The Muscle Specific Chloride Channel Clc-1 and Myotonia Congenita in Northern Finland

D959etukansi.kesken.fm Page 1 Monday, December 10, 2007 4:31 PM

apponen OULU 2008 D 959

UNIVERSITY OF OULU P.O. Box 7500 FI-90014 UNIVERSITY OF OULU FINLAND UNIVERSITATIS OULUENSIS ACTA UNIVERSITATIS OULUENSIS ACTA D SERIES EDITORS Hinni Papponen MEDICA

ASCIENTIAE RERUM NATURALIUM Professor Mikko Siponen THE MUSCLE SPECIFIC BHUMANIORA CHLORIDE CHANNEL Professor Harri Mantila CLC-1 AND MYOTONIA CTECHNICA Professor Juha Kostamovaara CONGENITA IN NORTHERN DMEDICA Professor Olli Vuolteenaho FINLAND

ESCIENTIAE RERUM SOCIALIUM Senior Assistant Timo Latomaa

FSCRIPTA ACADEMICA Communications Officer Elna Stjerna

GOECONOMICA Senior Lecturer Seppo Eriksson

EDITOR IN CHIEF Professor Olli Vuolteenaho

EDITORIAL SECRETARY Publications Editor Kirsti Nurkkala FACULTY OF MEDICINE, DEPARTMENT OF ANATOMY AND CELL BIOLOGY, DEPARTMENT OF NEUROLOGY, ISBN 951-951-42-8691-9 (Paperback) FACULTY OF SCIENCE, ISBN 978-951-42-8692-6 (PDF) DEPARTMENT OF BIOCHEMISTRY, ISSN 0355-3221 (Print) UNIVERSITY OF OULU ISSN 1796-2234 (Online)

ACTA UNIVERSITATIS OULUENSIS D Medica 959

HINNI PAPPONEN

THE MUSCLE SPECIFIC CHLORIDE CHANNEL CLC-1 AND MYOTONIA CONGENITA IN NORTHERN FINLAND

Academic dissertation to be presented, with the assent of the Faculty of Medicine of the University of Oulu, for public defence in Auditorium A101 of the Department of Anatomy and Cell Biology, on January 18th, 2008, at 12 noon

Supervised by Docent Kalervo Metsikkö Professor Raili Myllylä Professor Vilho Myllylä

Reviewed by Docent Antero Salminen Professor Timo Takala

ISBN 951-951-42-8691-9 (Paperback) ISBN 978-951-42-8692-6 (PDF) http://herkules.oulu.fi/isbn9789514286926/ ISSN 0355-3221 (Printed) ISSN 1796-2234 (Online) http://herkules.oulu.fi/issn03553221/

Cover design Raimo Ahonen

OULU UNIVERSITY PRESS OULU 2008 Papponen, Hinni, The muscle specific chloride channel ClC-1 and myotonia congenita in Northern Finland Faculty of Medicine, Department of Anatomy and Cell Biology, Department of Neurology, University of Oulu, P.O.Box 5000, FI-90014 University of Oulu, Finland, Faculty of Science, Department of Biochemistry, University of Oulu, P.O.Box 3000, FI-90014 University of Oulu, Finland Acta Univ. Oul. D 959, 2008 Oulu, Finland

Abstract Functional defects in the muscle specific chloride channel ClC-1 result in reduced chloride conductance and electrical hyperexcitability, which in turn impairs muscle relaxation and leads to myotonia. The gene CLCN 1 codes for ClC-1 in humans, and mutations in CLCN 1 cause the disease known as myotonia congenita. Worldwide over 80 mutations in CLCN1 have been described, but only three were found in patients in Northern Finland. These included two missense mutations and a nonsense mutation. The behavior and localization of the normal and mutated ClC-1 mRNA and protein were analyzed in muscle cell cultures. In intact muscle the ClC-1 protein was seen in the sarcolemma, but after myofiber isolation the protein was located intracellularly. Sarcolemmal localization was restored when myofibers were electrically stimulated or treated with a protein kinase C inhibitor. When mutated ClC-1 proteins were examined in a myofiber cell culture system, retardation in the ER was observed with the two missense mutations. The nonsense mutation did not have an effect on the transport from the ER to the Golgi elements, but the mutated ClC-1 was degraded more rapidly than the wild type ClC-1, at least in myotubes. Both retardation and degradation of the mutated ClC- 1 are likely to result in too few channels present at the plasma membrane of the muscle cell to maintain normal physiological function. A very strict quality control in muscle cells was observed. The behavior and survival of multinuclear skeletal muscle cells is dependent on innervation and muscle activity, and the balance between the phosphorylation and dephosphorylation pathways modulates the function of muscle chloride channels.

Keywords: chloride channels, Finland, muscle fibers, mutation, myotonia congenita, protein transport, rats, sarcolemma

Papponen, Hinni, Lihasspesifinen kloridikanava ClC-1 ja myotonia congenita Pohjois-Suomessa Lääketieteellinen tiedekunta, Anatomian ja solubiologian laitos, Neurologian klinikka, Oulun yliopisto, PL 5000, 90014 Oulu, Luonnontieteellinen tiedekunta, Biokemian laitos, Oulun yliopisto, PL 3000, 90014 Oulun yliopisto Acta Univ. Oul. D 959, 2008 Oulu

Tiivistelmä Lihasspesifisen kloridikanavan ClC-1:n toiminnalliset virheet johtavat alentuneeseen kloridin johtumiseen solukalvon läpi ja lihassolun ylieksitoitumiseen. Tämän seurauksena lihaksen rentoutuminen vaikeutuu ja havaitaan myotoniaa, lihasjäykkyyttä. Pohjoissuomalaisesta potilasmateriaalista tautiin johtavia geenimutaatioita löytyi kolme erilaista. Poikkeuksellista havainnoissa on erilaisten mutaatioiden vähyys, mikä on tyypillistä suomalaiselle tautiperinnölle. Yhteensä tämän kloridikanavan mutaatioita on julkaistu yli 80 erilaista. Tutkiessamme normaalin ja mutatoidun ClC-1 lRNA:n ja proteiinin käyttäytymistä ja sijaintia lihassoluviljelmissä. Havaitsimme eron lihasleikkeiden ja eristettyjen myofiibereiden välillä. Lihasleikkeissä ClC-1 paikantui solun pinnalle sarkolemmalle, mutta eristetyissä myofiibereissä lähinnä solun sisälle. Stimuloimalla eristettyjä myofiibereitä sähkövirralla tai käsittelemällä proteiini kinaasi C inhibiittorilla, saimme kloridikanava-proteiinin siirtymään takaisin solun pinnalle. Proteiinitasolla kuljetuksessa on havaittavissa eroja. Aminohappomuutokseen johtavat pistemutaatiot aiheuttivat proteiinin jäämisen endoplasmiseen kalvostoon, kun taas ennenaikaisen stop-kodonin johdosta lyhentynyt proteiini kuljetetaan eteenpäin Golgin laitteeseen. Myotuubeissa tämä lyhentynyt proteiini kuitenkin hajotettiin nopeammin kuin normaali kloridikanavaproteiini. Sekä kuljetuksen hidastuminen että nopeampi hajotus johtavat tilanteeseen, jossa lihassolun solukalvolla on liian vähän kloridikanavia ylläpitämään lihaksen normaalia fysiologista toimintaa. Monitumaisten lihassolujen laaduntarkkailu havaittiin vielä monitahoisemmaksi kuin yksitumaisilla. Monitumainen lihassolu on riippuvainen hermoärsytyksestä ja lihasaktiivisuudesta. Lisäksi fosforylaatioon liittyvä signalointi on tärkeää ClC-1 proteiinin oikealle paikantumiselle lihassolussa.

