabilizing effect of NBF2 on ADP binding at NBF1. This rnay add to ment data that suggests that the mechanism of action for locking open by AMP-PNP is far fiom king understood completely and may involve multiple sites within CFTR (Mathews et al., 1996).

Correlation of Obsemed Effects to Patient Phenotype - Consistent with previous reports (Kerem et al., 1989; Welsh and Smith, 1993), mutations in CLs 3 & 4 followed the trend that lack of CFTR maturation to the ce11 surface produced a more severe phenotype Fig. 7.6. Single-ehamei activity associated with processed CL 1 & 2 mutants. (A) Upon addition of 180 nM PKA and 1 mM MgATP to the cytosolic side of hide-out membrane patches excised fkom CHO cells, channel activity was observeci for wild-type 0and al1 maturation-competent CL 1 & 2 mutated CFT&. Cmtswere recordeci at a membrane potential of -30 mV. The closed state is indicated by a horizontal line on the right (B)Mean single-channel current-voltage relationship of wild-type CFTR and 11481-CF7R as a representative CL 1 & 2 mutant. Each point represents the mean i SE. (where this is Iarger than the size of the symbol) of data fiom 4 (I148T) or 5 (WT)patches. (C) Mean single- charme1 conductance of those CL 1 & 2 mutants studied, showing mean daia fiom 3-5 patches. In each case error bars represent one S.E.. None of the conductances was significantly different f?om wild-type @ > 0.95, two-tailed t-test).

Fig. 7.7. Mean kinetic properties of single CL 1 & 2 mutant channeis. Open Robability Po

(A), mean binst duration To (B), and mean intehurst duration Tc (C)were calcuiated as descxibed in chapter 2. In each case the data are the mean f SE.hm 4-5 patches as indicated by the N-value. Parameters that are si@candy Werent hmwild-type (WT) are rnarked (*. p < 0.005; **, p c 0.000 1; two-tailed t-test). Note that the I148T- and G178R-CFTR variants couid not be locked open with AMP-PM, so that for these mutants the number of channels in each patch may have been underestirnated. than a situation in which a partially functionai channel could reach the surface. Table 7.1 illustrates that this was not the case for CLs 1 & 2. Of the three misprocessed variants for which a phenotype was reporteci, two allowed pancreatic sufEciency. Even more surprising, of the three mutants that were indistinguishable fiom wild-type in terms of processing capability or anion translocation characteristics (1148T-CFTR, I175V-CFTR, R297Q-

CFTR), dl three caused a severe pancreatic insufficient phenotype. This makes CLs 1 & 2 very distinct fiom CLs 3 & 4 and rnay even suggest that a chloride channel defect is not the oniy parameter that should be shidied when investigating the effect of CL 1 & 2 mutations on CFTR function. Table 7.1. Rocessing characteristics versus patient phenotype. The nine mutations within

CLs i & 2 are groupecl according to their impact on processing. The original paper describing each mutation is indicated. PS, pancreatic sufncient; PI, pancreatic insufncient; '-', unspeci fied pancreatic status. Mutation Pancreatic Reference S tatus

Processed:

Bozon et al., 1994 Romey et al., 1994 Zielenski et al., 199 1 Mercier et al., 1995 Graham et al., 1994

Mis-Processed:

Ferec et ai., 1995 Mercier et al., 1995 Audrezet et ai., 1994 Mercier et al., 1995 Discussion and Future Directions Discussion Studying the regulatory rnechanisms of CFTR is an important endeavor in the interests of basic sciences and medical research. CFTR is a peculiar molecule that captures the scientific imagination because it is unique in various aspects when compared to related proteins. Thus far, CFTR is the only chloride channel identified îhat is not regulated by voltage gating or ligand gating, but rather through cellular energy levels and hormonaily induced phosphorylation. Phosphoxylation and dephosphorylation are also a common means of moddating the function of other ion channels (Walaas and Greengard, 1991; Riordan, 1992; Raymond et al., 1993), however, this modulation is usually secondary to the primary gating events, instead influencing channel properties such as inactivation kinetics (Numan. et al., 1991; West et al., 1991, 1992; Ismailov and Benos, 1995). Furthemore, CFTR resembles transporters of the ABC-superfamily more than known chloride charnels not ody in its regulatory properties but also in structure, with one major difference - the R- domain. The R-domain connects two stnicturally similar halves in the same way as the linker region in P-glycoprotein, but it is a more elaborate, space-filling structure that is highly charged and rich in consensus sites for various kinases. Since this domain is unprecedented in other proteins it will be interesting and important to elucidate its functional role, itç rnechanisrn of action, and its collaboration with additional regulatory domains of CFTR such as the NBFs. Complete understanding of the regdation of this chloride channel will also be significant in view of our hope to cure CF with gene and protein therapy. Both processes presently are inefficient and high activation of relatively low amounts of CFTR, delivered to the site of action, will be beneficial. For CFTR, phosphorylation by PKA seems to exert the primary control of activation of the chlonde conductance (Tabcharani et al., 1991). Previous studies showed that mutagenesis of al1 ten dibasic consensus sites for PKA interactions (IOSA-CFTR) drastically diminishes phosphorylation of the CFTR variant by the kinase, but only decreases PKA-mediated activation to 30% of the wild-type level (Chang et al., 1993). One aim of this thesis was to address the mystey of PKA activating CFTR without apparent phosphorylation. Since PKA-phosphorylated sites do not always adhere to the

dibasic consensus (Kemeliy and Krebs, 1991 ), we hypothesized that additional sites might be present in CFTR that are phosphorylated at relatively low levels but still account for the residual sensitivity of IOSA-CFTR. The experiments described in Chapter 3 codmed the validity of this hypothesis. Using large amounts of protein we were able to

