Mutations in the PAS Domain of the HERG Impacts Cell Surface Expression and Stability

By

Natasha Alana Holder

Department of Physiology Faculty of Medicine McGill University Montreal, Quebec, Canada

August 2004

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science

© Natasha Alana Holder, 2004 ABSTRACT

Long QT syndrome (LQTS) is a congenital disorder characterized by a prolongation of the QT interval on an electrocardiogram (ECG) and a propensity to ventricular

tachyarrhythmias. Defective trafficking of mutant HERG (Human Ether a go-go Related

Gene) protein is becoming increasingly recognized as an underlying mechanism for

LQTS. Trafficking deficient mutations in HERG have been identified in the

transmembrane domains, the pore region and C-terminus of HERG. However, little is

known about the role of theN-terminus in cell surface expression of HERG. To examine

the function of the PAS domain in HERG processing and its implication in LQTS, we

have characterized three long QT-associated mutations (G53R, E58K, and 196T) within

this domain. Western blot analysis shows that these point mutations are not expressed at

the cell surface due to endoplasmic reticulum (ER) retention and not as a result of

disrupted of tetrameric assembly. Functional studies of these mutations reveal a marked

decrease in current amplitude that can be restored by reducing incubation temperatures.

Removal of the HERG PAS domain enhances surface expression; however, the stability of the channel at the cell surface is impaired. Thus, we demonstrate that by altering the conserved a/~ fold of the PAS domain, cell surface expression and stability of the mutant

HERG channel is reduced.

ii , , RESUME

Le syndrome du long QT (SLQT) est un desordre congenital du systeme electro- physiologique du creur caracterise par un intervalle QT allonge et une propension aux tachyarythmies ventriculaires. Le trafic defectueux du canal potassique HERG (Human

Ether a go-go Related Gene) devient de plus en plus reconnu comme mecanisme fondamental responsable du SLQT. Des mutations qui generent un trafic defectueux ont ete identifiees dans les domaines transmembranaires, la region du pore et la partie C- terminale du HERG. Cependant, tres peu est connu au sujet du role de la partie N- terminale dans !'expression du HERG a la surface cellulaire. Pour examiner la fonction du domaine PAS de la partie N-terminale dans la biosynthese du HERG ainsi que son implication dans SLQT, nous avons caracterise trois mutants associes avec le SLQT

(G53R, E58K, et I96T) qui presentent des mutations dans ce domaine. L'analyse par immunobuvardage demontre que ces mutants ne sont pas localises a la surface de cellules due a leur accumulation dans le reticulum endoplasmique et non pas a leur assemblage tetramerique defectueux. L'etude electrophysiologique sur ces mutants indiquent une diminution marquee en I' amplitude des courants qui peuvent etre reconstituee en reduisant les temperatures d'incubation. L'ablation du domaine PAS augmente !'expression du

HERG a la surface de cellules; malgre cela, la stabilite du canal a la surface est compromise. Ainsi, no us demontrons qu' en changeant le pli conserve a/~ du domaine

PAS, !'expression des mutants HERG a la surface des cellules et la stabilite du canal sont reduites.

111 ACKNOWLEDGEMENTS

I was confronted with the perils of graduate school from the very beginning. However, with the support of numerous individuals I made it through successfully. I would like to take this opportunity to thank everyone that made this possible. Without the continued support of my supervisor, Dr. Alvin Shrier, my success in graduate studies would not be possible. He afforded me the opportunity to work in his lab freely and allowed me to find a project suitable to my strengths. I was given room to learn the skills I needed at my own pace and a nice budget to carry out my experiments! I would also like to thank Armin Akhavan for teaching me all the skills I needed to carry out this project. Your sweat and perseverance enabled me to accomplish many things. Roxana Atanasiu was the first to take me under her wing and moulded me into a Western blot machine! I'm sorry our initial project did not see the light but those rats had to go! I would like to thank Tomohiro Noguchi for all the patch clamp work needed for this project. Without you this project could not have wrapped up as nicely as it did. V alerie Walker taught me everything I need to know about the Zeiss LSM 510 confocal microscope. Thank you for your patience and encouragement. I would like to thank the rest of the Shrier lab for their assistance and friendship.

I would like to thank the members of my supervisory committee for their support and suggestions that have guided me through my studies. The support staff of the Physiology department has been tremendously helpful over the last 2 years. You are a wonderful bunch of ladies. Thanks for all your help (and the occasional free meal!).

There were days when I wanted to throw in the towel but I had a good group of friends carrying me through. Thanks guys for your encouragement, advice and rock solid friendship. JD you helped me through some frustrating moments, calmed me down and relieved my worries. Thanks for your support. I owe you a massage!

Most importantly, I would like to thank my family for their continued support and confidence in my abilities. Thanks for always being there and never faltering in your faith in me. Mom, you were my light when things got dark and you still are. Without you this couldn't have been possible. I dedicate this thesis to you.

Natasha Holder August 2004

iv CONTRIBUTIONS OF AUTHORS

Chapter 2 is based on the manuscript: Holder, N., Noguchi, T., Atanasiu, R., Akhavan,

A., and Shrier, A. (2004). Long QT syndrome-associated mutations in the Per-Amt-Sim

(PAS) domain of the HERG potassium channel impacts cell surface expression and stability. (In preparation). T. Noguchi performed all patch clamp recordings. R. Atanasiu carried out the sucrose gradient experiments. A. Akhavan provided technical support. N.

Holder performed all other experiments.

V LIST OF ABBREVIATIONS

3D Three-dimensional CNBD Cyclic nucleotide-binding domain CNX Calnexin CRT Calreticulin E Glutamate EAG Ether-a-go-go EndoH Endoglycosidase H ER Endoplasmic reticulum ERAD ER-associated degradation ERGIC ER-Golgi intermediate compartment G Glycine HERG Human Ether-a-go-go Related Gene Hsp Heat shock protein I Isoleucine Ikr Delayed rectifier potassium current (rapid component) K+ Potassium kDa Kilo Daltons LQT2 Long QT Syndrome type 2 that results from HERG Channel Dysfunction LQTS Long QT Syndrome N Asparagine PAS Per-Arnt-Sim PK Proteinase K PNGase N-glycosidase F QC Quality control QTc Corrected QT interval s Serine SDS-PAGE Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis T Threonine TdP Torsades des pointes UGGT Uridine 5' -diphosphate-glucose: glycoprotein glucosyltransferase

vi TABLE OF CONTENTS

ABSTRACT ...... ~ ...... ii RESUME ...... iii ACKNOWLEDGEMENTS ...... iv CONTRffiUTIONS OF AUTHORS ...... v LIST OF ABBREVIATIONS ...... vi TABLE OF CONTENTS ...... vii

CHAPTER 1: Inroduction ...... :...... 1

1. The HERG Potassium Channel ...... 2

1.1 Structural Characteristics of HERG ...... 2 1.2 HERG Protein Processing ...... 3 1.2.1 HERG Glycosylation ...... 3 1.2.2 Quality Control Mechanisms Involved in HERG Processing ...... 4 1.2.3 ER Associated Degradation ...... 6 1.3 Physiological Function of HERG in the Heart ...... 7 1.4 Biophysical Properties of HERG Channels ...... 7 1.5 Pharmacological Properties of HERG Channels ...... 8

2. Long QT Syndrome ...... 9

2.1 Introduction ...... 9 2.2 Diagnosis of long QT syndrome ...... 10 2.3 Molecular genetics of long QT syndrome ...... 10 2.4 Mechanisms underlying long QT syndrome type 2 ...... 13 2.4.1 Dominant Negative Suppression ...... 13 2.4.2 Abnormal Current Function ...... l4 2.4.3 Defective Protein Trafficking ...... 15

3. The PAS domain ...... 17

3.1 Introduction ...... l7 3.2 Structure of the PAS fold ...... 17 3.3 The HERG PAS domain ...... 18

4. Rationale and Objectives ...... 19

Figure 1. Schematic representation of the HERG potassium channel and pore ...... 21 Figure 2. The and the surface electrocardiogram ...... 22 Figure 3. Crystal structure and structure based alignment of known PAS domains ...... 23

Vll CHAPTER 2: Long QT syndrome-associated mutations in the Per-Arnt­ Sim (PAS) domain of the HERG potassium channel impacts cell surface expression and stability ...... 24

SUMMARY ...... 25 INTRODUCTION ...... 26 EXPERIMENTAL PROCEDURES ...... 29 RESULTS ...... 34 DISCUSSION ...... 40

Figure 4. The HERG potassium channel...... 45 Figure 5. Western blot analysis ofLQTS mutants in the PAS domain ...... 46 Figure 6. Pulse-chase analysis of PAS domain mutants ...... 47 Figure 7. Effect of temperature on cell surface expression ...... 48 Figure 8. Voltage clamp recordings of wild-type HERG and mutants at 37°C ...... 49 Figure 9. Voltage clamp recordings of wild-type HERG and mutants at 27°C ...... 50 Figure 10. Comparison of tail current density at 37°C and 27°C ...... 51 Figure 11. Sucrose gradient analysis ...... 52 Figure 12. Subcellular distribution of HERG and mutant channels ...... 53 Figure 13. Western Blot analysis of deletion mutants ...... 54 Figure 14. Functional analysis of the ~PAS mutant...... 55 Figure 15. Pulse-chase analysis of the &AS mutant...... 56

CHAPTER 3: GENERAL DISCUSSION AND CONCLUSIONS ...... 57

REFERENCES ...... 62

APPENDIX ...... 77

Vlll CHAPTER!

INTRODUCTION

1 1. The HERG Potassium Channel

1.1 Structural Characteristics of HERG

The human ether-a-go-go related gene (HERG) was first cloned from a hippocampal cDNA library. This gene was found to encode a distinct voltage-activated potassium channel belonging to the ether a-go-go (EAG) family. The EAG gene was identified when disruption of this gene in the melanogaster (fruit fly) resulted in an ether-induced leg-shaking phenotype [1, 2]. HERG is located on 7q35- q36 [3] and is expressed in both cardiac and noncardiac tissue [4]. HERG is predominantly expressed in the heart, however, HERG transcripts can also be found in the brain, retina, cornea, thymus, adrenal gland, skeletal muscle and lung tissue [4]. This voltage-gated potassium channel is comprised of six-transmembrane helices, an rich voltage­ sensor located in the fourth helix (S4) and a potassium selective pore between helices five

(S5) and six (S6). Each a.-subunit also contains large amino and carboxyl terminal tails that reside in the cytoplasm. A functional channel consists of four a.-subunits that assemble to form a pore. (Figure 1).