Asiasanat: kloridikanavat, lihassolut, mutaatiot, myotonia congenita, proteiinien kuljetus, rotat, solukalvot, Suomi

Acknowledgements

This work was carried out at the Department of Biochemistry and the Department of Anatomy and Cell Biology, University of Oulu. I owe my deepest thanks to my supervisor-triplet; Docent Kalervo Metsikkö for introducing me the challenging world of muscle and cell biology, Professor Raili Myllylä for teaching me molecular biology research, and Professor Vilho Myllylä for bringing a clinical aspect to this work. I have been very lucky to have three distinguished supervisors who always have had time for me and my research, but who also gave me a lot of freedom to do my own decisions and this way have helped me to become a better researcher. I wish to express my sincere gratitude to the former head of the Department of Anatomy and Cell Biology, Professor Hannu Rajaniemi for providing me this opportunity to work at his department and to the present head Professor Juha Tuukkanen for his positive attitude towards everything. I also want to thank Professor Petri Lehenkari, Docent Anthony Heape and Doctor Ulla Petäjä-Repo for creating an excellent research environment. I am very grateful to Docent Antero Salminen and Professor Timo Takala for the efficient review of the manuscript and Doctor Deborah Kaska for revision of the language. I would also like to thank my co-authors and all colleagues, both former and present ones. The skilful staff of the two departments, Biochemistry and Anatomy, is warmly acknowledged, especially Paula Salmela who let me into her clean and well organized lab and has helped me many ways during these years. The two most recent doctors of our department, Satu Jalonen and Vesa Aaltonen deserve special thanks for sharing many feelings and for several good advices and hints during this process. Mikas next door (Kaakinen and Nevalainen) and our “coffee room occupants” are thanked for both scientific and non-scientific discussions during the lunch and coffee breaks. A bunch of girls/ladies/wives/mothers/biochemists/doctors: Tuula Kaisto, Minna Männikkö, Marja Nissinen, Heli Ruotsalainen, Tuire Salonurmi and Minna Valtavaara, are warmly acknowledged for sharing a lot of things. I have had so many precious and unforgettable moments with you, your friendship is invaluable. I want to thank my parents Ulla and Jorma, my sister Heli, my brother Jussi- Pekka and my cousin Meliina, the backround support has always been available. Finally, my deepest gratitude belongs to the three men of my life: Anssi, Sami and Teemu, you keep me going, or should I say running.

7 This work was financially supported by the Academy of Finland, the Emil Aaltonen Foundation, the Finnish Cultural Foundation and the Finnish Konkordia Foundation. Oulu 10.12.2007 Hinni Papponen

8 Abbreviations

9AC 9-anthracene-carboxylic acid Ach Acetylcholine ATP adenosine triphosphate BFA Brefeldin A BHK Baby hamster kidney cells BSA Bovine serum albumin BTX α-bungarotoxin C carboxy- Ca2+ calcium ion CBS Cystathionine-β-synthetase CF Cystic fibrosis CFTR Cystic fibrosis transmembrane conductance regulator Cl- Chloride ion ClC Chloride channel Clcn Gene name for murine chloride channel CLCN Gene name for human chloride channel COPII Coat protein II CPP p-chloro-phenoxy-propionic acid CSQ Calsequestrin DHPR Dihydropyridine receptor cDNA complementary deoxyribonucleic acid DMEM Dulbecco’s minimal essential medium DTT dithiothreitol EDL Extensor digitorum longus EndoH Endoglycosidase H ER Endoplasmic reticulum FDB Flexor digitorum brevis GABA Gamma amino butyric acid GLUT4 glucose transporter 4 GTPase guanosine triphosphatase Hek human embryonic kidney IF Immunofluorescence kDa kilodalton mRNA Messenger ribonucleic acid mAb Monoclonal antibody

9 MHC Myosin heavy chain MTJ Myotendous junction MW Molecular weight N amino- NBD Nucleotide binding domain NCL neuronal ceroid lipofuscinosis NMJ Neuromuscular junction OST Oligosaccharyl transferase pAb Polyclonal antibody PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PCR Polymerase chain reaction PFA Paraformaldehyde PKC protein kinase C PT Proximal tubule Sol Soleus SR Sarcoplasmic reticulum SFV Semliki Forest virus SDS Sodium dodecyl sulfate T- transverse TGN Trans Golgi network WT Wild type

10 List of original publications

This thesis is based on the following publications, which are referred to in the text by their Roman numerals:

I Papponen H, Toppinen T, Baumann P, Myllylä V, Leisti J, Kuivaniemi H, Tromp G & Myllylä R (1999) Founder mutations and the high prevalence of myotonia congenita in northern Finland. Neurology 53: 297-302. II Papponen H, Kaisto T, Myllylä VV, Myllylä R & Metsikkö K (2005) Regulated sarcolemmal localization of the muscle specific ClC-1 chloride channel. Exp Neurol 191: 163-173. III Papponen H, Nissinen M, Kaisto T, Myllylä VV, Myllylä R & Metsikkö K (2007) F413C and A531V but not R894X myotonia congenita mutations cause defective endoplasmic reticulum export of the muscle-specific chloride channel ClC-1. In press, Muscle Nerve.

12 Contents

Abstract Tiivistelmä Acknowledgements 7 Abbreviations 9 List of original publications 11 Contents 13 1 Introduction 15 2 Review of the literature 17 2.1 Skeletal muscle ...... 17 2.1.1 Myogenesis...... 17 2.1.2 General structure and function of myofibers...... 19 2.1.3 Muscle cell culture ...... 24 2.2 Chloride channels...... 25 2.2.1 Voltage-gated chloride channels (ClC)...... 25 2.2.2 Cystic fibrosis transmembrane conductance regulator (CFTR) ...... 36 2.2.3 Volume regulated chloride channels (VRAC) ...... 38 2.2.4 Calcium-activated chloride channels (CaCC) ...... 38 2.2.5 Intracellular chloride channels (CLIC)...... 39 2.2.6 Ligand gated chloride channels ...... 40 2.3 Myotonia congenita...... 40 2.3.1 Autosomal dominant form...... 41 2.3.2 Autosomal recessive form ...... 41 3 Aims of the study 43 4 Materials and methods 45 4.1 Patients...... 46 4.2 Myofiber cell culture...... 46 4.3 Confocal microscopy ...... 46 5 Results 49 5.1 Mutation analyses of the patients (I)...... 49 5.2 ClC-1 mRNA localization (III) ...... 50 5.3 ClC-1 protein (II, III) ...... 52 5.3.1 ClC-1 peptide antibody...... 52 5.3.2 ClC-1 protein in myoblasts...... 53 5.3.3 ClC-1 protein in myotubes ...... 54

13 5.3.4 ClC-1 protein in myofibers...... 55 6 Discussion 59 6.1 Tools used in the thesis...... 59 6.2 Myotonia congenita in Northern Finland ...... 60 6.3 ClC-1 mRNA in myofibers ...... 61 6.4 Sarcolemmal ClC-1...... 61 6.5 ClC-1 degradation in myotubes...... 63 6.6 Transport of ClC-1 from the ER to the Golgi elements ...... 64 7 Conclusions 65 References 67 Original publications 81