show that IOSA-CFTR is still phosphorylated in viîm by PKA. We traced a considerable amount of this phosphorylation to residue Ser 753 and demonstrated a substantial decrease in the PKA activation of IOSA-CFTR upon removal of Ser 753. The observed radiolabeling of a residue is the equilibriurn between the rate of its phosphorylation and the rate of its dephosphorylation. Thus, low leveis of labeling ai Ser 753 could be due to very fast rates of dephosphorylation. Such a scenario may even be imaginable in the applied in vitro phospholabeling procedures since CFTR appears to be tightly linked to endogenous phosphatases (Tabcharani et al., 199 1; Becq et al., 1994) so that CO-precipitationof such phosphatases is possible. However, since a less than perfect consensus site is utilized, the alternative scenario may be more applicable that low levels of labeling of Ser 753 are the result of less favorable phosphorylation kinetics (Kemelly and Krebs, 199 1). What advantage may the presence of inefficiently labeled residues provide to CFTR? The structural conseqsences of phosphorylation-induced changes in the R-domain for the entire molecule are not understood thus far, so that there is little insight as to why and how a multiple-site mechanism is employed. It has been speculated (Riordan et al., 1993) and experimentally reinforced (Fischer and Machen, 1994) that a multiple-site mechanism provides for a graded response to hormonal stimulation of chloride secretion. Such a rnechanism could be more fmely regulated by the various phospholabeling kinetics of different sites. Indications that greater activation can be achieved by higher levels of phosphorylation were first reported by Drumm et al. (1 99 1), who showed that in Xenopus oocytes elevations of intracellular CAMP resulted in an increase in the overall chloride conductance of CFTR, presurnably due to the stknulation of PKA-mediated hyperphosphorylation. The observed increase in total ce11 chloride conductance should correspond to an increase in the average number of channels open at any given instant, since none of the phosphorylation site mutants studied thus far have altered single channel conductances (Chang et ai., 1993; Rich et ai., 1993a), suggesting that once a channel is opened its conductance is not influenced by the degree of phosphorylation. For sites such as Ser 753 at which it is dficult to detect labeling, conditions of elevated cellular CAMP levels (Dnunm et al., 1991) which increase the potential of phosphorylation events to occur could overcome an unfavorable phosphorylation/ dephosphorylation equilibrium. The utilization of sites such as Ser 753 to contribute to channel activation might in this way increase the potential for a metered response of total ce11 chloride conductance in situations of extrerne stimulation. However, under normal cellular conditions, the possibility exists that a site with unfavorable phospholabeling kinetics seldom contributes to function of the wild-type molecule. By the time Ser 753 is labeled as the result of hormonal stimulation, the wild- type channel often may already be open due to phosphate binding at other major sites. In contrast, in molecules such as IOSA-CFTR, the major sites are missing so that the channel remains closed until phosphorylation at Ser 753 andor other unfavorable sites eventually occurs. in such a setting, these sites gain great fundional importance. We note that phosphorylation of Ser 753 was observed in the lOSA background after mutating al1 dibasic consensus sites. In the case of P-glycoproteh, Ser 683 is PKC phosphorylated, but only deradditional major PKC sites are removed (Chambers et al., 1995). A similar situation could possibly hold for IOSA-CFTR.The lOSA mutations may have sufficiently altered the conformation of the R-domain to make additional sites available for phospholabeling, such as Ser 753, which normally would be hhidden from the kinase and not contribute to £Ùnction13. Conformational changes due to the lOSA mutations have been observed on a recombinant R-domain (Dulhanty and Riordan, 1994a), but it is not known if these alterations occur in the stabilizing environment of the whole protein and whether they would affect function. In fact, since the R-domain is not very strongly conserved between species (Diamond et al., 1991), it is possible that small conformational changes do not make a significant difference in its functional properties. In any case, with our approach it is extremely difncult to prove labeling of Ser 753 in the wild-type molecule. The low levels of phosphorylation will be overpowered in autoradiography studies by other highly labeled sites in close physical proximity, so that Ser 753 labeling cannot be estirnated in wild-type CFTR. In addition, it has generally been considered infeasible to evaluate individual phosphate acceptors with a functional approach since CFTR phosphorylation sites appear to act in a redundant manner so that removal of a single site has little or no effect on function (Cheng et al., 199 1). This redundancy may explair. the intriguing observation that to date none of the mutations reported to cause CF have mapped to a CFTR phosphorylation site Uicluding Ser 753 (Tsui, 1995). However, the problems mentioned above were partially overcome through the recent application of a matnx-assisted laser desorptiodionization-ion trap mass spectrometry to phospholabeled R-domain which allowed the evaluation of in vitro labeling of specific sites (Naim et al., 1996). This study demonstrated that the relative labeling eficiency of various serine sites by PKA is 768>737»700=795>712=660= 8 l3»670=753, i.e. confirming the detection of Ser 753 labeling in a wild-type sequence. Upon removal of Ser 753 in the IOSA-CFTR background it was seen that the resulting 11SA-S753A-CFTR variant was still phospholabeled and activated to some degree by PKA. Since the phosphorylation could be traced to a distinct -9 kDa band in a

-- - 13 In addition, potentid conformational changes due to the IOSA mutations could also exaggerate the activatibility of the IOSA-CFTR variant similar to the hyperactivation by pre-treatment with PKC which elevates PKA-mediated activation (Tabcharani et al., 199 t ) possibty due to ïnduced confornationai changes. CNBr cleavage, it is likely that the phosphates are accepted by specific sites, rather than occming randomly on various sites in different molecules. However, the hypothesis that a11 residual sites are monobasic consensus sites sirnilar to Ser 753 did not hold up. The crude investigation of Chapter 4 indicated that only a hction of this labeling occurs on monobasic consensus sites and that al1 residual phosphorylation that could have any functional significance resides on cryptic sites that utilize positive charges distal in the primary sequence for their interaction with PKA. One or several sites may be involved,

but it will become increasingly difficult to identiQ the true phosphate acceptors with mutagenic mapping methods since even our highly sensitive techniques are approaching the limit of detection. We tried to utilize a report that many kinase phosphorylation sites

occur in predicted P-tum secondary structures (Small et al., 1977) and were hoping that secondary structure predictions would highlight potential cryptic candidates by placing

them into P-tums. Yet, it becarne apparent that most serines and threonines that are

located in the phosphorylated -9 kDa band are part of a predicted P-tum, which is not surprishg in view of the high likelihood that these residues, especially serine, act as P-