Full-length HERG is comprised of 1159 amino acids. The first 135 amino acids encompass a highly conserved Per-Arnt-Sim (PAS) domain. Morais-Cabral et al. determined that the crystal structure of residues 26 to 135 in HERG is similar to other PAS domains involved in sensing and signal transduction. This is the first eukaryotic PAS domain to be crystallized [5]. The C-terminus of HERG bears high sequence similarity to a putative cyclic nucleotide-binding domain (CNBD) present in cyclic nucleotide-gated cation channels [ 1].

2 1.2 HERG Protein Processing

1.2.1 HERG Glycosylation

HERG is initially synthesized in the endoplasmic reticulum (ER) where it is cotranslocationally modified by the addition of asparagine (N)-linked oligosaccharides.

The addition of a 14-saccharide unit from a lipid-linked precursor in the ER is known as core glycosylation. The initial transfer of the lipid-linked 14-saccharide unit to the nascent

HERG polypeptide is facilitated by an oligosaccharyltransferase enzyme that recognizes N­ linked glycosylation sites in HERG [6]. These consensus sites have the following sequence: N-X-T/S, where X is any amino acid except proline [8]. Warmke et al. identified two potential N-linked consensus sites at residues N598 and N629 [1]. Gong et al. have shown that the addition of core and complex glycans only occurs at position N598 [8].

Once the core glycosylated protein is properly folded and oligomerized, it is directed to the

Golgi apparatus where it undergoes complex glycosylation. That is, the glycan undergoes structural diversification to acquire complex terminal glycosylation. Finally, this mature and fully glycosylated HERG protein is inserted into the . Gong et al. determined that glycosylation is not required for the transport of HERG to the cell surface but it does play a key role in conferring stability to the channel at the cell surface. This was demonstrated through pulse-chase experiments which showed that non-glycosylated HERG channels had a more rapid turnover than glycosylated channels [8]. When HERG is expressed in mammalian cell lines it has two distinct bands on Western blot. The immature core glycosylated form of HERG is 135 kDa and the mature complex glycosylated form is

155 kDa [9].

3 1.2.2 Quality Control Mechanisms Involved in HERG Processing

In order for HERO to be expressed at the cell surface, it must successfully pass the

ER quality-control system. The ER has stringent quality control (QC) mechanisms in place to ensure that only correctly folded and assembled proteins can reach their final destinations. The ER provides a specific environment for appropriate folding and maturation of newly synthesized proteins by providing molecular chaperones and folding enzymes [10]. It is presumed that HERO begins folding cotranslocationally with the assistance of chaperones as it integrates into the ER's lipid bilayer. Once translocation is complete, theN- and C-terminal tails are exposed to the cytoplasm where they are believed to interact with two cytosolic chaperones, heat shock protein (hsp) 70 and Hsp90. Picker et al. have shown through immunoprecipitation assays that Hsp70 and Hsp90 only interact with the core gylcosylated form of HERO and that this interaction is crucial for the maturation of HERO. Inhibition of Hsp90 with geldanamycin prevents maturation and efficiently targets HERO to proteosomal degradation [11]. Other ER chaperones such as

BiP and Protein Disulphide Isomerase (PDI) also assist in the folding of glycoproteins. BiP is an abundant ER chaperone that is closely related to Hsp70. It assists in the folding process by recognizing incorrectly folded proteins via a specific peptide binding site. This site recognizes exposed hydrophobic residues on the protein's surface and binds these residues to create an energetically favourable environment comparable to the hydrophobic interior of a folded protein [12]. PDI is an ER resident protein that catalyzes disulphide bond formation and isomerization. Puig and Gilbert have shown that PDI exhibits chaperone activity that prevents protein aggregation and promotes correct folding [13].

The isomerase and chaperone activities of PDI are required to assist in protein folding [14].

4 ER retention signals are also important in QC because they prevent proteins from exiting the ER until the correct native conformation is attained. A putative retention signal has the amino acid sequence R-X-R where X is any amino acid [7]. Kuperschmidt et al. have identified an ER retention signal, R-G-R, in the C-terminus of HERG at positions

1005-1007. In order for HERG to exit the ER it must attain the proper conformation to ensure that its C-terminal ER retention signal is masked within the protein [15].

Within the lumen of the ER, the glycans attached to HERG play a pivotal role in folding and oligomerization. This N-linked "tag" allows glycoproteins to be recognized by the lectin-based chaperones, Calnexin (CNX) and Calreticulin (CRT) in the ER. CNX is a transmembrane protein, whereas, CRT is a soluble protein. Both chaperones form complexes with ERp57, a thiol oxidoreductase [16]. Once core glycans are added to the newly synthesized proteins, glucosidases I and II remove two glucoses to generate a monoglucosylated glycoprotein that can interact with CNX and CRT. This interaction prevents aggregation and export of incompletely folded proteins. It also allows the glycoprotein to interact with ERp57, which assists in the formation of disulphide bonds.

Cleavage of the remaining glucose residue by glucosidase II enables disassociation of the protein from CNX and CRT. If upon dissociation the protein is misfolded, it is recognized by a soluble enzyme, uridine 5' -diphosphate (UDP)-glucose:glycoprotein glucosyltransferase (UGGT). UGGT adds a single glucose residue onto the protein to regenerate the monoglucosylated form, which can now re-enter the CNX/CRT cycle [17].

Once HERG is correctly folded, four subunits must correctly associate to form a functional channel. Oligomerization depends upon the subunits' capacity to recognize each other and to localize spatially and temporally [18]. HERG tetramerization is mediated by a

5 short C-terminal domain [19, 20]. Improper assembly of the subunits can lead to ER retention and degradation.

Two mannose-specific lectins, ER-Golgi intermediate compartment (ERGIC)-53 and VIP36, in the ER act as transport receptors for glycoproteins to facilitate ER-to-Golgi traffic [21]. The transport of HERG from the ER to the Golgi has been shown to be mediated by GM130/golgin-95, a Golgi-associated protein. GM130 is involved in vesicular transport to the Golgi from the ERGIC. Roti et al. have shown that the C­ terminus of HERG interacts with GM 130. They speculate that this interaction tethers

HERG in the Golgi, thereby acting a molecular checkpoint in HERG trafficking [22]. The role GM130 plays in HERG maturation and glycosylation is still unknown.

1.2.3 ER Associated Degradation

A protein's function is highly dependant on its three-dimensional (3D) fold achieved during the normal folding process. Persistently misfolded or unassembled proteins are targeted for destruction via ER-associated degradation (ERAD). Signals of degradation include: N-linked glycans, exposed ER retention signals or hydrophobic residues, unpaired cysteines, prolonged association with chaperones, misfolded proteins or incompletely assembled oligomers. ER-associated degradation of glycoproteins involves the recognition of the protein as misfolded, their retrotranslocation to the cytosol, ubiquitination and subsequent degradation by the 26S proteasome [23-25]. ERAD ensures that improperly folded proteins do not reach their target destination.

6 1.3 Physiological Function of HERG in the Heart

Once HERO is successfully folded and assembled its function at the cell surface is to encode a voltage-gated potassium (K+) channel that has properties similar to the rapidly activating delayed rectifier current (I~cr) [1-3]. I~cr is expressed in both human atrial and ventricular myocytes [26, 27]. However, HERO expression is more abundant in ventricular, than atrial, tissue [28]. In the sinoatrial node, HERO current is believed to play a role in pacemaking [35]. The cardiac action potential is the result of several ion channels with different biophysical properties working in concert. The slow and rapid components of the delayed rectifier K+ current [29] contribute to the repolarization of the heart (Figure 2). In the ventricular myocyte, Ikr contributes to the repolarization phase of the cardiac action potential. Thus, it plays a significant role in determining the duration and shape of the action potential.

1.4 Biophysical Properties of HERG Channels

HERO channels exhibit two distinct biophysical properties: rapid inactivation and slow deactivation [29]. This I~cr channel displays voltage-dependant activation followed by rapid inactivation that causes marked inward rectification. Upon depolarization of the membrane, HERO rapidly activates and reaches maximum current amplitude between -10 to 0 m V. At voltages positive to 0 m V the channels rapidly inactivate. This non-conductive state greatly reduces the outward current causing inward rectification. As the membrane repolarizes the inactivated channels return to an open, conductive state that further facilitates repolarization. The channels then slowly return to a closed state by a process known as deactivation. In voltage clamp experiments this slow deactivation step results in large "tail" currents. [30-35].

7 Comparison of native Ikr and HERO current expressed in heterologous systems reveals a difference in kinetics. Ikr activates and deactivates more rapidly than HERG channels [9, 31, 36]. A plausible explanation for this can be that regulatory ~-subunits that are present in cardiomyocytes are absent in heterologous cells. Abbott et al. suggest that

HERG assembles with a small transmembrane protein, MinK-related Peptide 1 (Mirpl).

The complexes formed by the association of HERG and Mirpl are speculated to resemble native Ikr properties [37]. However, a study conducted by Weerapura et al. using similar conditions did not show any changes to HERG current that made it resemble native Ikr when eo-expressed with Mirp 1 [38]. The role of Mirp 1 in modulating HERO channels remains to be elucidated.

1.5 Pharmacological Properties of HERG Channels

Ikr was originally identified by its sensitivity to the class Ill antiarrhythmic agent, E-

4031 [29]. Blockade of HERO by methanesulfonanilides, such as , E-4031 and

MK499, prolongs the cardiac action potential duration and the refractory period of the ventricles. Excessive prolongation of the action potential can lead to acquired long QT syndrome, a cardiac disorder that predisposes the affected individuals to fatal cardiac [39]. Acquired long QT syndrome is commonly associated with administration of class lA and class Ill antiarrhythmic drugs [45]. Acquired long QT syndrome is often caused by both cardiac and non-cardiac drugs that selectively block HERG K+ channels

[40]. These drugs are believed to block HERO by being trapped in the large inner vestibule of the channel [41]. Studies using alanine-scanning mutagenesis suggest that blockade occurs through favourable binding of the drug to the aromatic residues tyrosine 652 and phenylalanine 656 located within the pore of the channel [42, 43]. The large number of

8 drugs that bind to HERG and are associated with increased susceptibilities to cardiac arrhythmias and sudden death has made HERG a major concern for drug development and approval.