14 1 Introduction

Skeletal muscles are composed of long, multinucleated cells called myofibers, which are highly differentiated cells and therefore unique in structure. Sarcolemma, the plasma membrane of the myofiber is divided into different structural and functional domains. To organize and maintain muscle specific membrane structures and the mosaic structure of the sarcolemma, protein transport pathways are even more specialized in muscle cells than in normal mononucleated cells. The ClC-1 channel is expressed in skeletal muscle. It has a single channel conductance of 1 pS and it contributes to about 75% of the resting conductance of the muscle membrane because of its high open probability at negative membrane - potentials. Because Cl is passively distributed, i.e. ECl equals the resting potential, ClC-1 suppresses depolarizing inputs and stabilizes the membrane potential at its negative level, but contributes to depolarization during action potentials. ClC-1 exists as a dimer that forms a double-barreled channel. Each barrel, or pore, of ClC-1 has its own gate ('fast' or 'single pore' gate), and both pores are also gated simultaneously by another mechanism ('slow' or 'common' gate). The expression of ClC-1 in skeletal muscle appears to be dependent on the extent of innervation and muscle electrical activity. The channel is regulated by the extracellular and intracellular anion binding sites, extracellular and intracellular pH, intracellular nucleotides and phosphorylation/dephosphorylation pathways. Functional defects in ClC-1 result in electrical hyperexcitability (spontaneous, repetitive action potential firing), which in turn impairs muscle relaxation and leads to myotonia. Myotonia congenita can be inherited both in an autosomal recessive form (Becker type) and in an autosomal dominant form (Thomsen type). Over 80 mutations in this gene have been described, including point mutations, frame shifts, splice site mutations, deletions and premature transcription terminations and these are quite evenly scattered throughout the protein. The experimental part of this work was carried out to identify the mutations causing myotonia congenita in Northern Finland and to analyze the molecular basis behind the defects in muscle function in these patients. Myofiber cell culture was used to study the behavior and localization of normal and mutated ClC-1. A very strict quality control in myofibers was demonstrated. The wild type ClC-1 protein was not present on the sarcolemma until treated with protein kinase C (PKC) inhibitor or stimulated electrically. The mutations were shown to influence protein transport from the ER to the Golgi element and the stability of the protein.

16 2 Review of the literature

2.1 Skeletal muscle

All three muscle types, skeletal, cardiac and smooth, are composed of elongated cells specialized for contraction. Cross-striated skeletal muscle is the most abundant tissue in the vertebrate body. It is organized into muscles that are responsible for all voluntary movement (Tortora & Grabowski 2003). Each muscle is comprised of a variable number of contracting fibers formed by the fusion of a large number of myogenic progenitors and thus containing up to many thousands of nuclei. Most vertebrate muscles are composed of variable proportions of different (fast or slow) fiber types determining the appropriate force and duration of contraction. Connective tissue surrounds myofibers, bundles of myofibers and whole muscles. At the end of the muscle, the connective tissue continues as a tendon or some other arrangement of collagenous fibers that attaches the muscle to other structures, typically those of the skeleton, forming a myotendous junction (MTJ). Skeletal muscles are well supplied with nerves and blood vessels. Each myofiber receives a single terminal branch of an alpha efferent axon, which ends at a neuromuscular junction (NMJ). (Alberts et al. 2002, Tortora & Grabowski 2003.)

2.1.1 Myogenesis

All the skeletal muscles in the vertebrate body, with the exception of some craniofacial muscles, derive from progenitors present in somites. Each newly formed somite rapidly differentiates into a ventral sclerotome and a dorsal dermomyotome from which myogenic precursors originate. Some myogenic precursor cells give rise to the terminally differentiated, mononucleated muscle cells (myocytes) of the primary myotome. Only a fraction of myogenic progenitors terminally differentiate during primary myotome formation, steps involving different type of myoblasts (embryonic, fetal myoblasts and satellite cells) are needed to establish skeletal muscle as schematized in Fig.1. (Biressi et al. 2007.) Myoblasts fuse into myotubes in which the assembly of myofilaments, as well as the production of muscle specific proteins begins. During differentiation the organization of the cellular organelles and the plasma membrane of the myoblasts changes dramatically. This process involves

17 reorganization of the microtubular network characterized by the relocalization of microtubule nucleating sites at the surface of the nuclei in myotubes, in marked contrast with the classical pericentriolar localization observed in myoblasts. (Tassin et al. 1985.) Also new muscle specific organelles such as the sarcolemma, the sarcoplasmic reticulum (SR) and the transverse (T-) tubules are formed (Yuan et al. 1991, Flucher 1992). The innervation of muscles starts while fibers are still forming. Each muscle fiber is initially innervated by multiple axons, all but one of which are subsequently eliminated. Postnatally, all the muscle fibers that remain contacted by axon branches of an individual motor neuron are of the same type. It has been assumed that the nerve plays a role in generating fiber type (fast/slow) diversity. Adult muscle fibers are highly heterogeneous when classified based on speed of contraction (one slow/three fast, see Fig.1). (Biressi et al. 2007.) The boundaries between these different classes of adult fibers are not absolute and intermediate fibers are common (Bottinelli & Reggiani 2000). Furthermore, fiber types are not fixed, but can change in response to several stimuli (Pette & Staron 1997).

Fig. 1. A proposed schematic model of skeletal muscle development. (Modified from Biressi et al. 2007).

Populations of muscle progenitors reside as quiescent satellite cells in the basal lamina of adult muscles. These adult progenitors arise from somite-derived progenitor populations in embryonic and neonatal muscle. In their nonproliferative state they express regulatory genes, which are essential for satellite cell formation. These cells can be activated if the muscle tissue is

18 damaged or in response to further growth demands. In these cases satellite cells activate differentiation genes and undergo a number of cell divisions producing fusion competent cells that can either fuse with damaged fibers or form new ones, and other cells that return to quiescence, thus maintaining the progenitor pool. (Pownall et al. 2002, Biressi et al. 2007.)

2.1.2 General structure and function of myofibers

Skeletal muscle consists of myofibers that are large, cylindrical multinuclear cells surrounded by a plasma membrane, the sarcolemma. Myofibers are 10 to 100 μm in diameter and up to several centimeters long. Most of the nuclei of the myofiber are located peripherally, just under the sarcolemma. The structure of the myofiber and the sarcomere is illustrated in Fig. 2. The fibers are further composed of myofibrils, membranes and a cytoskeletal network, which anchor the contractile fibrils to the sarcolemma. The basic contractile unit of myofibrils is the sarcomere that includes the thin, thick, titin and nebulin filaments. In skeletal muscle the sarcomeres of adjacent myofibrils are aligned, which generates the striated appearance of these tissues in microscopic preparations. The light band is termed the I-band because it is isotropic in polarized light; the dark band is known as the A-band because it is anisotropic. The principle components of striated muscle sarcomeres include actin-containing thin filaments that span the I-band and overlap with myosin-containing thick filaments in the A-band. The third filament system is made up of single molecules of titin (the largest vertebrate protein identified to date), which span half sarcomeres. Another giant protein, nebulin, spans the length of the actin filaments and forms the fourth filament system in skeletal muscle. The Z-lines (also called Z-band or Z- disc) represent the lateral boundaries of the sarcomere where the thin, titin, and nebulin filaments are anchored. One sarcomere is the area between two Z-lines, and thus the Z-line connects the actin filaments from neighboring sarcomeres. (Clark et al. 2002, Kierszenbaum 2007.)