tum formers (Reithrneier and Deber, 1992). During the investigation of monobasic consensus sites, two observations were made that deviated fiom predictions of current models for CFTR function. Hwang and coworkers (1994) had applied either the phosphatase inhibitor okadaic acid or continued run-down to put CFTR into a low-phosphorylation state. The 'low' phosphorylated CFTR molecules differed fiom highly phosphorylated channels in that they could not be locked open by AMP-PNP and were unable to stabilize the open state towards full-length channel openings (Fig. 1.4). In sharp contrast, the ISSA-CFTR mutant of chapter 4 was locked open by AMP-PNP and the calculated mean open tirne was similar to reported mean open times of wild-type CFTR. It is not clear what is the basis for this difference, but the various approaches may look at different phosphorylation sites which have different contributions to CFTR function. Although very little phosphorylation remains, it is possible that the fifteen Ser to Ala mutations of the ISSA-variant do not include the one site that is responsible for the switch fiom the low to the high phosphorylation state in the Hwang et al. (1994) data. It has been speculated that phosphorylation of the R-domain rnay be necessary to allow functional maturation of CFTR (Lukacs et al., 1994). Our findings do not support this theory since decrease of phospholabeling of the rnolecule to minute levels does not significantly alter the processing capability. Even the 1SSA-CFTR variant mahired to levels that are comparable to wild-type. While answering one problem, the second airn of this thesis was to raise more questions. We hypothesized that in addition to the NBFs and the R-domain, the CLs may represent a third set of cytoplasmic domains which are important to the hction of CFIX. This theory was partly based on the assumption that it is unlikely that these fairly large domains (prediction: 55-65 amino acids; Riordan et al., 1989) would just protrude into the cytoplasm without any contribution to CFTR-mediated processes. In addition, the high occurrence of CF-associated mutations in these domains indicated that the CLs may have some significance, however, the efTect of these mutations on the CFTR molecule had never been investigated (Ferec et al., 1995). Reconstruction of the mutations demonstrated that the gene alterations generally affected CFTR by either dismpting its processing or its hction, so that it is now possible to say that these mutations are not only disease-associated, but disease-ca~sin~'~.The observed processing and functional effects for the various mutations could be consistently reproduced when studied in transient and stable expression systems utilizing various ce11 lines (CHO, HEK-293, COS-1) which strengthens the significance of the data. In fact, a recent publication by Cotten et al. (1996) expressed 9 of the 18 point

" Welsh and Smith (1 993) have grouped CF-causing mutations into four classes. Within this scheme we foimd that the point mutations of the CLs fa11 into either class II mutations that cause disease beause of an effect on protein maturation or class III mutations that cause disease because of an effect on rpguiation of the chioride channel. However, this grouping is not rnutually exclusive, since some rnisprocessed mutants were also observed to bave altered replation mutations in CUin HeLa celis and described findings almost identical to our observations. Only the results for mutant R1 O7OQ dinered, wtiich properly matured in our studies but was found to be misprocessed by Cotten et al. (1996). This variation could coincide with the report that the R1070Q mutation results in a pancreatic suficient phenotype in some patients and in a pancreatic ins6cient phenotype in others (Mercier et al., 1993), indicathg that it may be highly dependent on the genetic/envimnmental background. The most obvious effect due to point mutations in the CLs was a disturbance of processing of the channel. Of the 30 mutations studied, 18 disallowed significant production of the mature, fully glycosylated band C of CFTR. In every case the immature, core- glycosylated band B was 100% sensitive to cleavage by endoglycosidase H, consistent with the concept that these CFTR variants were unable to mature fiom the ER. Mutations in CU especially afTected processing, so that it appears that the high fiequency of mutations in this domain is not a reflection of its functional importance but rather of its structurai role.

Although never proven directly for CFTR, it is presently assumed that lack of processing is the result of retention in the ER due to protein misfolding. Within this mode1 it appears that correct folding of the CLs is crucial for correct overall folding of the rnolecule or misfolding of the CLs is sufficient to maintain interactions with ER quality control components.

When correlating the phenotype of patients with the processing status of CFTR we found that for mutations in CLs 3 & 4 the general notion was true that lack of CFTR on the ce11 surface results in more severe disease than presence of a partially functional channel. Not too much significance can be attributed to this generalization since many of the mutations were observed in only one or two patients. However, since the same trend occurs collectively in al1 the different mutations, it rnay still be valid. If rnisprocessing occurs, it appears to be quite irrevenible since out of al1 the point mutations and deletion mutants (Xie et al., 1995) midied, only one mutant (H949Y)could be partially rescued by conditions of lower temperature or increased levels of glycerol. It is not obvious why some amino acid substitutions in the CLs may compromise CFTR topology sufnciently to inhibit maturation hmthe ER quality control machinery, while others appear not to. However, some trends could be observed. Except for two mutations, al1 of the substitutions resulting in misprocessing involved either charge changes or the introduction of a proline. Interestingly, this is not an isolated finding, since for the anion transporter band 3, Jarolim et al. (1 995) described three disease-causing mutations, al1 of which appear to result in misprocessing, and al1 of which involve charge changes. Thus, our initial suggestion that the many charge changes of CF-associated mutations in the CLs may reflect their involvement in ion attractiodselectivity does not hold up, but rather the charge changes often cause CF because they produce common misprocessing of these mutants. Note that some charge changes did allow processing, e.g. G970R Additional observed trends were that aiterations at all of the positions in the C-temiinal half of CU resulted in misprocessing, but only half of those in the N-terminal half of CLA did so. The processing effect is very site specific since it was seen that in some cases mutations in neighboring residues had opposite effects on CFTR tranicking. For example, R1070Q allowed maturation of the charnel whereas Q 1O7 1 P inhibited maturation. Sirnilar situations were observed for residues K 1O6O/G 106 1 and R 1066/A 1067. Furthemore, different alterations of the sarne residue always produced the same efect, such that R1066C. R1066H, R1066L as well as Ml 101 K, Ml 1O 1R al1 inhibited proper maturation of CFTR, whereas R1 O7OW, R1 O7OQ were both normally processed. Also, none of five different substitutions in residue G970 influenced the processing capability of the CFTR variants. Overall, the rising number of identified rnisprocessed mutants suggests that a method of therapy that promotes proper protein maturation would not oniy be beneficial to AF508 patients, but possibly also to patients who carry other mutations resulting in niisprocessing. In a cornparison of human, bovine and mouse CFTRs al1 af5ected residues in al1 four loops were 100% conserved, except Gly 1069 (Diamond et al., 1991), Mersuggesting that these amino acids are relevant for proper function andor processing of CFTR". In the

alignment of the amino acid sequences of various ABC transporten (Manavalan et al.,