2. Long QT Syndrome

2.1 Introduction

Congenital long QT syndrome is a rare disorder of cardiac action potential repolarization. LQTS clinically manifests itself on an electrocardiogram (ECG) as a prolonged QT interval where Q and T denote characteristic peaks on the ECG (Figure 2)

[44]. Affected individuals are predisposed to syncope and sudden death due to the development of termed "torsades des pointes" (TdP). Syncope occurs due to the onset of TdP and sudden death ensues if TdP degenerates into ventricular fibrillation. In 1966, Fran~ois Dessertenne coined the term TdP to describe the appearance of the QRS complex twisting around the isoelectric line of the ECG [45].

LQTS can be classified as two main types: familial and acquired. Jervell and

Lange-Nielsen first described LQTS in a family that had several children who suffered from congenital deafness, syncope and a prolonged QT interval [46]. This condition was characterized by an autosomal recessive mode of inheritance. A few years later, Romano et al. and Ward described a similar disease in two separate families that was inherited in an autosomal dominant manner [47-48]. The children experienced syncope and sudden death however, the disease was not associated with congenital deafness.

Acquired LQTS is the most common form of the disease. Pathological conditions that can lead to this form of the disease include: cardiomyopathy, cardiac ischemia,

9 bradycardia, and metabolic abnormalities such as reduced potassium serum levels.

Treatment with numerous medications such as antiarrhythmics, antibiotics, psychoactive agents and antimicrobials is the most common cause of acquired LQTS [40, 49].

2.2 Diagnosis of long QT syndrome

LQTS is a rare disorder with an estimated frequency of approximately 1 in 5000 people. Patients usually present with sudden death or unexplained syncope as a result of exercise or emotional excitement. The clinical hallmark of LQTS is the prolongation of the corrected QT interval (QTc) on an ECG, which is a measure of the duration of cardiac repolarization [50]. During adolescence the probability of experienqing a cardiac event increases significantly. The risk of cardiac events in is higher in males during puberty and higher in females during childhood [51]. LQTS is variable and influenced by the length of the QTc interval, sex, environmental factors, therapy and genotype. Upon diagnosis, the initial current therapy of choice is the use of ~-blocking drugs. ~-blockers reduce the frequency of syncopal events without affecting the QTc interval, however, they do not provide protection from fatal cardiac events [51, 52]. Other therapeutic options include: pacemakers, sympathectomy and the use an implantable cardiac defibrillator [52].

Whenever possible these forms of treatments may be used in conjunction with ~-blockers

[52].

2.3 Molecular genetics of long QT syndrome

LQTS is caused by mutations in 6 defined genes and in the gene encoding the adapter protein, ankyrin B. This disorder has variable expression, that is, the severity of the LQTS phenotype is dependant on the type and location of the genetic

10 mutation [51]. The genes identified in LQTS are assigned a LQT number that reflects the chronological order in which the gene locus was identified. More than 200 mutations have been identified in the 7 LQTS genes. In 1991, Keating et al. mapped the first LQT gene to chromosome llpl5.5 [53]. Shortly thereafter several studies demonstrated that multiple genes are implicated in LQTS [54-56]

LQTl - KCNQl (KVLQTl)

KCNQl encodes a potassium channel that coassembles with MinK to form a slowly activating K+ -delayed rectifier current, lks· lks is important for repolarization of the cardiac cell. Mutations in this gene reduce lks thereby resulting in prolongation of cardiac repolarization [50, 57]

LQT2-HERG

HERO is the gene responsible for -linked LQTS. The majority of

LQTS mutations identified reside in HERO. HERO encodes the rapidly activating K+­ delayed rectifier current, I~a, which is the major contributor to repolarization of the myocyte.

Mutations in HERO reduce I~a which consequently delays repolarization and action potential duration [3].

LQT3-SCNSA

SCN5A encodes a cardiac implicated in chromosome 3-linked

LQTS [58]. Sodium ions depolarize the cardiac membrane via the brief opening of the Na+ channel. The channel then inactivates and closes for the remaining duration of the cardiac action potential. Mutations in SCN5A cause a gain of function abnormality. SCN5A mutations interfere with the inactivation of the channel thereby increasing the influx of Na+ ions during the action potential. LQTS results because this increased influx prolongs action potential duration and delays repolarization [59].

11 LQT4- Ankyrin B

Ankyrin B is an adaptor protein required for the assembly of the sodium pump, the sodium/calcium exchanger, and inositol-!, 4, 5-trisphosphate receptors. Ankyrin B dysfunction is clinically manifested by bradycardia, ventricular fibrillation, ventricular tachycardia and sudden death. Unlike other classic long QT syndromes, ankyrin B mutations are not commonly associated with a prolonged QT interval [60, 61]. Mohler et al. have identified eight mutations in ankyrin B that are associated with cardiac arrhythmias.

Patients with these mutations experience arrhythmias, syncope or sudden death in response to exercise or emotional stress [60]. Ankyrin B dysfunction is due abnormal coordination of the sodium pump, the sodium/calcium exchanger, and inositol-!, 4, 5-trisphosphate receptors in cardiomyocytes [60, 61].

LQTS- KCNEl (minK)

MinK encodes a transmembrane protein that coassembles with KCNQ 1. Splawski et al. have identified mutations in the MinK gene that cause LQTS. Mutations in MinK reduce lks and results in delayed cardiac repolarization [62].

LQT6- KCNE2 (MirPl)

The mink related peptide 1, MirPl, is a single transmembrane potassium channel mapped to chromosome 21 q22.1. Ab bott et al. identified mutations in this gene associated with LQTS and demonstrated that these mutations diminish potassium currents by the slow opening and rapid closing of mutant channels [37].

LQT7-KCNJ2

KCNJ2 encodes the inward rectifier K+ channel Kir2.1, which is expressed in skeletal and . Andersen syndrome is a disease that arises due to mutations in this gene. This disease in characterized by periodic paralysis, prolongation of the QT

12 interval with ventricular arrhythmias, and skeletal developmental abnormalities. Tristani­

Firouzi et al. have identified mutations in the KCNJ2 that suppress the Kir2.1 current in a dominant negative manner. The reduction in Kir2.1 current prolongs the terminal phase of the cardiac action potential thereby leading to LQTS [63].

2.4 Mechanisms underlying long QT syndrome type 2

In 1995, Curran et al. mapped the LQT2 locus in affected families to the chromosome 7q35-36. They also identified six mutations in HERG that are responsible for

LQT2 [3]. Since then several studies have been published elucidating the various mechanisms by which mutations in HERG cause LQTS. Single amino acid substitutions account for the majority of the mutations in the LQT2 gene; however, mutations that result in truncated proteins, splice-acceptor sites and frame shifts have also been implicated in

LQT2. The LQT2 phenotype is a consequence of the reduction of I~cr which prolongs action potential depolarization and delays myocyte repolarization. Several studies have expounded multiple mechanisms by which mutations in HERG reduce I~cr. These mechanisms can be classified into three categories: dominant negative suppression, abnormal current function and defective protein trafficking.

2.4.1 Dominant Negative Suppression

A functional HERG channel in the cell membrane is comprised of four a.-subunits that coassemble. Therefore, the manner in which mutant a.-subunits interact with normal

HERG subunits is important to the understanding of LQTS. There are two general mechanisms for HERG channel eo-assembly with mutant subunits. In one mechanism,

13 termed haploinsufficiency, mutant subunits are unable to coassemble with wild-type. This results in a 50% reduction in the number of functional HERO channels assuming that both wild-type and mutant channels are translated and processed equally in the cell. In the other mechanism, mutant subunits can randomly assemble with wild-type in various ratios. The presence of even a single mutant subunit can result in a mutant channel phenotype, known as the dominant negative effect. Dominant negative suppression of wild-type function by a mutant subunit(s) results in more than a 50% reduction of functional HERO channels [64].

Electrophysiological analysis of the LQT2 mutants A561V, 0628S and N470D demonstrate that these mutants exert a dominant negative effect of wild-type current expression [65-67].

2.4.2 Abnormal Current Function

Several LQT2 mutant channels expressed at the cell surface generate an abnormal current that gives rise to the LQTS phenotype. In heterologous systems when the mutant channel is expressed alone it does not generate HERO current. Most of the LQT2 mutants that lack normal channel function reside near or in the pore region which has been implicated in normal channel gating [68]. Missense mutations in the pore region of HERO produce a more severe clinical phenotype than mutations in the N- or C-terminus [69].

LQTS-associated mutations in HERO are not restricted to any one region of the channel and can affect channel current by various means. Several mutations in the N-terminus of

HERO have been shown to accelerate channel deactivation [70, 71]. Mutations in the S4-

S5 linker also accelerate the rate of channel deactivation [72]. Channel mutations that cause current activation at more negative voltages than wild-type have been identified and studied in both oocyte and mammalian expression systems [65, 73]. Studies using the

14 Xenopus oocyte expression system have characterized LQT2 mutations that shift the voltage dependence of HERO inactivation [74, 75]. Interestingly, the mutation N629D abolishes C-type inactivation and reduces the channel's selectivity for K+ over Na+ ions.

This produces a novel gain of function where the channel's outward repolarizing current is replaced by an inward depolarizing sodium current [76].

2.4.3 Defective Protein Trafficking

Defective protein trafficking and processing of mutant protein is becoming increasingly recognized as an underlying mechanism of human disease. Trafficking deficiencies are associated with a significant group of LQT2 mutants. Several studies have linked the LQT2 phenotype to defects in the processing of the mutant HERO channel.

These mutations give rise to abnormally folded or assembled channels that are retained in the ER by quality control mechanisms [11, 66, 67, 73, 77-81]. Disruption of the interactions between HERO and its protein partners that are involved in its maturation also contribute to the absence of the mutant protein from the cell surface [11, 22]. Chaperone binding sites in HERG include its N-terrninal PAS domain and C-terrninal cyclic nucleotide binding domain (CNBD).