19 Fig. 2. The structure of a skeletal muscle cell, a myofiber. Sarcomeres composed of myofibrils that are surrounded by sarcoplasmic reticulum. Near A-I junctions terminal cisternae and T-tubules form triads. (Modified from Rogers 1983).

Filamentous actin and myosin are responsible for contraction. In sarcomeres, the fast-growing barbed ends of actin polymers point toward the Z-lines (Ishikawa et al. 1969). In muscle, actin interacts with tropomyosin and troponins, which are important in the regulation of muscle contraction (Poole et al. 2006). It is essential that the actin length remains the same in sarcomeres. In the myosin superfamily there are 15 classes. As a general feature, myosins can bind actin and act as motor proteins. Head and tail regions constitute the myosin molecule. The head region is the motor domain binding to actin, hydrolyzing ATP and generating movement. The myosin tail region contains a coiled-coil forming region, which enables the dimerization of two myosin heavy chains in a bipolar order. In striated muscle, these hexameres are bundled together by parallel interactions along the filaments to form thick myosin filaments. A single thick filament in a sarcomere can contain up to 600 myosin molecules. Binding of myosin to actin is controlled

20 by the troponin complex in a calcium-dependent manner. (Clark et al. 2002, Au 2004.) Myosin heavy chain (MHC) isoforms appear to represent the most appropriate markers for fiber type delineation. Pure fiber types are characterized by the expression of a single MHC isoform whereas hybrid fiber types express two or more MHC isoforms. In adult mammalian skeletal muscle, the following pure fiber types exists: slow type I with MHCIβ, and three fast types, type IIA with MHCIIa, type IID with MHCIId and type IIB with MHCIIb. Muscle fibers are dynamic structures capable of altering their phenotype under various conditions. (Pette & Staron 2000.)

Sarcolemma and its specific compartments

The plasma membrane of the myofiber, the sarcolemma, is divided into different structural and functional domains. Lipid rafts are small heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that are formed by lipid-lipid interactions that compartmentalize cellular processes. (Simons & Ikonen 1997.) Caveolae, flask-shaped invaginations of the plasma membrane, are particularly abundant in muscle cells, and their coat contains a specific isoform of caveolin, caveolin-3. Caveola formation involves oligomerization and association with cholesterol-rich lipid-raft domains. Accordingly, caveolae represent a specialized, morphologically distinct sphingolipid-cholesterol microdomain, which is stabilized by the caveolin protein. (Parton & Simons 2007.) Caveolin-3 is associated with T-tubule formation during muscle development (Parton et al. 1997) and also with T-tubules of mature muscle fibers (Ralston & Ploug 1999). The T-tubule system is a network of tubular invaginations of the sarcolemma that forms specific, functional associations with the sarcoplasmic reticulum (SR). These anatomical junctions are called triads. The T-tubule system of mammalian cells is an extensive membranous system that penetrates the entire muscle fiber but is continuous with the muscle plasma membrane. (Flucher 1992.) The T-tubule system is responsible for the rapid inward spread of excitation from the sarcolemma to the inside of the muscle fiber as well as for signaling to the SR, which initiates calcium release that, in turn, activates the contractile elements (Block et al. 1988, Stephenson 2006). T-tubules, like caveolae are known to be enriched in cholesterol (Hidalgo et al. 1983, Parton & Simons 2007). Both caveolae and T-tubules represent membrane systems that are continuous with the plasma membrane but have a distinct composition. The entire muscle sarcolemma

21 seems to be an organized array of caveolin-associated raft and nonraft domains comprising the mosaic T-tubule domains, sarcolemmal caveolae and β- dystroglycan domains. (Rahkila et al. 2001.) The neuromuscular junction (NMJ) is a complex structure that serves to efficiently communicate the electrical impulse from the motor neuron to the skeletal muscle to signal contraction via the chemical transmitter acetylcholine (ACh). The NMJ has three major structural elements: the presynaptic nerve terminal that is capped by a terminal Schwann cell, the synaptic basal lamina that occupies the synaptic cleft and the specialized postsynaptic membrane of the muscle. ACh-receptors are anchored at the crest of the secondary synaptic folds, the deep infoldings of the sarcolemma. ACh-receptors are connected to the sarcolemma by α- and β-dystroglycans, two members of a complex group of dystrophin-associated proteins, which are concentrated at the NMJ. The proteins rapsyn and utrophin and the sodium channels are present at the NMJ. (Lomo 2003, Hughes et al. 2006.) The myotendous junction (MTJ) is a functionally distinct compartment of the subsarcolemmal and extracellular matrix domains. The MTJ is specialized to transmit the force generated by the contracting sarcomeres to the adjacent tendon, and requires extreme structural stability. The MTJ sarcolemma is extensively folded into digit-like processes that increase the surface of lateral contacts between the myofilaments and the membrane and also reduce the tension per unit area. (Berthier & Blaineau 1997.)

Protein transport to the plasma membrane

The secretory membrane system allows cells to regulate delivery of newly synthesized proteins, carbohydrates, and lipids to the cell surface. This system consists of the endoplasmic reticulum (ER), Golgi complex, plasma membrane, and tubulovesicular transport intermediates between the aforementioned structures. (Lippincott-Schwartz et al. 2000.) The ER is the starting point of the secretory pathway. It is subdivided into three domains, the nuclear envelope (NE), the smooth ER (sER) and the rough ER (rER), the latter being involved in the synthesis of secretory and membrane proteins. Besides the synthesis and quality control of membrane and secretory proteins, the ER participates in the regulation of intracellular calcium, as well as the synthesis of phospholipids, cholesterol and steroids. The nascent polypeptide chain is translocated across the ER membrane via the translocon, the highly

22 conserved macromolecular machinery. (Baumann & Walz 2001.) Newly synthesized proteins encounter luminal chaperones (e.g. binding protein BiP, calnexin, calreticulin and protein disulfide isomerase PDI), whose role is to facilitate the folding reactions necessary for protein maturation and oligomerization (Helenius et al. 1992). Folding begins cotranslationally and this means that for a typical protein, folding occurs vectorially from the N-terminus toward the C-terminus (Fedorov & Baldwin 1997). Completion of folding and oligomerization usually occurs after the release of the fully synthesized polypeptide from the ribosome (Chen & Helenius 2000). The ER plays an important quality control role in protein transport. Incorrectly folded and assembled proteins are retained in the ER or translocated to the cytosol. (Helenius 2001.) The precise composition of the ER channel(s) used to retrotranslocate terminally misfolded polypeptides into the cytosol is still unknown (Ruddock & Molinari 2006), but it has been shown that the retrotranslocation and proteasome- mediated degradation are coupled reactions (Mayer et al. 1998, Mancini et al. 2000, Molinari et al. 2002). After proper folding and oligomer assembly, the secretory cargo is selectively separated from ER resident proteins at ER exit sites. The cargo is packaged into COPII-coated vesicular carriers that are competent for fusion with Golgi membranes. (Bannykh et al. 1996.) The COPII coat complex mediates the formation of transport carriers. It consists of the Sar1 GTPase and the Sec23/Sec24 and the Sec13/Sec31 subcomplexes. Both stimulate the GTPase activity of Sar1, which itself triggers coat disassembly. (Lippincott-Schwartz et al. 2000, Aridor & Traub 2002, Tang et al. 2005.) The Golgi complex occupies a central position in the secretory pathway. Its main function is the addition of oligosaccharides to proteins and lipids. Transport across the Golgi elements is a discontinuous process involving vesicle budding and fusion between stationary Golgi compartments (cis-, medial-, and trans- Golgi). As the last station of the Golgi complex, the trans-Golgi network (TGN) plays a major role in directing proteins in the secretory pathway to the appropriate cellular destination (cell surface, intracellular endosomal membrane system). Proteins are carried to the plasma membrane in vesicular or tubular structures, which have an as yet unknown protein coat. Whereas vesicles containing material for constitutive release fuse with the plasma membrane once they arrive there, secretory vesicles in the regulated pathway wait at the membrane until the cell receives a signal to secrete and then fuse. (Lippincott-Schwartz et al. 2000, Alberts et al. 2002.) This directional membrane flow is balanced by retrieval