1993), R1066 is the most conserved residue of ail sites studied. Three mutations have ken published for this residue (Fanan et al., 1992; Ferec et al., 1992; Mercier et al., 1993) and a fourth was recently identified (CF Genetic Anaiysis Consortium, personai communication), dl of which result in fdure of maturation (Cotten et ai., 1996). The conservation of this residue and potentially of many of the other affected sites rnay thus be a result of their

importance for the proper processing of the protein. In the case of R1066 it has been recently suggested that this residue rnay be so fiequentiy mutated because the CpG dinucleotide at position 3328/9 is a 'hotspot' for mutations, but no evidence cm presently

be provided to support such a theory (Cremonesi et al., 1996) To address our hypothesis that the CLs may be involved in CFTR function, we were

especiaily interested whether any of the CL alterations wouid affect activity of the chloride channel. Interestingly, mutations in the various loop regions produced different trends, highiighting that each of the four interna1 loops may be involved in diEerent hctions. For CU,six variants were able to escape the quaiity control machinery of the ER. When

expressed in COS4 cells, the channel activity of dl six mutants was modified to such a small degree that the whole-ce11 conductance was not significantly different fiom wild-type

as evaluated by iodide efflux, agreeing well with the observation that these gene alterations

generally aliow the less severe pancreatic sunicient phenotype. When analyzed on the single-charnel level, ail CL4 mutants couid be activated by PKA and ATP and demonstrated an unaltered conductance. This indicates that despite its predicted close proximity to the pore entrance, CUis most likely not involved in handling the peneîrating

15 Interestingiy, in murine CFTR Giy 1069 is replaceci by Arg, a human mutation that we found to lave biosynthetic processing unaffected but to reduce channel open probabiiity mainly due to a decreased channel mean open time. This coincides with the description ht, in cornparison to human CFTR, murine CFTR shows a decreased Podue to a decreased mean burst duration with no diffaence in the mean closed the (Robinson et ai., 1996). anion. However, ail of the mutants had a somewhat decreased Po which was apparently suf5ciently low to cause the mild foxm of CF. A trend codd be observed that for mutations towards the N-terminus of the loop the decrease in the Powas more the result of a decrease

in the rnean burst duration and that for mutations towards the C-tenninus of the loop the decrease in the Po was more the result of an increase in the rnean interburst duration. The changes in the Po were not ciramatic, although statisticdy significant, and certainiy conceptually sigdicant since deletion of 19 residues in CL2 did not alter Po (Xie et al., 1995). Thus, mutations in CL4 do not affect pore properties of CFIR, but to some degree can influence both the rate of channel opening as well as the stability of the open state.

The altered channel gating of CL4 mutants at Uiis point is only an observation and identification of the underlying molecular rnechanisms awaits fûrther shidies. Sirice the changes were not extensive, it is possible that response tirnes to regulatory stimuli were slowed because the mutations disturbed the ability of the molecule to perform global rearrangements necessary for anion transport. Altematively, direct interactions with regulatory domains could be infiuenced which may be implied by the effects on the open and closed tirnes of the muiated channels. Cotten et al. (1996) even suggested that their data indicates that CL4 mutations dEect the ability of the loop to interact with the NBFs, particularly NBF2. In their opinion, the observation that the A1067T-CFTR variant responds to elevations in ATP concentrations with an elevation of the Pothat resembles that of variants carrying NBF mutations G55 1D, G1244E, G1349D indicates that CUand the NBFs rnay interact. They go on to demonstrate that locking open with PPi is less effective for R1066L and F1052V variants than for wild-type CFTR. Since this locking open is suggested to occur via NBF2 (Carson et al., 199%) it indicates to Cotten et al. (1996) that CL4 interacts with NBFZ. However, the interpretations should be viewed with care since the PP, finding is only observed in two mutants and the locking open is not inhibited, but only decreased. Furthemore, the effect is observed as a change in the percentage of the induced increase in activity and it is not clear whether the results fiorn different staiùng

levels can be compared this easily.

Mutations in CL3 had much more extensive effécts on CFTR activity than CU mutations and were easily detected as changes in the iodide efflux level of transfected cell populations. When analyzed by singie-channe1 patch-clamping, the most striking result of

the amino acid substitutions was a strongiy altered Po of the mutant Cmrelative to wild- type CFTR, with S945L and G970R decreasing the Po of the channel and II949Y doubhg its

Po. For ail three mutations these effects were largely the resdt of a change in the mean open time of the altered channels. Evidence has been put forward that ATP hydrolysis at NBFl is associated with activation of CFTR and ATP hydrolysis at MF;! provides a mechanism for

timing the duration of the open state (see Chapter I; Gunderçon and Kopito, 1994; Hwang et al., 1994; Carson et al., 199%; Carson and Welsh, 1995; Winet al., 1996). Within the fiamework of this model, the altered duration of the open state consistently observed for al1 CL3 variants rnay indicate that mutations in CL3 can atfect events at NBF'2 or affect communication fÎom NBF2 to the pore. Mutations within CL3 can have opposite effects of either prolonging (H949Y) or dramatically decreasing (S945L, G970R) the duration of the

open me. A sirnilar fincihg was obtained for mutations within the NBF2 domain, with

removal of Lys in the Walker A motif and removal of Asp in the Walker B motif prolonging activity after forskolin removal, whereas replacement of the conserved G1349 markedly destabilises the active state (Wilkinson et al., 1996).