Trafficking deficient mutations reduce ltcr due to a defect in the normal folding and maturation process that prevents their export from the ER to the plasma membrane. The strategies developed for rescuing trafficking deficient LQT2 mutants have been primarily based on previously reported strategies that have successfully rescued the most common mutation, AF508. This mutation has been rescued by lowering incubation temperature or by the application of chemical or pharmacological chaperones [82-85].

15 The majority of the LQT2 trafficking deficient mutants are temperature-sensitive.

These mutants appear to undergo normal intracellular folding when the incubation temperature is reduced to 27°C [77-79]. Chemical chaperones, such as glycerol and dimethylsulfoxide (DMSO) have been used to stabilize the mutant HERG protein conformation during maturation. However, not all trafficking deficient mutations in HERG can be rescued by these non-specific chemical chaperones [77]. Thapsigargin, a calcium pump inhibitor, is another chemical chaperone used to restore intracellular trafficking of

HERG [86]. It is presumed that by inhibiting calcium release from the sarcoplasmic/endoplasmic reticulum calcium ATPase the activity of calcium-dependant chaperones is altered. Inhibition of calcium release by thapsigargin consequently disrupts

HERG protein/chaperone interactions.

Pharmacological chaperones have been shown to restore the intracellular transport to LQT2 mutants [71, 79-81]. These drugs that are specific blockers of HERG channels include: E-4031, astemizole, terfenadine, cisapride and fexofenadine. Not all mutations can be rescued by externally applied drugs [67, 77, 80]. Picker et al. have shown that restoration with channel blockers is dependant on the protein domain affected. Mutations in the CNBD of HERG seem to be insensitive to pharmacological rescue [80]. Restoration of trafficking requires that the drug bind to the inner vestibu]e of the HERG channel.

However, the mechanism by which these drugs stabilize the mutant conformation to facilitate its processing remains to be elucidated.

16 3. The PAS domain

3.1 Introduction

PAS (Per-Amt-Sim) domains are sensor modules that monitor changes in light, oxygen, redox potential and the overall energy of the celL PAS domains also mediate protein-protein interactions such as the dimerization of numerous transcription factors. PAS is the acronym for the names of the proteins in which this domain was first identified:

Drosophila period clock protein (PER), vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT), and Drosophila single-minded protein (SIM). These three proteins are involved in the .regulation of circadian rhythms, activation of the xenobiotic response and cell fate determination, respectively. PAS domains are found in all kingdoms of life and in many proteins including histidine-kinases, clock proteins, light receptor and regulator proteins, chemoreceptors, cyclic nucleotide phosphodiesterases and ion channels. Unlike other sensing modules, PAS domains are located in the cytosoL These domains sense changes to the extracellular environment directly through stimuli that enters the cell and indirectly through changes within the cell. Detection of sensory signals occurs through the binding of cofactors or ligands to the PAS domain. [87-89].

3.2 Structure of the PAS fold

Structural studies have shown that PAS domains contain a highly conserved a/~ fold and exhibit very little similarity at the amino acid sequence level [90, 91].

Crystallographic structures have identified the photoactive yellow protein (PYP), a bacterial blue-light photoreceptor receptor, as a major structural prototype for the three-dimensional fold of PAS domains [92]. At the functional level, PYP exhibits many of the characteristics

17 of PAS domains: sensing (intense blue light), protein-protein interactions that facilitate signal transduction and binding to ligands/cofactors (chromophore p-coumaric acid). The characteristic PAS/PYP fold is comprised of four segments: the N-terminal cap, the PAS core, the helical connector and the ~-scaffold [89, 92]. The emergence of crystal structures for the PAS domains in the heme-binding domain of FixL [93], LOV (light, oxygen, or voltage) domain family [94] and the HERG potassium channel [5] confirms that PAS domains share a predictable ai~ fold (Figure 3). PAS domains are not only structurally conserved; they share a common flexibility [90]. Within the common fold of PAS domains only a few residues (mostly leucines, isoleucines and valines) are conserved at the sequence level and they reside in the conserved hydrophobic core. Several glycines also show a large degree of conservation within PAS domains. These conserved glycines are believed to serve as hinge points that confer similar flexibilities to PAS domains [90].

3.3 The HERG PAS domain

The N-terminal HERG PAS domain is the first eukaryotic PAS domain to be

crystallized [5]. Unlike other sensing proteins, the PAS domain of HERG is involved in

regulating channel deactivation. The hydrophobic core is believed to interact with the S4-

S5 linker to mediate the slow deactivation of the channel [5, 95]. Several deletion and

point mutations in the HERG PAS domain impact channel deactivation. These mutants

accelerate deactivation; thus leading to a reduced outward current that prolongs action

potential duration [96-98]. Accelerated deactivation is believed to occur due to the

disruption of the interaction between the PAS domain and the S4-S5 linker.

18 Twenty-five mutations in the HERG PAS domain have been associated with LQT2

[99]. Chen et al. have extensively studied eight of these mutants functionally in Xenopus oocytes. They attribute the LQT2 phenotype to accelerated deactivation of the mutant channels. However, this study does not rule out the possibility of trafficking deficiencies associated with these mutants. A study has identified a trafficking deficient mutant in the

HERG PAS domain however; the role of the PAS domain in HERG maturation remains to be elucidated [71].

4. Rationale and Objectives

Several human diseases are associated with aberrant trafficking or defective processing of the mutant protein [100, 101]. Trafficking deficiencies are present in numerous HERG mutants associated with LQT2. The majority of these mutations have been identified in the transmembrane domains, the pore region and C-terminus of HERG.

Recently, our lab has shown that the C-terminus of HERG is important for channel maturation [102]. Mutations in theN-terminal PAS domain of HERG have been identified and implicated· in LQTS however, the effect of these mutations may have on channel maturation remain to be elucidated. Functional studies on several PAS domain mutations using Xenopus oocytes have demonstrated that the LQTS phenotype is due to the altered current kinetics of the channel. However, the use of this type of expression system may mask possible trafficking deficiencies associated with these mutants. Currently, little is known about the role of the PAS domain in HERG processing and trafficking to the cell surface. A novel LQT2 mutation within the PAS domain has been associated with a

19 trafficking defect. This discovery leaves the field with an unanswered question: Does the

PAS domain play a role in the cell surface expression of HERG?

The objective of this project is two-fold:

1. To examine the effects that LQTS-associated mutations in the PAS domain have on

cell surface expression of HERG.

2. To investigate the role of the PAS domain in the biosynthetic processing of HERG.

20 A

COOH NH2

B 0~0

008>~<300 PORE 0 0 0 0:0 0 oOo 0

Figure 1. Schematic representation of the HERG potassium channel and pore.

A. The HERG a-subunit consists of six-transmembrane helices (S 1-S6), an arginine rich voltage-sensor located in the fourth helix (S4) and potassium selective pore between helices five (S5) and six (S6). There are two cytoplasmic domains in the channel: the N-terminal Per-Arnt-Sim (PAS) domain and the C-terminal Cyclic Nucleotide Binding Domain (CNBD).

B. A functional HERG channel consists of four assembled a-subunits which forms a central pore.

21 •• • lks (KvLQT1 /minK) _,.._~·... >:: \ lkr (HERG) ~ •• AP •• •• • • • • • • • •

R

•• •••• • ECG .• •· .....

Q s T

QT Interval

Figure 2. The cardiac action potential and the surface electrocardiogram.

The top trace depicts the shape of a normal ventricular action potential (solid line). The rapid Okr) and slow (hs) components of the delayed rectifier are involved in the repolarization phase of the action potential (indicated by the arrows). The lower trace depicts a typical ECG recording (solid line). The QRS complex represents ventricular depolarization and the T wave (broad positive deflection) represents ventricular repolarization. The QT interval is a measure of the duration of cardiac repolarization. Mutations in the KvLQTl and HERG genes result in Long QT Syndrome which is characterized by an increase in the duration of the action potential and QT interval (depicted by the dotted lines).

22 A

HERG pyp FIXL LOV2

B

HERG 26 77 pyp 1 74 FIXL 152 ;;;;~;;;;;;;;;~~;;;;;;~;;ill: ~A iit~~,'~l~~~~~~~~~~:~!!!;;!! 199 LOV2 929 ------KS ILP p FAS RIL E E ~- --·R-- IRGIDII 977 ~C ~D ~E

HERG 78 ...K------135 pyp 75 :FYGKFKEGIASGNLNT----MF:IAAOOAOIILGAE-ER----~- Y D --QM CPTKV~LC.PI KKAL-~IDIIiliiii- -S -S V KRE ~ ------125 FIXL 200 :HDSY. SRYRTTSD-PHIIGIGRI -K ~P MH s Elos-- EP RD IHQQTQARLQELQ 270 LOV2 978 :IVQL RDIIKEQR-DV---- LN-DT G RAiWNIF VIRDIN DIQ VQQE ------1032

Figure 3. Crystal structure and structure based alignment of known PAS domains.

A. Ribbon diagrams of the crystallized PAS domains of HERG, Photoactive Yellow Protein (PYP), FixL heme domain and Light (FIXL), Oxygen and Voltage photoreceptor domain (LOV2) [110] .

B. Structure-based alignment of crystallized PAS domains. Black corresponds to identical residues in at least 3 proteins and structurally similar residues are in grey. Arrows indicate ~-sheets and bars indicate a-helices [ 111, 112].