23 pathways that bring membrane and selected proteins back to the compartment of origin (Lippincott-Schwartz et al. 2000). Skeletal muscle cells have the usual membrane traffic pathways for partitioning newly synthesized proteins, internalizing cell surface receptors for hormones and nutrients and mediating membrane repair. However, in muscle, these pathways must be further specialized to deal with targeting to and organizing muscle-specific membrane structures, satisfying the unique metabolic requirements of muscle and meeting the high demand for membrane repair in a tissue that is constantly under mechanical stress. Specialized regulation of the membrane traffic of the GLUT4 glucose transporter, the function of dysferlin in muscle membrane traffic and repair, and the formation of NMJ and MTJ are examples of this. (Towler et al. 2004.)

2.1.3 Muscle cell culture

Satellite cells can be induced to proliferate by muscle injury and thus make up an enriched source of myoblasts that can be extracted from the muscle tissue and propagated in culture. Numerous muscle cell lines have been established from these stem cell populations, all of which maintain the ability to undergo myogenic differentiation in serum-deficient media. Established muscle cell lines like rat L6 or mouse C2C12 are commercially available. Such muscle cultures can be induced to differentiate into multinucleated myotubes that become striated. However, in general, they do not fully mature and therefore do not model mature muscle. (Neville et al. 1998.) Recently it was shown that electric pulse stimulation -induced repetitive Ca2+ transients accelerated de novo assembly of functional sarcomere structures in C2C12 myotubes (Fujita et al. 2007). Since differentiation in these cell lines is not complete, the situation in myotubes cannot totally correspond to that in mature myofibers. A method to isolate and culture mature myofibers on dishes was developed by Bekoff and Betz (1977). The expression of developmentally mature proteins and the absence of fetal proteins, in addition to the maintenance of normal calcium handling, highlights the FDB culture system as a more mature and perhaps more relevant culture system for the study of adult skeletal muscle function (Ravenscroft et al. 2007).

24 2.2 Chloride channels

Chloride ion (Cl-) is the most abundant anion in organisms. Because of this, anion channels are usually called chloride channels although these channels may - - - conduct other anions such as iodide (I ), bromide (Br ), and also nitrates (NO3 ), - - phosphates (PO4 ) and negatively charged amino acids. Cl channel functions range from ion homeostasis to cell volume regulation, transepithelial transport and regulation of electrical excitability. Cl- channels are regulated by a variety of signals and they may perform their functions in the plasma membrane or in membranes of intracellular organelles. (Nilius & Droogmans 2003.) Cl- channels may be classified according to their localization (plasma membrane versus vesicular), single channel conductance or mechanism of regulation. The most logical classification of Cl- channels will be based on their molecular structure. However, Cl- channels and genes have been identified that are not yet matched. So far, the molecular structures of three Cl- channel family members are known: ClC-, CFTR- and ligand gated Cl- channels. (Jentsch et al. 2002.)

2.2.1 Voltage-gated chloride channels (ClC)

It is known that ClC genes are present in organisms as diverse as animals, plants, yeast, archaebacteria and eubacteria. Some bacteria such as Escherichia coli have two ClC genes (Blattner et al. 1997) in contrast to the single ClC gene (gef1) in the eukaryote Saccharomyces cerevisiae (Greene et al. 1993). The mammalian ClC family consists of nine members. They show different tissue distribution and function both in plasma membranes and in intracellular organelles. Based on sequence homology they can be grouped into three branches (Figure 3). The first branch comprises plasma membrane channels, and, in the other two branches, channels predominantly reside in intracellular membranes but may be trafficked to the plasma membrane under special circumstances. (Jentsch et al. 1999). The ClC-family can also be grouped into channel and transporter subtypes, the former residing in plasma membranes and the latter in acidifying intracellular membranes (Miller 2006). Although channel-like properties in transport families or vice versa have been described before, the ClC proteins are the first family where channels and transporters coexist with a very similar and conserved structural core (Gadsby 2004). Table 1 summarizes the mammalian ClC family: the gene and its location in human chromosomes, tissue distribution, presumed functions and disease associated with the channel, respectively.

25 Fig. 3. The dendogram of the nine members of the mammalian ClC family.

Table 1. The mammalian ClC family of chloride channels.

Subtype Gene(location) Expression Function Human(murine)phenotype ClC-1 CLCN1 (7q35) skeletal muscle Sarcolemmal excitability Myotonia congenita ClC-2 CLCN2 (3q26- broad Epithelial Cl- transport? (retinal degeneration, qter) pH, volume regulation? male infertility) ClC-3 CLCN3 (4q23) broad synaptic vesicle (neurodegeneration, (brain, kidney, acidification, cell volume retinal degeneration) liver…) regulation ? ClC-4 CLCN4(Xp22.3) broad unknown (brain, muscle…) ClC-5 CLCN5 (Xp11.22) kidney Endosomal acidification Dent’s disease (intestine, liver…) ClC-6 CLCN6 (1p36) broad unknown ClC-7 CLCN7 (16p13) broad Bone resorption by Infantile malignant osteoclasts, lysosomal osteopetrosis acidification ClC-Ka CLCNKA (1p36) kidney, inner ear renal Cl- reabsorption (nephrogenic diabetes insipidus) ClC-Kb CLCNKB (1p36) kidney, inner ear renal Cl- reabsorption Bartter’s syndrome

All ClC proteins contain a conserved membrane-inserted catalytic pore domain, which is in many cases, followed by a large cytoplasmic domain. In vertebrates, the length of the cytoplasmic domain varies from 160 to 315 amino acids. (Meyer & Dutzler 2006.) The cytosolic carboxy terminus contains two CBS subdomains, CBS1 and CBS2 (Estevez & Jentsch 2002). A variable linker region is located between the two CBS domains and a C-peptide sequence that follows CBS2

26 (Meyer & Dutzler 2006). The crystal structure of bacterial ClC reveals that the protein is composed of 18 helices, 17 of which are membrane-associated. Most of these helices are not perpendicular to the membrane, but severely tilted (Fig. 4). The majority of mammalian ClC channels are glycosylated. The ClC proteins are homodimers of two identical subunits, and each subunit forms its own independent pore. (Dutzler et al. 2002, Dutzler et al. 2003.) The channels (most, but possibly not all) are gated in a voltage-dependent manner.