CL3 mutations af3ected channel Po mainly by alte~gthe mean burst duration, whereas previous stiidies showed that other factors that auence CFTR channel activity, such

as altered ATP concentrations (Winter et al., 1994) or removal of phosphorylation sites by site-kted mutagenesis (Chang et al., 1993; Rich et al., 1993a), tend to affect the interburst duration much more than the burst duration. CL3 mutations also influence the mean closed time of CFTR, however, these changes are not very sigdicant. The slightly altered conductances due to mutations S945L and G970R show that mutagenesis of residues in CL3 ha a subtle influence on pore propereies, suggestùig that CL3 may contribute to a region around the inner mouth of the pore. However, neither CU nor CU muîations altered conductance of the channel to such a degree that it would be indicative of the cytoplasmic loops being a part of the ion conductive pathway. This fundion may in fact be carried by the extracellular loops (EL) of CFTR since Price et al. (1996) jut demonstrated that residues in

EL1 innuence the selectivity of CFTR When the three residues that are different in EL1 between human CFTR and Xenopur Cmwere changed to the Xenopur versions in the hurnan background molecule, the altered human ClTt showed selectivity characteristics of Xenop CFTR. Notably, one of the CL3 mutations, H949Y, resulted in a channel that is more active than the one produced by nature. A similar effect was noted previously for the CF-associated NBF1-mutation P574H. Both Sheppard et al. (1995) and Champigny et al. (1995) found that this mutation demes processing of CFTR, but increases the channel's activity. The occurrence of substantial activity for these mutants comlates weli with the observation that patients canyhg the P574H and H949Y mutations saer fiom a less severe fom of CF and are pancreatic su£Ecient (Kerem et al., 1990; Ghanem et al., 1994). Residue substitutions that increase channel activity may eventually be helpfbl in gene or protein therapy by extracthg maximal chloride transport fiom molecules introduced into target cells.

An evaluation of the fimctional importance of CLs 1 & 2 proved to be much more difficult than was the case for the C-terminal loops 3 & 4, maidy due to the smaller number of CF-associated point mutations published for these domains. Within CL1 we identified four mutations and within CL2 one mutation which did not affect processing of CFTR. Since these gene alterations are found in CF patients, we expected them to somehow alter chloride movement via this channel, either on a regdatory level or on a conductive level. Yef none of the mutations influenced the conductance of CFTR and only two of them, G178R and El 93K, had an effect on the anion translocation capabilities of the protein. In the case of E193K- CFTR, singlechamel patch-clarnping demonstrated an extensive decrease in the Po of the channel that was equally signincant as changes in the Po resulting hmmutations in CL3. Interestingly, whereas CL3 alterations affected mainly the mean open time of Cmthe

E193K-variant demonstraîed a large increase in the mean closed time of the molecule. As descRbed above, cmtrnodels of CFTR finiction (Gunderson and Kopito, 1994; Hwang et

al., 1994; Carson et al., 1995~;Carson and Welsh, 1995; WiUcinson et al., 1996; Fig. 1.4) indicate that channe1 opening occurs via ATP hydroiysis at NBFI, whereas the duration of the open state is tirned by ATP bindinghydrolysis at NBF2. Thus, ahhough the çample size is small, the much larger time periods between charnel openings and an unchangeci duration of the open state may indicate that a mutation in CL1 can affect events at NBFl or affect the communication from NBFl to the pore of the channel. Altematively, inhibition of phosphorylation was also seen to increase the mean closed time of CFTR (Chapter 4), so that the observeci effects could as well aise fiom an altered interaction with the R-domain. A proposal, based on our fünctional data, that CL1 may interact with NBFl and CL3 may interact with NBF2 would agree weU with the biochemical hding in the related P- glycoprotein that NBFl ceprecipitates with the N-temiinal transmembrane domain and MF2 co-precipitates with the C-terminai transmembrane domain (Loo and Clarke, 1995).

The G 1 78R mutation of CL 1 also reduced anion translocation in the iodide efflwc, but this modified function could not be detected by single-charnel patch-clamping. A reason for this codd be a second hding for this CFTR-variant, which is its inability to be locked open by AMP-PNP. This negated our standard procedure of evduating the number of channels in the patch and may have led to an overestimation of the Poof this mutant. The fact that two N- temiinally CL 1 located gene alterations (1 l48T, G 178R) cm inhibit locking open by the nucleotide AMP-PNP, a mechanism which presently is thought to occur via the C- terminally located NBF2 (Hwang et al., 1994) reiterates that the concept of 'iocking open' is far fiom king understood completely and rnay involve more than one site as recently proposed by Mathews et al. (1996). Three muiations of CLs 1 & 2 (I148'1; I17N, R297Q) do not modulate processing of

the protein and show the same regdation and anion conductance as wild-type CFnt Superficiaily, this codd indicate that theçe amino acid substitutions lie in areas of the N- temiinai CLs that are not important for CFTR fimction. However, it is intnguing that despite an unaltered chioride channel activity and normal tratficking of these CFTR-variants, in patients they ail cause the very severe pancreatic insufficient phenotype. Therefore, the influence of these mutations on the chlonde channel activity of CFTR may not be the only

parameter that should be evduated in ûying to elucidate why these gene alterations cause

CF. Possibly some mutations in CLs 1 & 2 actually aEea a CFTR function other than chloride movement, such as regulation of the sodium channel or bctional interaction with the outward rectifier; a kidney isofom that only produces the N-temünal half of CFTR still regulates the outward rectifier (Morales et al., 1996). When digning the two halves of CFTR it is noted that mutagenesis of paraIlel residues has different effects on the CFTR rnolecule. I148T in CL1 allows processing and

has minimal affects on chloride movement, whereas S945L in CL3 significantly decreases both processing and fûnction of the CFTR channel. Even more comparable are the two mutations at sites 178 in CL1 and 970 in CL3 becaw both residues are glycines and both are mutated to arginine. Both mutations allow processing of the CFTR protein to the ce11 surface, but G970R eliminates chloride channel function almost completely, whereas

G178R has a smaller effect on the activation via CAMP. This is a fûrther indication that the aligning loops are not functionally equivalent. Interestingly, in P-glycoprotein, glycines 169 and 8 12 align with the CFTR glycines 178 and 970, respectively. Mutation of either of the P-glycoprotein residues allows processing of the protein (Loo and Clarke, personal communication). Interestingly, of the processed mutations that did not affect chloride channel activity, the I175V substitution occurs at an amino acid which aligns with one of four conserved residues within CL1 of P-glycoprotein that alter the dmg transport profile of this protein lrpon mutagenic substitution (Currier et al., 1992). In addition, 1175 borders a second

transporter motif, that has not ken associated with CFTR previously: CLs 1 & 3 of the

metal-tetracyche/H' antiporter (ET)of E. coli (Yamaguchi et al., 1993) and of glucose transporters (Henderson, 1990) contain the motif m-X-X-X-RK-X-G-R/K/E-W. Site-

directeci rnutagenesis indicated that the ht glycine of the motif and the charge of the second

Iast residue of the motif are most crucial for transport activity of ET(Yamaguchi et al..