23 CHAPTER2

Long QT syndrome-associated mutations in the Per-Arnt-Sim (PAS) domain of the HERG potassium channel impacts cell surface expression and stability

24 SUMMARY

Mutations in the human ether a-go-go related gene (HERG) have been implicated in long QT syndrome (LQTS), a associated with fatal ventricular arrhythmias. To date, several mutations within theN-terminal PAS (Eer-Arnt-.S.im) domain of HERG have been identified in patients with LQTS. To examine the function of the PAS domain in HERG processing and its implication in LQTS, we have characterized three long

QT -associated mutations (G53R, E58K, and 196T) within this domain. These mutations were selected on the basis of their structural conservation within other PAS domains with known crystal structures. Western blot analysis shows that these point mutations are not expressed at the cell surface due to endoplasmic reticulum (ER) retention and not as a result of disrupted tetrameric assembly. Functional studies reveal a marked decrease in current amplitude that can be restored by reducing incubation temperatures. Examination of channel stability by pulse-chase analysis reveals a more rapid turnover rate for these mutants when compared to wild-type channels. Disruption of the structurally conserved a/~ fold by removal of any or beta sheet produces the same ER retention phenotype as the point mutations. Interestingly, complete removal of the PAS fold

(residues 26-135) enhances cell surface expression of HERG; however, the stability of the channel at the cell surface is impaired. Thus, we demonstrate that by altering the conserved a/~ fold of the PAS domain, cell surface expression and stability of the mutant HERG channel is reduced.

25 INTRODUCTION

Defective processing of a channel from the endoplasmic reticulum to the Golgi and ultimately to the cell surface, can result in human disease [100, 101]. An example of the consequences of defective protein processing are seen in mutations in the human ether a-go­ go related gene (HERG) potassium channel that results in long QT syndrome (LQTS).

LQTS is an inherited cardiac disorder that predisposes affected individuals to fatal cardiac arrhythmias. It is characterized by a prolonged QT interval on an electrocardiogram and ventricular tachycardia termed "torsades des pointes" which results in syncope and sudden death [45]. Over 200 mutations in seven genes, including HERG, have been implicated in

LQTS. HERG encodes a six-transmembrane potassium channel that has properties similar to the rapidly activating delayed rectifier current (I~cr) [1-3]. In the ventricular myocyte, IKr contributes to the repolarization phase and duration of the cardiac action potential [29].

Defective trafficking of mutant HERG protein is becoming increasingly recognised

as an underlying mechanism for LQTS. Trafficking deficient mutations in HERG have

been identified in the transmembrane domains, the pore region and C-terminus of HERG

[11, 66, 67, 73, 77-81]. The ER retained phenotype arises due to abnormal protein

processing of the mutant channels which results in their absence from the cell surface.

Recently, a C-terminal segment has been identified as a crucial element required for HERG

maturation [102]. However, little is known about the role of theN-terminus in cell surface

expression of HERG. The N-terminus of HERG has been implicated in gating via an

interaction with its S4-S5 linker. More specifically, the region of interaction resides within

the Per-Arnt-Sim (PAS) domain of the HERG N-Terminus [5]. This domain exhibits high

structural similarities to other PAS containing proteins. PAS domains are found in all

26 kingdoms of life where they act as sensors in signal transduction [87, 88]. At the amino­ acid sequence level there is very little homology between domains, however, their three­ dimensional structure is highly conserved. This high degree of structural similarity confers a common flexibility to PAS domains [90]. Twenty-five mutations within the PAS domain of HERG have been identified and implicated in LQTS (Fig. 4 and Refs. 70, 71, 99). Eight of these mutations have been characterized in the Xenopus oocyte expression system and found to accelerate deactivation [70]. The accelerated deactivation of these channels reduces IKr and increases action potential duration, thus leading to LQTS. Functional characterization of these mutations in oocytes at room temperature may not reveal possible trafficking or stability defects that manifest in a mammalian expression system at physiological temperature. A novel mutation (T65P) within the PAS domain of patients with LQTS has been associated with defective HERG trafficking [71]. However, it remains to be determined if this applies to other PAS domain mutants and whether the PAS domain itself plays a role in HERG trafficking.

In this study we use biochemical and electrophysiological methods to investigate the function of the PAS domain in the intracellular processing and stability of HERG channels. We studied the surface expression and stability of previously uncharacterized

HERG LQT-associated mutants, E58K [103] and I96T [104], as well as the functionally characterized G53R mutant [70, 105]. Analysis of mutations at these structurally conserved residues in PAS domains demonstrates that preservation of the conserved PAS fold is required for HERG efficient trafficking to the cell surface. Moreover, we show that disruption of the conserved a/~ fold of the PAS domain, by removal of any alpha helix or beta sheet, impairs cell surface expression of HERG. Surprisingly, complete deletion of the

27 PAS fold facilitated surface expression; however, the mutant exhibited stability defects once it reached the cell surface. This study is the first to associate stability defects with

LQT -associated mutations in the PAS domain of HERG in addition to the trafficking defects observed.

28 EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis and Construct Generation

The generation of N-terminal Myc-tagged HERG has been previously described

[102]. To eliminate unwanted mutations in the 7.8 Kb cDNA of Myc-HERG a truncation was made using the unique BamHI and Bglii restriction sites. Restriction enzymes were purchased from New England Biolabs. The truncated HERG was then ligated into the pSG5 vector (Stratagene) using the same restriction sites. Deletions and point mutations were engineered using the QuickChange XL Site-directed mutatgenesis kit (Stratagene).

Synthetic primers used for site-directed mutagenesis were obtained from Invitrogen. The truncated HERG, which contained the mutation of interest, was then subcloned back into its original vector. All plasmids were purified using Qiagen' s purification kits. Deletion mutants APAS, AaA, AaB, Aa310, AaC, A~A. A~B. A~C, A~D and A~E correspond to deletions of the following residues: 26-135, 45-53, 55-60, 66-70, 75-89, 28-34, 40-44, 92-

100, 104-117 and 121-135 respectively.

Stable and Transient Transfection in HEK 293 Cells

Transient transfections were performed in HEK 293 cells for Western Blots and in

M2 cells for electrophysiology. HEK 293 cells were cultured in a-minimal essential medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. M2 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were cultured at 37°C under a

5% C02 atmosphere. Transfections were carried out using Lipofectamine (lnvitogen) as described by the manufacturer. Stable cells lines for wild-type HERG, G53R, E58K and

196T were generated using the G-418 selection method. Transfected cells were maintained

29 in a-minimal essential medium supplemented with 800 ug/ml G-418 (lnvitrogen) for 10 to

15 days. G-418 resistant colonies were selected by their protein expression levels and

purity, as determined by western blot analysis and immunocytochemistry.

Western Blot Analysis

Cells growing on 35mm dishes were washed two times with cold PBS and

membrane proteins were solubilized in a lysis buffer containing 50 mM Tris (pH 8), 75mM

NaCl, 0.5% Nonidet P-40 and a protease inhibitor cocktail (Roche). Cells were harvested

. and homogenized by pipetting. Detergent insoluble material was sedimented at 16, 000 x g

for 30 minutes. The detergent soluble fraction (cell lysate) was resuspended in 2X Laemli

buffer (0.5M Tris pH 6.8, 4% SDS, 5% ~-mercaptoethanol, 20% glycerol and 0.001%

Bromophenol blue). Protein concentration was determined using a detergent compatible

assay (Bio-Rad). Samples were resolved on a 7.5% polyacrylamide SDS gel (unless

indicated otherwise) and transferred onto nitrocellulose membranes (Bio-Rad). The

membranes were then blocked with 5% nonfat dry milk and 0.1% Tween-20 in PBS for one

hour. The membranes were incubated with monoclonal anti-Myc (Santa Cruz

Biotechnologies) or Anti-Cask (Chemicon) at a 1:1000 dilution for one hour at room

temperature and then washed extensively with 2% nonfat dry milk and 0.1% Tween-20 in

PBS. This was followed by incubation with goat anti-mouse lgG conjugated to horseradish

peroxidase (Jackson lmmunoResearch Labs) at a dilution of 1:15,000 for one hour. After

extensive washing, the horseradish peroxidase-bound proteins were visualized on X-ray

films using the ECL Plus detection kit (Amersham Biosciences ).

30 Glycosidase Treatment

Celllysates were incubated with N~glycosidase F (PNGase) and Endoglycosidase H

(EndoH) as recommended by the manufacturer (New England Biolabs) at 37°C for 24 hours. The samples were then analyzed by Western blot as described above.

Proteinase K Treatment

Cells were washed with ice~cold PBS and incubated in Proteinase K buffer (10 mM

HEPES, 150 mM NaCl, 2 mM CaCb) with or without 20 ug/mL of Proteinase K (BioShop,

McGill University) at 37°C under a 5% C02 atmosphere for 30 minutes. The cells were harvested and the activity of the proteinase K was stopped using a buffer containing 10 mM

HEPES, 25 mM EDTA and 20 mM Pefabloc SC (Roche Diagnostics). The cells were washed with 25 mM EDTA and a protease inhibitor cocktail (Roche Diagnostics) in PBS.

The cells were lysed and then analyzed by Western blot.

Pulse-chase Metabolic Labelling and Immunprecipitation

Transiently transfected cells or stable cell lines were starved for 1 hour in

Dulbecco's modified Eagle's medium depleted of cysteine and methionine. Cells were metabolically labelled with 150 uCi/ml of e5s] cysteine/methionine and chased at different time intervals with Dulbecco's modified Eagle's medium containing 2 mM unlabelled cysteine and methionine. At .the end of the chase time interval the cells were lysed, as previously described, and precleared using Protein G agarose beads (Amersham

Biosciences). The celllysates were then immunoprecipitated with an anti-MYC antibody

(1:250, Santa Cruz Biotechnologies). Protein A agarose beads (Amersham Biosciences) were used to isolate the antigen-antibody complexes. The beads were washed with lysis

31 buffer and the complexes were solubilized in 2X Laemli buffer. The samples were subjected to 7.5% SDS-PAGE and visualized using autoradiography.

Sucrose Gradient Sedimentation

Cell lysates were centrifuged 150,000 x g for 45 minutes and layered on a 5-40% continuous sucrose density gradient. Molecular weight markers, dehydrogenase

(150 kDa) and thyroglobulin (669 kDA), were layered on a separate gradient. The gradients were spun at 220,000 x g for 16 hours at 4°C and 12 fractions were then collected from each gradient using an AutoDensi Flow automatic device (Buchler Instruments).

Samples from each fraction were resuspended in Laemli buffer, subjected to SDS-PAGE and analyzed by immunoblotting.