ClC-0

The activity of this Cl- channel was discovered and characterized by reconstituting electric organ membranes into lipid bilayers (Miller 1982). This first channel of a family of voltage-dependent chloride channels, ClC-0, was identified by expression cloning of the electric organ of the electric ray Torpedo marmorata. The clone is predicted to encode 805 amino acids and the calculated

MR of the protein is 89K. Amino acid sequence revealed several putative membrane-spanning domains, a hydrophilic N-terminal without a typical cleavable signal peptide, two potential N-linked glycosylation sites and a consensus phosphorylation site for cAMP-dependent kinase. (Jentsch et al. 1990.) The structure of the cytoplasmic domain of the Cl- channels was resolved from ClC-0 domain crystals. The strong conservation within CBS domains is evident, although there are large differences in the linker and the C-peptide, both being significantly longer in ClC-1 (Figure 4). (Meyer & Dutzler 2006.) The linker and C-peptide regions are extended in the closely related chloride channels ClC-0, ClC-1 and ClC-2 and show a high probability of being disordered. NMR spectroscopy experiments revealed that these two large sections of the protein, the linker and the C-peptide, show large conformational heterogeneity and fast dynamics on a nanosecond scale. Although the role of the two regions is currently still not understood, mutations were shown to have significant effects on channel function and on the interaction with regulatory proteins. (Alioth et al. 2007.) Gating of the channel is both voltage and Cl- dependent and the voltage dependence of the fast gate arises from interactions with the permeating anion rather than through an intrinsic voltage-sensing mechanism (Pusch et al. 1995, Chen & Miller 1996). The channel model is highly unusual; each of these pores can be gated independently by a process that is fast (millisecond scale = fast gating) and in addition, there is a common gate that closes both pores at the same time (second-to-minute scale = slow gating). Slow gating is highly temperature

27 dependent. (Jentsch et al. 2002.) A large movement in the C-terminus is associated with the slow gating of ClC-0. A physical separation of the C-termini between the two subunits is associated with the closure of the slow gate, but the reason why this movement affects the gating is not known. One possibility is that it is coupled to conformational changes in helix R. (Bykova et al. 2006.) The fast gating is suggested to be controlled via three ion binding sites of the narrow channel (Bisset et al. 2005, Cheng et al. 2007).

ClC-1

The skeletal muscle Cl- channel ClC-1 was cloned by homology to ClC-0 (Steinmeyer et al. 1991). It is probably the closest mammalian ortholog of the Torpedo channel since the electric organ is evolutionally derived from skeletal muscle. The large resting chloride conductance in skeletal muscle helps to stabilize the membrane potential and contributes to the repolarization current that terminates the action potential. ClC-1 contributes to the majority, about 70-80 %, of this resting membrane conductance of muscle ensuring its electrical stability. (Jentsch et al. 2002.) The predicted protein consists of 994 amino acids and a relative molecular mass MR is 110K, making it larger than the ClC-0. ClC-1 is predominantly expressed in skeletal muscle. Northern analysis also detected some expression of ClC-1 mRNA in kidney, liver, heart and smooth muscle. (Steinmeyer et al. 1991.)The expression of ClC-1 is normally lower in slow- twitch muscles than in fast-twitch muscles (Pierno et al. 2002), and during aging the expression of ClC-1 is reduced (De Luca et al. 1994, Pierno et al. 1999). The single cell RT-PCR technique has demonstrated ClC-1 expression in outer hair cells of rat cochlea (Kawasaki et al. 1999), and multiple isoforms of ClC-1 were identified in rat glioma C6 cells and astrocytes in primary culture (Zhang et al. 2004). Physiological experiments on skeletal muscle localize Cl- conductance to the T-tubular system (Dulhunty 1979, Coonan & Lamb 1998), but on the other hand immunocytochemistry has shown it to be specifically localized in the sarcolemmal membrane (Gurnett et al. 1995).

28 Fig. 4. Subunit organization and a model of domain structures of the ClC (Modified from Dutzler et al. 2002 and Meyer and Dutzler 2006).

The gating mechanism of ClC-1 is similar to ClC-0 (Accardi & Pusch 2000) including both fast and slow gating and three distinct Cl- ion binding sites in the selectivity filter (Aromataris & Rychkov 2006). Despite these similarities, the common gates in ClC-1 and ClC-0 have opposite voltage dependence, different time scales of operation and different temperature dependence (Jentsch et al. 2002). It has been suggested that the highly conserved amino acid residue glutamate (E232) may correspond to the fast gate of the channel. From analysis of the crystal structure, it appears that this gate must swing out of the way and this occurs when a Cl- ion from the extracellular solution enters the pore and displaces the glutamate side chain as a result of charge interactions. (Estevez & Jentsch 2002, Dutzler et al. 2003.) All three ion binding sites become occupied only upon opening and facing the high extracellular Cl- concentration (Lobet & Dutzler 2006). No structure has yet been identified that could represent the common gate. However, it is clear that common gating involves significant structural rearrangements of the protein, because the temperature dependence of this type of gating is unusually high. (Pusch et al. 1997, Duffield et al. 2003.) It is also clear that it involves interactions between two subunits and possibly between the carboxy tails. Mutations affecting the common gate have been found in different parts of protein including the carboxy tail, but such mutations seem to cluster on the interface between the subunits where the interaction is likely to occur. (Duffield et al. 2003.)

29 The carboxy tail of human ClC-1 (the CBS domains, the linker and the C- peptide) contains more than 390 amino acids, approximately 40% of the total, and many different functions are proposed for this part of the protein (Dutzler 2007). Only one CBS domain seems necessary for the basic ClC-1 function (Hryciw et al. 1998, Estevez et al. 2004), and a short sequence (amino acids 863-888) seems to be important for trafficking and plasma membrane insertion (Wu et al. 2006). The C-peptide has been shown to have an influence on channel function (Hebeisen et al. 2004), and truncation mutations deleting the C-peptide affect the structure of the poly-proline helix and the flexibility of the C-terminus (Macias et al. 2007). Research with yeast chloride channels has suggested that CBS domains could be involved in trafficking, oligomerization, and allosteric enzyme regulation (Schwappach et al. 1998, Jhee et al. 2000, Shan et al. 2001). ATP modulates the common gating of ClC-1 and the regulatory effect of ATP is mediated by the CBS domains. The putative ATP binding site is in a cleft between the two C-terminal CBS domains. ATP may exert its effect on common gating by allosterically modulating the interaction between the CBS and channel domain. (Bennetts et al. 2005.) Low intracellular pH also inhibits ClC-1 and in the presence of ATP intracellular acidosis inhibits ClC-1 by enhancing both ATP sensitivity and the maximal effect of ATP on common gating. ATP and protons act independently by stabilizing the closed state of the common gate, but also act cooperatively via a separate mechanism. H847 in the carboxy-terminal CBS domain of ClC-1 is a critical residue for the effects of both protons and ATP. The primary physiological significance of ATP modulation of ClC-1 may be in maintaining excitability and opposing fatigue by potentiating the inhibitory effect of acidosis on ClC-1. (Tseng et al. 2007, Bennetts et al. 2007.) There are ten potential sites for protein kinase C (PKC)-mediated phosphorylation in the human ClC-1 sequence and several of them are located in the C-terminus. 4β-phorbol esters, direct activators of protein kinase C, reduce macroscopic ClC-1 currents in heterologous expression systems specifically and this effect can be prevented with chelerythine, a specific blocker of protein kinase C. (Brinkmeier & Jockusch 1987, Rosenbohm et al. 1999.) The PKC activation can also be induced via cholera-toxin sensitive G-protein. The effects of both the phorbol ester and cholera toxin were inhibited by staurosporine, thus indicating that either direct or indirect (via G protein) activation of PKC accounts for the decrease of chloride conductance. (De Luca et al. 1994.) ClC-1 channel activity can be elevated by the protein kinase inhibitor, staurosporine, in isolated muscle fibers (Chen & Jockusch 1999). It has also been shown that activity of a