1992). GeneraUy it was observed that one of the loops (CL1 in TET) forms the 'active' leafiet

wtiile the other (CL3 in TET) only provides the 'silent' cornterpart (Yamaguchi et al., 1993).

In CFTR, a sequence can be identifid in CL1 ,*ch resembles the motif: 165 L-K-L-S-S-R-

V-L-Q-K 174; the £ktresidue which is normally a Gly was also found to be a Leu in two of

the glucose transporten (Henderson, 1990). In addition, the sequence following G970 of CL3

is as follows: 970 Q-G-1-LN-R-F-S-K-D 979, so that the most important residues of the motif are present. However, recurrhg suggestions of CFïR also acting as a transporter are only

based on its structural similanty to other tramporters and thus far are purely speculative. Overall, the CLs are starting to emerge as potentially significant contributors io the fùnction of CFTR, with site-directed mutagenesis indicating each loop to be very distinct. Mutations in CLs 1 & 3 were observed to have drastic effects on the ability of CFTR to respond to regdatory stimuli. The E l93K mutation of CL1 decreased the ability of CFTR to

respond to an activating stimulus, in agreement with the reduced iodide efflux activity of

G178R-CFTR, whereas mutaticns in CL3 af5ected the duration of CFTR rernaining in the open state. In contrast, the only CF-associated mutation that could be evaluated in CL2 plus a deletion of CL2 (Xie et ai., 1996) had little effect on the chloride channel activity of CFTR and mutations in CLA also produced relatively small functional alterations. The correct folding of the CLs appears to be cmcial for achievement of a correct overall conformation since many mutations, especially in the C-terminal loops, caused rnisprocessing of CFTR. More detailed Mesof each loop will be necessary to obtain a deeper understanding of the overlap in function between the diffèrent CLs and their potential physical interaction with the regdatory domains and with each other. Tao et ai. (1996) suggested that such

interactions are likely to occur in view of the hydrophiiic nature of the CLs. The signincant decrease in CFTR fhction due to the G970R mutation is consistent with this point of view,

since the loss of activity was due to the charge raîher than the size of the introduced Arg. This, and the observation that mature G970R-CFTR is properly trafEcked to the plasma membrane,

suggests that G970R affects activity through an altered electrostatic interaction within the

protein or with other molecules rather than through a gros change in CFTR structure. Interaction between the CLs and the NBFs has been suggested to occur in the reiated histidine

periplasmic permease via an E-A-A motif (Kerppola and Ames, 1932), but this sequence appears to be conserveci neither in Cmnor in P-glycoprotein. Future Directions

Phosphoryiation - Mutagenic studies of Cheng et al. (1991), Chang et ai. (1993),

Rich et al. (1 993a), and this thesis have firmly established several aspects of PKA-mediated regulation of CFTR - the necessity of phosphorylation for activation, a graded response due to various levels of phosphorylation, and lack of correlation between the amount of observed labeling of a site and the resulting activation. At this point, identification and Mer removal of additional phosphorylation sites would be interesting, but will only

reiterate these facts. One of our initial motivations to eliminate al1 phosphorylation sites was to obtain a non-responsive CFTR protein into which individual sites could be reintroduced to study their contribution to funaion. Such an approach was taken because, ever since the cloning of the CFTR gene, various experiments indicated that removal of an individual site ha little, if any, effect on fiuiction. However, a preliminary report by Winter and Welsh (1996) suggested that careful analysis of these small effects may be s-cient to evaluate the relative contribution of individual sites; stimulatory and inhibitory sites could be disthguished 6th this method. An approach that mutates only one residue is less likely to produce artinciai resdts than an approach in which 1 1+ residues are modified and is thus the method of choice for future hvestigations. In fact, using this experimentd route, the effect of potential phosphorylation sites in NBFl may aiso be studied if individual mutants are properly processed. Some of the individual NBFl mutants are still expected to be mislocalized and can be evaluated by reconstitution into a planar Lipid bilayer. Analysis in the bilayer would also be the next necessary step to study the misprocessed 4SA mutants of Chapter 4.

CytopIasmic Lwps - By establishing the effect of naturally occhg point mutations in the various loops this thesis has laid the groundwork for future approaches to further investigate function of the CLs. Such investigations could be aimed in several directions. One of the most striking features of CL alterations is the fact îhat a majority retards maturation of CFTR. Attempts to understand the inability of AF508-CFTR to escape fiorn the quality control machinery of the ER have been very hstrating and, despite intense studies, no concrete findings cm be reported to date about altered folding of CFTR, altered interactions with chaperones, or any other criteria that would target the mutated protein for retention. The many CL mutations allow different degrees of processing, so that they provide 'graded' tools to study misprocessing. If some of the AF508 experiments are applied to CL mutations, new concepts rnay develop. Avenues that could be taken are pulse- chase radiolabelhg to observe the stability of the mutants, CO-precipitationexperiments to address interactions with various chaperones, and non-reducing gel electrophoresis to obtain indications of misfolding. The 'graded' tools may also give more information regardhg the validity of the hypothesis in chapter 1 that the proteolytic event which is blocked by MG- 132 (Jensen et al., 1995), the ATP dependent step that is needed to move core-glycosylated

CFTR into a protease resistant state that eventually cm mature fiom the ER (Lukacs et al., 1994), and the core-glycosylated wild-type CFTR that shows a shifi on the glycerol gradients, but is still associated with calnexin (Pind et al., 1994), are al1 linked to each der. Furthemore, it is commonly stated that mutations may affect CFTR activity by altering the recycling rates on the membrane, but this has never ken studied. Introduction of a newly described extraceiidar epitope (Howard et ai., 19%) into the many CL mutants that reach the surface wiil facilitate study of such eEécts and an estimation of the degree to which altered recycling rates influence overaii activity. The many misprocessecl CFTR-variants may also be utilized in a screen for substances that will promote maturation to the ce11 surface. Such an approach has recentiy gained more relevance due to a finding in P- glycoprotein research where ail processing-defective mutants examuled could be promoted to the ce11 surface by exposure to substrates and modulators of the protein (Loo and Clarke, 1997).