Immunofluorescence Microscopy

The stable cell lines were plated on Fibronectin coated coverslips. The cells were then fixed using 2% paraformaldehyde and permeabilized for 30 minutes using 0.1% Triton and 10% goat serum (lnvitrogen) in PBS. The coverslips were incubated with monoclonal anti-MYC for 1 hour and washed extensively with PBS. The coverslips were then incubated with an Oregon Green conjugated secondary antibody (Molecular Probes) for 45 minutes and washed extensively. The slides were mounted using Immunofloure Mounting media (ICN Biomedicals) and imaged using the Zeiss LSM 510 Confocal Microscope.

Patch Clamp Recordings

The whole-cell patch clamp technique was used to record membrane currents as previously described [106]. Stable cell lines were cultured at 37°C or 27°C where indicated. M2 cells were cultured at 37°C. M2 cells were transiently transfected with the cDNA of interest along with the surface antigen, CD8. Transfected cells were selected

32 using immunomagentic Dynabeads (Dynal) coated with a monoclonal anti-CD8 antibody.

Patch clamp electrodes were filled with medium containing (mM): 130 KCl, 1 MgC}z, 5

EGTA, 5 MgATP and 10 HEPES (pH 7.2). The external medium contained (mM): 137

NaCl, 4 KCI, 1.8 CaC}z, 1 MgC}z, 10 glucose and 10 HEPES (pH 7.4). Membrane currents were recorded using an Axopatch amplifier (Axon Instruments). A two-step voltage clamp protocol was used to asses HERG current. From a holding potential of -80 mV, HERG currents were activated by a 4 second depolarizing pulse to potentials between -60 m V and

+50 m V in increments of 10 m V. To asses the characteristic HERG tail currents at the end of the depolarizing pulse, the membrane was repolarized to -60 m V for 2 seconds before returning to the -80 m V holding potential. Cell capacitance was estimated by analog measurements from the patch clamp amplifier. pClamp software (Axon Instruments) was used to analyze data and generate traces.

Densiometric and Statistical Analysis

Densiometric analysis was carried out using the Alpha Innotech Imaging System

(Alpha Innotech). Each band was quantified by an average pixel value which incorporated background subtraction. Data are presented as mean ± standard error of the mean.

Statistical significance was determined using Student's t test (p<0.05).

33 RESULTS

Western Blot Analysis of naturally occurring LQTS mutations

Wild-type HERO expression in HEK 293 cells reveals two distinct bands on a western blot which are indicative of N-linked glycosylation [9]. The lower band of 135 kDa is the immature core-glycosylated form of HERO retained in the ER that is sensitive to both EndoH and PNGase (Fig. 5B). The upper, more diffuse band of 155 k:Da that is sensitive to PNGase only, corresponds to the complex-glycosylated mature form of HERO located in the Golgi and ultimately expressed at the cell surface (Fig. 5B). Western blot analysis of the LQT mutants G53R, E58K, and I96T expressed at 37°C reveals the presence of only the ER retained form and the absence of the mature band (Fig. SA).

EndoH and PNGase digestions produce the expected shifts to a lower molecular weight

(132 kDa), confirming that the 135 k:Da band is indeed the core-glycosylated form of the channel (Fig. 5B). These results confirm that G53R, E58K, and I96T fail to exit the ER and acquire complex oligosaccharides in the Golgi apparatus.

Pulse-chase analysis of LQTS mutations in the PAS domain

The absence of the 155 k:Da band in the G53R, E58K, and I96T mutants is indicative of a defect in the biosynthetic processing of the mutant channel. To examine the stability of the mutants, pulse-chase experiments were performed using metabolic labelling.

Figure 6A shows that the mature form of HERO is synthesized from its 135 kDa precursor.

The transition of the 135 k:Da form to the mature 155 kDa form is gradual and the mature fraction remains relatively stable up to 24 hours. In contrast, both G53R and E58K fail to convert the immature fraction to the 155 k:Da form over a 24 hour period. Interestingly,

I96T is able to produce a mature band whose expression peaks at 4 hours. However, as

34 time progresses this mature fraction is rapidly degraded and absent from the cell surface by

24 hours (Fig. 6A). A comparison of the stability of the 135 kDa ER form in mutants to wild-type reveals that G53R, E58K, and I96T are degraded more rapidly than wild-type and are virtually absent at 24 hours (Fig. 6A). Densiometric analysis of the immature fraction in four independent experiments confirms that the mutants show more rapid turnover kinetics when compared to wild-type (Fig. 6B). This data demonstrates that mutations in structurally conserved residues in the PAS domain compromise the stability of the HERG channeL

Effect of Temperature on mutant channel surface expression and function

Numerous trafficking deficient mutations in HERG have been rescued by lowering incubation temperature to 27°C [71, 77-79]. Lower temperatures may facilitate improved channel folding by increasing ER retention times and inhibiting proteosomal degradation.

Incubation of G53R, E58K, and I96T stable cell lines at 27°C for at least 24 hours enabled a portion of the ER fraction to mature and traffic to the cell surface (Fig. 7). Western blot analysis shows the presence of a 155 kDa band for the mutants, which suggests improved folding and maturation at 27°C. These findings imply that misfolding could be an underlying mechanism of the impaired maturation and surface expression of these LQTS mutants.

To assess function and temperature-dependant maturation voltage-clamp experiments were performed on the mutants incubated at 37°C and 27°C. Characteristic

HERG tail currents were observed at both 37°C and 27°C for wild-type (Fig. 8 and 9).

G53R, E58K, and I96T had a marked reduction in tail current density at the physiological temperature (Fig. 8). When the mutants were incubated at 27°C there was a significant

35 increase in the recorded current amplitude (Fig. 9). Figure 10 illustrates the significant increase in tail current density for the mutants cultured 27°C when compared to cells cultured at the physiological temperature. These data further corroborate the results seen in figure 7 and demonstrate that lower temperatures facilitate the correction of the trafficking deficient phenotype of the mutant HERG protein.

Tetrameric assembly of mutant channels

Proteins that fail to properly assemble are recognised by the ER quality control and degraded [16, 23-25]. To determine whether or not defective tetramerization could explain the ER retention phenotype of our PAS domain mutants, sucrose gradients were performed.

Wild-type, G53R, E58K, and 196T cell lysates were layered on separate non-denaturing continuous sucrose gradients. The distribution profiles of the mutants were very similar to wild-type and the peak immunoreactivity occurred around fraction 7 (Fig. 11 ). Fraction 8 corresponds to the molecular weight of thyroglobulin, 669 k:Da. Therefore, the single peak at fraction 7 is consistent with the apparent molecular weight of tetrameric HERG which is approximately 540 k:Da. These data suggests that the impaired cell surface expression of

G53R, E58K, and 196T is not caused by improper tetrameric assembly of the channel.

Immunolocalization of mutant channels

Subcellular distribution of wild-type and mutant protein was examined at 37°C using confocal microscopy. Confocal images show that wild-type is primarily expressed at the cell surface, whereas G53R, E58K, and 196T were located in the perinuclear region of the cell (Fig. 12). This restricted localization to the perinuclear region is consistent with the ER retention phenotype indicated by the biochemical analyses in figure 5A.

36 Disruption of the structurally conserved a/~ fold

In order to delineate a region within the PAS domain that may be critical for HERG maturation we sequentially deleted each alpha helix and beta sheet. Deletion of the secondary structural motifs of the HERG PAS domain resulted in the presence of the ER retained form of the channel and the absence of the mature band (Fig. 13A). Upon removal of any of the alpha helix or beta sheet in the PAS domain, HERG is retained in the ER.

Taken together, the data indicates that altering the fold of the PAS domain by either disrupting a motif within the domain or by mutating a structurally conserved amino acid impairs maturation of HERG. To further validate this point, we created another LQTS mutant, R56Q [105], in the PAS domain that is not structurally conserved and found that it did not impact HERG maturation (Fig. 13B). This demonstrates that not all mutations within the PAS domain affect channel maturation. Mutations of structurally conserved amino acids are most likely to impact maturation and stability by altering the conserved fold of this domain thereby leading to ER retention.

Deletion of the PAS fold

We have shown that 3 structurally conserved point mutations and disruption of the secondary structural elements in the HERG PAS domain impact the protein's maturation and stability. To asses whether the PAS domain is critical for HERG's biosynthetic processing we selectively deleted the region from residues 26 to 135, that has high structural homology to other PAS domains. Deletion of the PAS domain (APAS) surprisingly revealed 2 bands on Western blot and produced the expected shift in the apparent molecular weight of HERG (Fig. 13A). To ensure that these bands were ER and

Golgi glycosyated, cell lysates were subjected to PNGase and Endo H treatment which

37 produced the expected shifts in molecular weight and confirmed that the M> AS mutant is indeed glycosylated (Fig. 13C). The presence of an intense Golgi glycosylated band demonstrates that deletion of the PAS domain does not impair HERG maturation. In fact,

~PAS may be more efficiently expressed at the cell surface. To further demonstrate that

M> AS is expressed at the cell surface, we use Proteinase K (PK), a serine protease that cleaves the extracellular ectodomains of membrane proteins. Treatment of cells transiently transfected with ~pAS results in the disappearance of the upper band and the appearance of a cleaved product at lower molecular weight (Fig. 13D). Sensitivity of the upper band to

PK treatment indicates that ~pAS is transported to the cell surface.

Functional characterization of Deletion mutants

Voltage-clamp recordings were performed on wild-type and M> AS that were transiently transfected in M2 cells. This melanoma cell line was chosen to facilitate the patch clamp recordings. Comparison of wild-type and ~pAS currents showed that ~PAS has more robust tail currents (Fig. 14A). The mean tail current density of M> AS is almost twice that of wild-type (Fig. 14B). The increased current density and the presence of a more intense Golgi glycosylated band for the ~PAS mutant suggest an increased number of channels at the cell surface.

Pulse chase analysis of Deletion mutants

The biosynthesis of the M>AS mutant was assessed using pulse-chase analysis.

When compared to wild-type, ~pAS underwent a similar transition from immature precursor to mature form. However, at 4 hours the mature band peaked in its intensity then steadily declined thereafter (Fig. 15A). Densiometric analysis of the immature fraction

38 showed an increased turnover rate for AP AS when compared to wild-type at the 8 and 24 hour time points (Fig 158). Taken together, the data indicate that APAS can mature to the cell surface but its stability is compromised and is subject to degradation.