30 phosphatase contributes to maintaining the chloride channel in a conductive state and this dephosphorylation is IGF-1 mediated. The balance between the phosphorylation and dephosphorylation pathways modulates the function of muscle chloride channels. However, it is still unknown if this phosphorylation site is in ClC-1 or in a closely associated subunit. (De Luca et al. 1998.) Niflumic acid promotes PKC activity by mobilizing mitochondrial calcium stores and it also blocks ClC-1 channels directly leading to ClC-1 current inhibition (Liantonio et al. 2006). Disuse of muscle reduces PKC activity and increases chloride conductance, which leads to a reduction of muscle excitability and further muscle impairment (Pierno et al. 2007). Changes in chloride conductance due to ClC-1 channels are also involved in myofiber type transitions (Pierno et al. 2002, Agbulut et al. 2004, Pierno et al. 2007). Taurine is known to directly modulate ClC-channel activity through interaction with a low affinity site. Taurine increases chloride channel conductance and also modulates gating and the kinetics of the voltage-dependent sodium channel, with the overall effect of stabilizing the sarcolemma. (Camerino et al. 2004.)The organic substances 9-anthracene-carboxylic acid (9AC) and p- chloro-phenoxy-propionic acid (CPP) are known to inhibit ClC-channels. 9AC acts strictly from the intracellular side and its action is strongly voltage dependent. CPP is known to inhibit chloride conductance in a stereospecific manner, S(-) enantiomers being much more effective than the R(+) enantiomers. (Pusch et al. 2002.) Zn2+ inhibits the resting membrane conductance of skeletal muscle, most of which is due to Cl-. Mutations of three cysteine residues (positions 242, 254 and 546) have been found to affect the Zn2+ block in ClC-0 and ClC-1 and despite a 100-fold difference in apparent affinity, this block seems mechanistically similar in these channels. The Zn2+ block is highly temperature dependent and affects the common gating. (Duffield et al. 2005.) Human mutations in the CLCN1 gene lead to a disease called myotonia congenita, which is reviewed later in this thesis. Transdominant alterations in CLCN1 mRNA have been observed in muscle obtained from human dystrofia myotonica (DM) patients (Mankodi et al. 2002). Both types of dystrofia myotonica (DM1 and DM2) are caused by the expansion of tandem repeats (Brook et al. 1992). At the RNA level these expansions form stable hairpins that alter the pre-mRNA splicing activities of two antagonistic factor families. One family promotes the inclusion of specific fetal exons in embryonic and neonatal tissues, whereas postnatal activation of the other family leads to fetal exon skipping and expression of adult protein isoforms. This leads to aberrant splicing

31 of CLCN1 mRNA. Some of these aberrantly spliced transcripts are degraded through the nonsense-mediated decay pathway while others are exported and direct the translation of nonfunctional truncated ClC-1 protein products. (Kanadia et al. 2006, Lueck et al. 2007a, Lueck et al. 2007b.) Given the crucial role of chloride conductance in muscle physiology, the availability of specific ligands that modulate ClC-1 could be of potential medical as well as toxicological importance (Liantonio et al. 2007). ClC-1 has also been shown to be a target of therapeutic molecules with different clinical uses such as statins (Pierno et al. 1995), taurine (Pierno et al. 1998, Camerino et al. 2004) and non-steroidal anti-inflammatory drugs (NSAIDs) (Astill et al. 1996).

Other ClCs

ClC-2 is a broadly expressed Cl- channel that was cloned by homology to ClC-1 (Thiemann et al. 1992). The protein is organized in N- and C-terminal regions with regulatory functions and a transmembrane region which forms the ring of the pore. It can be activated by hyperpolarization, cell swelling, and extracellular acidification. It can also be activated by protein kinase A, arachidonic acid, amidation and activated omeprazole. It inactivates very slowly. Northern analysis detected its mRNA in every tissue and cell line examined, albeit at different levels. Brain, kidney and intestine express high levels of ClC-2. The activity of ClC-2 determines intracellular Na+ and Cl- concentration, and it is assumed that ClC-2 plays a role in membrane stabilization with hyperpolarizing transmission by GABA and glycine in neurons. (Jentsch et al. 2002, Nilius & Droogmans 2003, Suzuki et al. 2006.) In this respect, ClC-2 mutations have been found in patients with idiopathic generalized epilepsy (Haug et al. 2003) and CLCN2 may be a susceptibility locus in a subset of cases of childhood absence epilepsy (Everett et al. 2007). ClC-2 has also been proposed to be the molecular candidate for the native cardiac inward-rectifying chloride channels (Duan et al. 2000). A severe degeneration of the testis and the retina (Bosl et al. 2001) and also a widespread, progressive spongiform vacuolation of the white matter of the brain and spinal cord that was accompanied by glial activation has been observed in ClC-2 deficient mice (Blanz et al. 2007). The internalization of ClC-2 is dynamin dependent and endosomal trafficking through the early endosomal and the recycling endosomal compartments is partially dependent on the activity of the small GTPases; rab5 and rab11, respectively. ATP depletion, an experimental maneuver modeling metabolic stress,

32 causes upregulation of ClC-2 channel activity at the cell surface. In addition to changes in gating, the upregulation of ClC-2 function reflects increased surface stability due to a decrease in its endocytosis as ClC-2 trafficking is altered by ATP depletion. There are several potential protein targets (e.g. dynein, Hsp90) which may contribute to the ATP-dependent trafficking of ClC-2. (Dhani et al. 2007.) In the plasma membrane ClC-2 is partially associated with lipid rafts and channel activity is increased upon displacement from the former by either cholesterol depletion or oxidative/metabolic stress (Hinzpeter et al. 2007). Endosomal trafficking does provide a mechanism for the regulation of the functional expression of ClC-2 during metabolic stress, but the physiological role of ClC-2 channels remains uncertain. The disordered regions in the C-terminus are responsible for kinase-dependent activation of the ClC-2 channel (Cuppoletti et al. 2004). The N-terminus is relevant to gating, but its deletion does not lead to total loss of voltage dependence (Varela et al. 2002). It is not yet clear whether the gating process for ClC-2 is similar to that in ClC-0 and ClC-1 (Zuniga et al. 2004, Yusef et al. 2006). ClC-3 is expressed in many tissues, prominently in brain. It has been shown to undergo N-glycosylation generating various tissue-specific glycoforms (Schmieder et al. 2001). ClC-3 activity is controlled by cell swelling, PKC activation and calmodulin kinase II, and the protein appears to be localized in intracellular organelles, such as synaptic vesicles and endosomes, and is coexpressed with the v-type H+ATPase. (Nilius & Droogmans 2003.) It is assumed that it contributes to vesicular acidification by providing an electrical shunt (Hara-Chikuma et al. 2005). ClC-3 exists in at least in two isoforms that result from splicing variations and. contains several potential trafficking motifs, but their significance has not been determined. Adaptor protein mediated mechanisms control the sorting of membrane proteins into synaptic vesicles, and it is shown that the adaptor complex AP-3 regulates the subcellular distribution of the ClC-3 channel (Salazar et al. 2004). The short form of ClC-3 is primarily intracellular (late endosomes and lysosomes), but a small fraction is present on the plasma membrane. The plasma membrane component is rapidly internalized in association with clathrin. This endocytosed ClC-3 either enters a recycling pool or slowly traffics to a distal compartment with characteristics of late endosomes or lysosomes. Internalization is dependent on the N-terminal dileucine cluster. (Zhao et al. 2007.) The N-terminus of the ClC-3 channel is also proposed to be important in regulation of the channel function (Rossow et al. 2006). The longer form of ClC-3 is localized to the Golgi apparatus (Gentzsch et al. 2003). ClC-3