A second ahfor the future will be to study Merthe functional importance of the

CLs. The obvious loops of choice for such a study are CLs 1 & 3 since the rnost significant alterations in the chloride channel activity of CFTR were observed in these domains. To obtain a clearer picture of the achial defect that causes disease, more mutations can be carried out. Similarly to (3970 (Chapter 6), residues G178, E 193, S945 and Hg49 can be replaced with various amino acids to observe if, for example, the decreased CFTR activity due to the E1 93K mutation onginates fiom a charge effect, or the increased CFTR activity due to the H949Y mutation is the result of an aromatic eRect. A requkement for such studies is that al1 mutations allow approximately equal levels of processing to permit fast evaluations by iodide efflux; altematively, the mutants have to be analyzed by single- channel patch-clamping. It ais0 shouid be investigated whether the functional consequence of the introduction of a positive charge at residues Glu 193 and Gly 970 is restricted to these residues, or can occur due to similar changes at smounding positions as well. Such an approach would map out a functional unit, but again the ease of such experiments depends on the processing capability of the various mutations. Recently, a disease causing H949R mutation has been identified in CL3 (CF Genetic Analysis Consortium, personal communication). It would be of great interest to evaluate whether such a mutation will allow hyperactivation of the molecule as observed for the second, published, mutation at this residue (H949Y) or eIiminate activation as observed for the introduction of positive charge at another site in CL3 (G970R).In addition, WAer 'random' mutations, especiaily in CLI, may paint a clearer pichire of the importance of these loops.

To complete information that comes hmthe deletion mutants of CLs 1 & 2

(Delaney et al., 1993; Xie et al., 1995), similar constructs should be prepared for CLs 3 & 4. It is expected that such mutants will also be rnisprocessed, but the protocol developed by Xie et al. (1995) allows reconstitution into Lipid biiayers and study of aitered chloride channel profiles. From our data we predict that with this method it may be found that only deletions of CLs 1 13 3ect anion translocation. Note that in the case of Rdomain mutants it has ken observed that deletion of a certain portion is compatible with processing of the protein, but Merdeletions inhibit maturation (Rich et ai., 1993b). Thus, various size deletions in the CLs may produce a mutant that is correctly processed. An alternative method of disrupting the CLs may be to replace the deletion approach with a combined approach of introduction of thrombin cleavage sites into each CL and preparation of membrane vesicles or study in excised membrane patches. If a deletioddisruption approach is applied, then the experiments with a recombinantly produced phosphoryIated/ dephosphorylated R-dornain of Ma et al. (1996) can be conducted on the mutants to observe if any of the R-domain effects are mediated via the CLs.

At this point our functional data is consistent with CL1 and CL3 king involved in the communication between, respectively, NBFl and NBF2 and the pore of the channel, but does in no way prove it. In fact, the final proof for domain interactions has to await the solving of CFTR's 3-D structure. Until then, indirect evidence will have to be accumulated. On paper, a method of choice to indicate possible intedons between CLs and NBFs or other domains is the yeast two-hybrid system; this approach is presently utilized by Kiser et al. (1 996). Another route may be the production of the various domains as recombinant peptides which, if sufficiently soluble, cm be utilized in co-precipitation experiments, protein affinity chrornatography, Wîy blotting on nitrocellulose, afEnity CO- electrophoresis, plasmon resonauce, or gel overlay assays. As done by Loo and Clarke (1995) these investigations should be initiated with large segments of CFïR, the size of which can then be reduced as the experirnents proceed. If interactions are observed, attempts can be made to disrupt them through the various CF-associated mutations, although it may be expected that point mutations will not be sufncient to disnipt such interactions, but that deletions may be required. In addition, introduction of the various mutations into recombinant loop peptides may give an indication of altered foldhg patterns if studied by C.D.. A process that is still poorly understood in CFTR function is the potentiation of PKA-mediated activation through PKC pre-treatxnent. Several of the CF-associated mutations in the CLs do affect consensus sites for potential interactions with PKC and we have observed labeling of CFTR by PKC outside the R-domain (data not shown). Since PKC potentiation can be observed by both iodide efflw and single-channel patch-clamping (Tabcharani et al., 1991; Chang et ai., 1993), it is worthwhile to take a hit and miss approach and use the produced loop mutants to investigate whether any of the afKected PKC sites are involved in this potentiation. Finally, in addition to further exploring processing effects and chloride channel contributions of the CLs, a third avenue can involve study of the effect of CFTR loop alterations on proposed interactions with other molecules, such as the sodium channel or the outward rectifier. Such studies would most likely involve CO-expressionof CFTR-variants with the molecules that are thought to be regulated by CFTR, but since the interactions at this point are understood poorly at bat, hope for success of such approaches is limited. REFERENCES Aguilar-Bryan, L., Nichols, C. G., Wechsler, S. W., Clment, J. T., Boycl, k R, GodesG., Herrera- Sosa, H., Nguy, K., Bqm, I., and Nelson, D. k (1995) Cloning of the beta ceIl hi&-affinity sulfonylurea receptor: a regulator of insuiin secretion. Science 268: 423-426.

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Zielenski, J., Bozon, D., Kerem, B., Markiewicz, D., Durie, P., Rommens, J. M., and Tsui, L. C. (1991) Identification of mutations in exons 1 through 8 of the cystic fibrosis transmembrane conductance regulator (CFTR)gene. Genomics 10: 229-235. Zielenski, J., Fujiwara, T. M., Markiewicz, D.,Paradis, A. f., Anacleto, A. X., Richards, B., Schwartz, R H., Klinger, K. W.,Tsui, L. C., and Morgan, K. (1 993) Identification of the Ml 1 O 1 K mutation in the cystic fibrosis transmembrane conductance reguiator (CFTR)gene and complete detection of cystic fibrosis mutations in the Hutterite population. Am J Hum Genet 52: 609-6 15.