39 DISCUSSION

Our results provide new information about the role of the PAS domain in HERG maturation and stability. We have characterized three LQTS mutations, G53R, E58K, and

196T, which are located in the PAS domain and have shown that they fail to be expressed at the cell surface due to ER retention (Fig. 5A). These mutants, which fail to become fully glycosylated in the Golgi, have very small tail currents, suggesting that a small fraction of the ER retained protein leaks to the cell surface and is detectable by patch clamp recordings. The failure of these LQTS mutants to escape the ER can be explained by their rapid degradation. Pulse chase-experiments revealed that the ER fraction of the mutants had an increased turnover rate. By 24 hours, the ER fractions of the mutants were barely detectible (Fig. 6A). These results are similar to those found in a study by Zhou et al. which show the ER retained LQTS mutants, Y611H and V822M, were rapidly degraded

[73]. It has been shown that glycosylation is not required for the transport of HERG to the cell surface but it does play a key role in conferring stability to the channel at the cell surface [8]. This was demonstrated through pulse-chase experiments that showed that non­ glycosylated HERG channels had a more rapid turnover than glycosylated channels [8].

G53R and E58K are unable to convert the 135 kDa precursor into its mature form, however, the mutant 196T manages to convert the precursor into its cell surface form by 4 hours. This mature band, which peaks at 4 hours, progressively declines and is virtually absent by 24 hours. This implies that once this mutant reaches the cell surface it is recognized for degradation by mechanisms not identified in this study. These mechanisms could possibly include lysosomal degradation or ubiquitin-mediated proteolysis.

40 The ER has stringent quality control mechanisms in place to ensure that only correctly folded and assembled proteins can reach their final destinations [10, 16]. When a protein fails to pass the ER quality control, the misfolded or unassembled protein is retained in the ER and subject to ER associated degradation (ERAD). ERAD ensures that improperly folded proteins do not make it to their target destination because protein function is highly dependant on its three-dimensional (3D) conformation achieved during the normal folding process. Glycine 53, glutamate 58 and isoleucine 96 in HERG are structurally conserved residues in PAS containing proteins with known crystal structures, namely, photoactive yellow protein (PYP), FixL heme domain (FixL) and light, oxygen and voltage photoreceptor domain (LOV2) [5, 90, 94]. These proteins, which are highly similar at the structural level, have several conserved glycines that confer a common flexibility to their PAS domains. Glycine 53 is one of these residues. Mutations in structurally conserved residues may alter the 3D fold of the PAS domain and therefore result in ER retention of HERG due to misfolding. Mutations, which lead to misfolding, have been identified as a mechanism of disease for several proteins such as CFTR and [107-109]. The trafficking deficiencies observed in these proteins and several

HERG mutations have been rescued by using lower cell incubation temperatures. Several

HERG mutations have also been rescued using the calcium pump inhibitor, thapsigargin

[86], the chemical chaperone, glycerol [79], and by channels blockers: E-4031 [79] and fexofenadine [81]. Western blot analysis shows that G53R, E58K, and I96T can be rescued by incubation at lower temperatures. Functional studies done on these cells cultured at

27°C confirm this result with a dramatic increase in tail current density. Rescue of these mutants could not be attained using E-4031, glycerol, DMSO, or by the calcium pump

41 inhibitors, Thapsigargin and (data not shown). Thus, our study identifies a class of temperature-sensitive mutants in the PAS domain that cannot be restored to the cell surface by osmolytes, calcium pump inhibitors or by channel blockers. These mutations impact maturation by ER retention due to misfolding and not by defective tetramerizaton.

We have shown that not all mutations in the PAS fold affect maturation. R56Q is a

LQTS mutation of a residue that is not structurally conserved in PAS containing proteins and it is expressed at the cell surface (Fig. 13B). The mutant, T65P, in the HERG PAS domain is also not structurally conserved, and has been show to be trafficking deficient

[71]. This can probably be attributed to the introduction of a kink in the chain by proline residue located before the single turn 310 helix. This change in structure is enough to render the channel misfolded. Mutations in structurally conserved amino acids are most likely to impact maturation and stability by altering the conserved fold of this domain thereby leading to ER retention. Our study shows that not all LQTS mutations within the PAS fold share the same underlying mechanism for the disease. The study by Paulussen et al. suggests that the PAS domain plays a key role in trafficking as evidenced indirectly though the characterization of a novel mutation, T65P [71]. In this study we further investigate this claim by studying the effects of deleting the conserved PAS fold in HERG. Recently,

Hefti et al. have suggested the use of the term "PAS fold" rather than domain to signify this structural element found in a variety of proteins [91]. In HERG, this fold is comprised of five ~-sheets (~A to ~E), a single turn of a 310 helix, and three a-helices ( aA to aC).

Removal of this PAS fold from HERG did not compromise channel maturation, that is, the

AP AS mutant was expressed as 2 distinct bands on a Western blot. The upper band, which is PNGase sensitive, corresponds to the mature fraction of APAS at the cell surface. This

42 band is consistently more intense than the mature band of wild-type which leads us to speculate that removal of the PAS fold allows HERG to be more efficiently expressed at the cell surface. Perhaps, the lack of this intricately folded structure enables the channel to pass through the ER quality control machinery and assemble more rapidly than wild-type.

The AP AS mutant may interact less with molecular chaperones thus enabling rapid exit from the ER. Further studies are required to explain this phenomenon.

Pulse-chase experiments showed that APAS was less stable at the cell surface than wild-type. Its expression peaks at 4 hours then steadily declines by 24 hours. This stability defect is similar to that observed for the I96T mutant. In this case also there seems to be a retrieval mechanism targeting APAS for degradation from the cell surface. The transient expression of these mutants at the cell surface further confirms the impact the PAS fold has on the stability of the HERG channeL

Patch clamp recordings support the argument that AP AS facilitates channel cell surface localization. Comparison of tail current density reveals an approximate 2-fold increase for APAS channels when compared to controls. Further functional studies are required to rule out the possibility that the increase in current density of APAS channels is not due to increased channel open time. Cabral et al. have studied deletion mutants in the first 25 amino acids of the HERG N-terminus that are disordered in the crystal structure [5].

The deletion of residues 2 to 9 (A2-9), 2 to 23 (A2-23), and 2 to 26 (A2-26) accelerated deactivation [5]. Several studies on N-terminal deletions and point mutations have found that these HERG mutants have a marked acceleration of deactivation when compared to wild-type [96-98]. Our study is the first to examine the biochemical significance of a deletion mutation in the HERG PAS domain using a mammalian expression system.

43 In summary, our data provides new information on the role of the PAS fold on

HERG maturation and stability. The LQTS-associated mutants (G53R, E58K, and I96T) and the deletion mutants of the alpha helices and beta sheets disrupt the conserved PAS fold which leads to ER retention of the channel. Temperature rescue· suggests the mechanism of retention is due to the overall misfolding of the tetrameric channel. These mutants, which are competent in homotetrameric assembly, are less stable than wild-type.

Interestingly removal of the PAS fold does not impede channel maturation but it does affect its stability at the cell surface. Together, these findings demonstrate that disruption of the conserved PAS fold in HERG gives rise to subtle folding defects that result in the channel's absence from the cell surface.

44 A

COOH G53R

B 175- ~ Mature ~ Immature 83-

Figure 4. The HERG potassium channel.

A. Schematic representation of the membrane topology of HERG illustrating the six­ transmembrane helices (S 1-S6). The functional channel is composed of four of these subunits. The LQTS mutations studied are depicted by black circles.

B. Western blot of Myc-tagged HERG expressed in HEK 293 cells. The mature band ( 155kDa) corresponds to the Golgi glycosylated form of the channel whereas the immature band (1 35 kDa) corresponds to the core glycosylated form of the endoplasmic reticulum.

45 A WT G53R E58K 196T 175 -

83-

B Wild-Type G53R E58K 196T

EndoH + + + + PNGase + + + + 175 -

83-

Figure 5. Western blot analysis of LQTS mutants in the PAS domain.

A. HERG, G53R, E58K and I96T were stably transfected into HEK 293 cells. 30 !lg of crude membrane proteins were subjected to 7.5% SDS-PAGE and detected by an anti­ MYC antibody. Similar results were obtained when HERG, G53R, E58K and I96T were transiently transfected into HEK 293 cells.

B. Membrane proteins of stably transfected HERG, G53R, E58K and I96T were treated (+)or not treated(-) with EndoH or PNGase.

46 A

MOCK WILD TYPE G53R

Chase Time (hrs) 0 0 2 4 8 24 0 2 4 8 24 175 -

83 -

MOCK E58K 196T

Chase Ti me (hrs) 0 0 2 4 8 24 0 2 4 8 24 175 -

83 -

B

Stability of the Immature Band

120

100

80 ·"';::c: • HERG 'jij •G53R 60 .,E Ill E58K a: D I96T ~ 40

20

0 0 2 4 8 24 Chase Time (Hrs)

Figure 6. Pulse-chase analysis of PAS domain mutants.

A. Stably transfected cells were metabolically labelled with e5S] cysteine/methionine for 1 hour and chased at the indicated time intervals. The cell lysates were immunoprecitated by an ant-MYC antibody and subjected to 7.5 % SDS-PAGE and autoradiography. Mock-transfected cells were also labelled for 1 hour and analyzed at the 0 hour chase time.

B. Densiometric analysis from four independent experiments illustrates the percentage of the immature fraction of the wild-type and mutant channels as a percentage of the expression level at chase time 0 hours. Asterisks (*) indicate statistically significant differences between the mutants when compared to wild-type (p < 0.05).

47 WT G53R E58K 196T WT G53R E58K 196T

175

83-

Figure 7. Effect of temperature on cell surface expression.

Stable cell lines were cultured at 37°C or 27°C for at least 24 hours. Celllysates were subjected to 7.5% SDS-PAGE and immunoblotted with an anti-MYC antibody.

48 Wild-Type G53R

100pA l§=====:a.--- 1 sec 1 sec

E58K 196T

-~._ ~

100 pA 100 pA l§ 1 sec 1 sec

Figure 8. Voltage clamp recordings of wild-type HERG and mutants at 37°C.

Patch clamp recordings of wild-type HERG, G53R, E58K and I96T stably transfected in HEK 293. Cells were cultured at 37°C. Wild-type HERG n = 15, G53R n = 25, E58K n = 17, I96T n = 19.

49 Wild-Type G53R

1 sec 1 sec

E58K 196T

1 sec 1 sec

Figure 9. Voltage clamp recordings of wild-type HERG and mutants at 27°C.

Patch clamp recordings of wild-type HERG, G53R, E58K and 196T stably transfected in HEK 293. Cells were cultured at 27°C. Wild-type HERG n = 8, G53R n = 8, E58K n = 8, 196T n = 9.

50 Tail Current Density

20 18 16 LL 14 a. 12 • 37 Degrees

Figure 10. Comparison of tail current density at 37°C and 27°C.

Mean tail current density was obtained from current recordings of wild-type HERG, G53R, E58K and I96T stably transfected in HEK 293cells at 37°C and 27°C (data derived from current recordings shown in Figures 8 and 9). Asterisks (*) denote statistically significant deviations from current recordings at 37°C (p < 0.05).

51 A

Fraction 1 2 3 4 5 6 7 8 9 10 11 12

WT

G53R

E58K

I96T

B

t 20

tOO

80 E - HERG ~ -..GS3R ·~ 60 ::E.. --E58K ... t 50 kDa -..196T 40

20

0 . 2 tO tt t2 t 3 Fraction Number

Figure 11. Sucrose gradient analysis.

A. Wild-type HERG, G53R, E58K and I96T stably transfected in HEK 293 cells were fractionated on 5-40% linear non-denaturing sucrose gradients. Samples were analyzed by SDS-PAGE and irnmunoblot.

B. Densitometry was used to measure the pixel intensity of each fraction as a percentage of the maximum intensity. The molecular weight markers used were alcohol dehydrogenase (150 kDa) and thyroglobulin (669 kDA).

52 Figure 12. Subcellular distribution of HERG and mutant channels.

Stably transfected HERO, 053R, E58K and 196T were grown on fibronectin coated coverslips. The cells were immunolabelled with an ant-Myc antibody followed by an Oregon green conjugated goat anti-mouse IgO antibody. Confocal imaging shows that wild type HERO is expressed at the cell surface whereas the mutants are located primarily in the perinuclear region (white arrows).

53 A Mock WT ~aA ~aB ~aC ~a3 10 ~PA ~PB ~pc ~pD ~PE LWAS

175

83 -

B Mock WT ilPAS R56Q G53R E58K 196T 175 -

83- c D ilPAS ~PAS Proteinase K Endo H + - + 175- PNGase + 83 175

62 - +--- Cleaved Product

83 48-

Figure 13. Western Blot analysis of deletion mutants.

A. Site-directed mutagenesis was used to generate the sequential deletions of all a-helices, p sheets and deletion of the entire PAS fold. The constructs were transiently transfected into HEK 293 cells and analyzed by SDS-PAGE. Cask, an endogenous protein, is used as a loading control.

B. Myc-tagged wild-type HERG, ~PAS and four naturally occurring LQTS mutants, G53R, R56Q, E58K and I96T were transiently transfected in to HEK 293 cells.

C. Cell lysates of transiently transfected wild-type HERG and ~PAS were treated ( +) or not treated (-) with EndoH or PNGase.

D. Cells transiently transfected with the ~PAS cDNA were treated with 20 ug/mL of Proteinase K (+) or vehicle (-).

54 A

Wild-Type L\ PAS

1oooAI sa::t~~~ 1 sec 1 sec

B

Tail Current Density

14

12

10 u.. Q. ~ 8 Q. 1-WTl c 6 ~

i 4

2

Figure 14. Functional analysis of the M> AS mutant.

A. The whole cell patch clamp technique was used to examine functional expression of HERG and L\PAS at 37°C. HERG n = 13, L\PAS n = 13.

B. Mean tail current amplitudes from the patch-clamp recordings were used to estimate channel density at the cell surface.

55 Mock Wild-Type LlPAS

Chase Time (hrs): 0 0 2 4 8 24 0 2 4 8 24 175 - ·

83

B

Stability of the Immature Band

120

100

80 Clc: '2 '(ij •WT E 60 Cl) D PAS a: 0~ 40

20

0 0 2 4 8 24 Chase Time (Hrs)

Figure 15. Pulse-chase analysis of the APAS mutant.

A. Transiently transfected cells were metabolically labelled with e5S] cysteine/methionine for 1 hour and chased at the indicated time intervals. The cell lysates were immunoprecitated by an ant-MYC antibody and subjected to 7.5% SDS-PAGE and autoradiography. Mock-transfected cells were also labelled for 1 hour and analyzed at the 0 hour chase time.

B. Densiometric analysis from four independent experiments illustrates the percentage of the immature fraction of the wild-type and L\PAS as a percentage of the expression level at chase time 0 hours. Asterisks (*) indicate statistically significant differences between L\PAS when compared to wild-type (p < 0.05).

56 CHAPTER3

GENERAL DISCUSSION AND CONCLUSIONS

57 The correct folding of proteins into their native three-dimensional conformation is fundamental to their biological function. This folding process is tightly coupled to the protein's stability and trafficking to its specific cellular location. Several diseases are caused by a loss of protein due to defective folding and degradation of mutant proteins

[100, 101]. The primary mechanism of disease causing mutations is disruption of protein folding or stability, which in turn leads to rapid degradation of the affected protein.

Defective folding and trafficking have been associated with several mutations in HERO that cause the long QT syndrome. The majority of these mutations have been identified in transmembrane domains, the pore region and the cyclic nucleotide binding domain of

HERO. However, relatively little is known about the mechanisms underling mutations in the PAS domain, aside from their affects on channel gating. Understanding the molecular mechanisms in which these mutations cause the LQTS phenotype is imperative to developing successful therapeutic strategies.

Novel elements and potential significance

In this thesis, we have associated three LQTS mutations in the PAS domain with defective trafficking due to misfolding. At physiological temperature the mutants, G53R,

E58Kand I96T, are retained in the ER and fail to acquire complex oligosaccharides in the

Golgi apparatus. Western blot analysis of the mutants incubated at 27°C reveals that partial restoration of the mature band can be attained. However, functional analysis depicts a significant increase in tail current density, which is not in complete accordance with the biochemical data. This discrepancy can be attributed to the limitation of Western blots (i.e. the antibodies) at quantitatively detecting protein levels. Patch-clamp analysis is a more sensitive technique that enables the detection of increased surface expression that is not

58 completely discernable by Western blot. Lower temperatures may facilitate improved channel folding by increasing ER retention times and inhibiting proteosomal degradation

[100]. These findings imply that misfolding may be an underlying mechanism of impaired maturation and surface expression of the LQTS mutants. 053R, E58K and 196T are capable of homotetrameric assembly; therefore, the mechanism of ER retention must due to the overall misfolding of the tetrameric channel. The absence of 053R, E58K and 196T from the cell surface suggested a defect in the stability of the channel. Through pulse chase experiments we were able to demonstrate that mutations in the PAS domain of HERO compromise the stability of the mutant channel. All three mutants had an increased turnover rate when compared to wild-type. Interestingly, the 196T mutant reaches the cell surface briefly before being targeted for degradation. This study is the first to associate stability defects to mutations in the PAS domain.

The QC mechanisms in place in the ER ensure that correctly folded proteins reach their final destination. Misfolded proteins are targeted to degradation by the 26S proteasome by mechanisms that are not fully understood. The PAS domain consists of a highly conserved a/~ fold. We demonstrate that LQTS-associated mutations that occur in structurally conserved residues disrupt the folded conformation of this domain which leads to ER retention and degradation of the channel. To further validate this argument, we systematically deleted each alpha helix and beta sheet that constitutes this fold. Deletion of any of the four alpha helices or five beta sheets resulted in ER retention of the mutant channel. This result confirms that a correctly folded PAS domain is required for cell surface expression of HERO. Surprisingly, complete removal of the PAS fold allows

HERO to be efficiently expressed at the cell surface. The question as to why .1PAS is more

59 efficiently expressed cannot be easily explained in the experiments. We speculate that

6PAS, which lacks an intricately folded domain, may bypass the ER quality control more readily than wild-type. However, once the 6PAS mutant reaches the cell surface it is subsequently targeted for degradation. Further experiments are required to investigate what signals AP AS for degradation when expressed at the cell surface.

Future Directions

We have demonstrated that mutations in the PAS domain are associated with defective protein processing, which results in their absence from the cell surface. ·

Functional studies using Xenopus oocytes to examine PAS domain mutations demonstrate that these mutants have an accelerated rate of deactivation [70]. It would be of interest to study the biophysical characteristics of G53R, E58K and I96T when restored to the cell surface at 27°C using a mammalian expression system.

From our results it is clear that the mutants G53R, E58K and I96T are retained in the ER due to misfolding. However, the exact mechanism in which these mutant proteins are recognized as misfolded in not evident. The cytosolic chaperones, Hsp 70 and Hsp 90 have been implicated in HERG maturation [11]. Characterization of the chaperone complexes with G53R, E58K and I96T is required to further elucidate the mechanisms behind ER retention of these mutants.

In chapter 2, we demonstrate that the mutants I96T and APAS are only temporarily expressed at the cell surface due to subsequent targeting for degradation. The mutants

G53R and E58K are not expressed at the cell surface due to rapid turnover kinetics in the

ER. Elucidation of the exact mechanism by which the protein quality control system

60 functions to recognize these proteins as targets would be useful in the generation of treatment strategies for LQTS.

Conclusions

The results described in this thesis make new contributions to the understanding of the mechanisms underlying LQTS-associated mutations in the PAS domain. Mutations that alter the conserved a/~ fold of the PAS domain lead to reduced cell surface expression and stability of the mutant HERG channeL The PAS domain is not required for cell surface expression of HERG; however, its absence comprises the stability of the channel. The data prese11ted in this thesis provides new information on the role of the PAS fold on HERG maturation and stability. These findings demonstrate that disruption of the conserved PAS fold in HERG gives rise to subtle folding defects that result in the channel's absence from the cell surface.

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76 APPENDIX

77