Zielenski, J., Markiewicz, D., Chen, H. S., Schappert, IC, Seller, A, Durie, P., Corey, M., and Tsui, L. C. ( 1 995a) Identification of six mutations (R3 1 L, 44 1 de& 681 defC, 146 lins4, W 1089% E 1 1MX) in the cystic fibrosis transmembrane conductance reguiator (CFTR)gene. Hum Murat 5: 43-47.

Zielenski, J., Paîrizio, P., Corey, M, Handelin, B., Markiewicz, D., Asch, R,and Tsui, L. C. (1 995b) CFTR gene variant for patients with congenital absence of vas deferens. Am J Hum Genet 57: 958-960. Pre-gene-discovery Findings in CF Research Cytoplasmic Metachromasia - The increased mucus secretion in CF organs prornpted Danes and Bearn (1968) to treat skin fibroblast cultures with the metachromatic dye toluidine blue O. With this method cytoplasmic intravesicdar metachromasia, resulting fiom a non-specifïc stainuig mechanism of intracellularly accurnulated negatively charged macromolecdes, can be observed in ail investigated CF children and in ahnost al1 parents. Cultures fiom healthy controls or patients of various other diseases show only occasional metachromatic cells. In addition, a subsequent shidy demonstrated that if metachromatic CF cells are grown in contact with normal cells, the metachrornasia disappears. Physical contact is required, since correction of the mutant phenotype is not observed if metachromatic CF cells are grown with normal cells on comrnon media but separated by a physical barrier, or if ametachromatic CF cells are used instead of normal cells (Danes, 1973). Unfortunately, more detailed studies showed that metachromasia is a fairly unspecific marker that gives positive results for several disorders besides CF (Taysi et al., 1969) and varies with tissue culture conditions (Spicer et al., 1980). The approach was abandoned and it was not until 10 years later that Tsui and Buchwald (1991) realized a possible link between the earlier metachromasia studies and the fmdings of Cheng et al.

( 1 989) that glycoconjugates of nasal epithelial cells experience increased sulfation in CF patients. CF Factor - One potential access for a CF treatment was to address the lack of mucus clearing. Spock et al. (1967) hypothesized that this deficiency is due to altered mucociliary movement. In fact, they were able to demonstrate that blood serum fiom CF patients disorganizes the cilliary rhythm in explants of rabbit tracheal mucosa. The disorganized beat is usually observed within 5-1 0 minutes and persists for a penod of up to one hour. The senim factor is heat labile, nondialysable, and precipitates with euglobulins, suggesting it to be a protein. It was observed at lower concentrations in obligatory heterozygotes, requiring concentration for detection, but in only 1 of 25 controls. Blitzer and Shapira (1982) purïfied the CF factor which turned out to be a glycopeptide that causes marked ciliq dyskinesia within 30 seconds of application, and gradua1 swelling and excessive secretion and accumulation of mucus-like droplets. They claimed that the normal couterpart of the CF factor can be purified fiom control individuals and has no inhibitory activity. However, the two peptides show slightly different molecular weights and chromatographie patterns, dthough preliminary data suggested that they have most of their antigenic detenninants in common. Since abnormalities in glycoprotein compositions had been observed in CF patients (Boat et al.,

1976), it was thus suggested that the CF factor activity may be a consequence of a generalized phenomenon of abnormal synthesis or degradation of glycoproteins. CF Antigen - In addition to the CF factor, a CF antigen was detected at elevated levels in CF patients (Hayward et al., 1986). The antigen is synthesized by granulocytes but regulated differently fiom other granulocyte proteins, indicating that the elevation in CF patients is not a compound effect of granulocyte proliferation and active tissue damage, but rather due to a specific association between this antigen and the CF gene (Hayward et al., 1987). More detailed analysis demonstrated that antibodies to the CF antigen recognize two proteins of 1 1 and 14 kDa (Wilkinson et al., 1988), whose genes localize to chromosome 1 and encode distantly related calcium binding proteins. Their expression is high in tongue, esophagus and buccal cells, but undetectable in tissues that are severely affected by CF such as lung or pancreas. A functional characterization of the CF antigen indicated that it iriiibits the activity of casein kinase 1 and II, but not CAMP- dependent protein kinase, protein kinase C, or various tyrosine kinases (Murao, et al.,

1989). Thus, chromosome location, tissue expression, and functional properties of the CF antigen are opposite to the characteristics of the CF protein eventually identified (see

Chapter 1 ), but this protein still is specifically elevated in CF patients. This gave an initial indication of the complex interplay of various processes that would eventually be found in CF research. Cellular Resistance Studies - mer the years there was a consistent trend that,

based on clinical findings, a certain hypothesis was produced. Often this theory could be confhned by one laboratory but subsequently was not reproducible for other groups. These hdings included increased cellular resistance to ouabain (Epstein and Breslow,

1977), dexamethasone (Breslow et al., 1978a,b), CAMP (Epstein et al., 1978), 17P- estradiol, dihydrotestosterone, progesterone (Breslow et al., l978a,b), or altered sodium transport of CF fibroblasts (Breslow, et al., 1981). In retrospect, more consistent results might have been obtained if tissues were utilized that are af3ected strongly by CF, rather than skin fibroblasts. For some studies fluids were used rather than tissues, such as the investigation of the reduced proteolytic activity of CF saliva (Rao et al., 1972a) and deficient kallikrein activity in CF plasma (Rao et al., 1972b). In both cases carriers showed intermediate values. However, again after great initial excitement and ten years of research, these projects were abandoned because they could not be reproduced consistently and showed great variability between subjects. More reliable results were obtained for y-glutarnyl transpeptidase, aminopeptidase M and alkaline phosphatase which are enzymes that are located in the surface of intestinal microvilli. In agreement with the observation that these areas are affected in CF patients it was observed that the levels of al1 three enzymes in the amniotic fluid of mothers carrying CF infants are significantly below the nom (Carbarns et al., 1983; Brock, 1983). These tests were used for pre-natal screening and had an estimated sensitivity of 90%. The only, thus far unexplained, fhding that withstood the test of tirne is an elevated trypsin level in the blood of CF new-boms. Although trypsin sinks to and even below nonnal levels if older than 1 year, the initial elevation is so universal that it has successfully been used to screen for CF prior to any symptomatic diagnosis (Crossley et al., 198 1). IMAGE EVALUATION TEST TARGET (QA-3)

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