33 knockout mice were viable but smaller than wild type littermates. They showed a selective degeneration of the hippocampus, but were able to acquire motor skills and survived for more than a year. In addition they completely lost their photoreceptors. (Stobrawa et al. 2001.) ClC-3 has biophysical properties similar to ClC-4 and ClC-5 that are known to operate as voltage-dependent Cl--H+ exchangers (Scheel et al. 2005, Picollo & Pusch 2005). ClC-4 behaves as a Cl--H+ exchanger (Scheel et al. 2005, Picollo & Pusch 2005). It has been reported to colocalize with the cystic fibrosis transmembrane conductance regulator in the brush border membrane of epithelial intestinal cells (Mohammad-Panah et al. 2002), with ClC-5 in early endosomes (Mohammad- Panah et al. 2003) and with the copper transporter ATP7B in the trans-Golgi network (Wang & Weinman 2004). Intracellular nucleotides are necessary for the full and continuous activity of ClC-4, but it is not known whether the nucleotide effect is directly on the ClC-4 channel or on another protein (Vanoye & George, Jr. 2002). In mice, expression is high in excitable tissues (nervous tissue, skeletal muscle) and the subcellular localization is ER/SR. In the N-terminus of ClC-4 is a stretch of amino acids that is both necessary and sufficient for targeting into the ER. (Okkenhaug et al. 2006). No disease causing mutations have been identified in the human ClC-4 gene (Ryan et al. 1999, Ludwig & Utsch 2004). ClC-5 belongs to the same sub-branch of ClC proteins as ClC-3 and ClC-4 and, as mentioned earlier, is a Cl--H+ exchanger and has functional properties similar to ClC-3 and ClC-4 (Picollo & Pusch 2005). ClC-5 is expressed in the proximal tubule and collecting duct of the kidney, although it has also been detected in the lung, liver, brain and intestine. Immunohistochemical studies show that ClC-5 is located in intracellular vesicles primarily of the endosomal and lysosomal pathway. (Hryciw et al. 2006, Jentsch 2007.) CLC-5 is N-glycosylated in a tissue specific manner. The non-glycosylated mutant is removed significantly faster from the cell surface compared to the glycosylated WT protein resulting in a reduced transport activity at the plasma membrane. (Schmieder et al. 2007.) The C-terminus of ClC-5 binds nucleotides (AMP and ATP), but does not hydrolyze ATP. As ClC-5 is believed to reside in endosomes, near the V-type ATPase, regulation in response to changes in the ADP/ATP ratio would be a possible scenario. (Wellhauser et al. 2006, Meyer et al. 2007.) The C-terminus has been shown to be important in trafficking of ClC-5 (the CBS2 domain) (Carr et al. 2003) and in internalization of the ClC-5 channel (proline-rich motif between CBS1 and CBS2 domains) (Schwake et al. 2001). Also a potential protein binding module (PDZ) resides in the C-terminus of ClC-5 (Hryciw et al. 2006). Human

34 mutations in CLCN5 lead to low molecular weight proteinuria, urinary loss of phosphate and calcium and frequently to kidney stones in a syndrome called Dent’s disease (Lloyd et al. 1996). A large number of disease-causing mutations have been identified and most of the missense mutations are found in the proposed dimer interface area. Hence, dimerization may be crucial for ClC-5 function. (Veizis & Cotton 2007.) In renal proximal tubule (PT) cells, ClC-5 colocalizes with a proton pump in apical endosomes.This suggests that a lack of ClC-5 might impair endosomal acidification and thereby compromise the reabsorption of filtered proteins that causes proteinuria. The lack of ClC-5 has an effect on the uptake of parathyroid hormone and vitamin D in PT; the outcome of this is two opposing mechanisms that determines whether there is an increase in serum vitamin D (active form) and hence hypercalciuria and kidney stones. (Maritzen et al. 2006, Jentsch 2007.) Two independent strains of ClC-5 knockout mice have been reported. Both lose low molecular weight proteins into the urine, but showed differences with regard to hypercalciuria. (Piwon et al. 2000, Wang et al. 2000.) The role of ClC-5 in the thick ascending limb and collecting duct have not been examined since tissue-specific knockouts are not yet available (Jentsch 2007). ClC-6 is the least understood mammalian ClC protein. It was cloned over 10 years ago (Brandt & Jentsch 1995) and its mRNA is broadly expressed (Brandt & Jentsch 1995, Kida et al. 2001). The ClC-6 protein is predominantly expressed in the nervous system, where it resides in late endosomes (Poet et al. 2006). It is N- glycosylated and associates with lipid rafts (Ignoul et al. 2007). In mice, disruption of ClC-6 gene leads to progressive damage of the central nervous system with features of neuronal ceroid lipofuscinosis (NCL), a subtype of human lysosomal storage disease. However, mutations in the ClC-6 gene were found in only 2 of 75 patients with late-onset NCL. (Poet et al. 2006.) Cloning of ClC-6 also led to the identification of ClC-7 (Brandt & Jentsch 1995). ClC-7 is mainly expressed in the osteoclasts in the bones and in neurons in the brain. Immunocytochemistry and subcellular fractionation demonstrated that ClC-7 is present in lysosomes and late endosomes in fibroblasts, which fits well with its presence in the ruffled membrane of osteoclasts. Mice deficient for ClC-7 develop severe osteopetrosis because their osteoclasts cannot secrete acid and thus cannot dissolve bone. These mice also display severe retinal degeneration. Mutations in the human CLCN7 gene cause a similar disorder, infantile malignant osteopetrosis. (Kornak et al. 2001.) ClC-7 requires Ostm1 as a β-subunit to support bone resorption and lysosomal function. Ostm1 requires ClC-7 to reach

35 lysosomes, and the stability of ClC-7 depends on its association with Ostm1. It is possible that the highly glycosylated Ostm1 protein shields ClC-7, the only mammalian ClC protein lacking N-linked glycosylation sites, from lysosomal proteases. (Lange et al. 2006.) The ClC-K (ClC-K1 and ClC-K2) channels were first identified in rat kidney (Adachi et al. 1994) and the human orthologues ClC-Ka and ClC-Kb were cloned shortly thereafter (Kieferle et al. 1994). Recently the structure of the cytoplasmic domain of the ClC-Ka channel was published. It revealed an interaction mode between two protein chains in a homodimeric molecule that is distinct from interactions seen in CBS motif containing domains from other protein families. (Markovic & Dutzler 2007.) Both channels require an accessory subunit (β- subunit) barttin for functional expression (Estevez et al. 2001). Barttin is found to bind to ClC-K2 via its transmembrane domains, which constitute the lateral surface of the ClC channel (Tajima et al. 2007). ClC-K channels are apparently the only ClC proteins that interact with barttin. Barttin carries a putative PY-motif in its C-terminus. Both channels are expressed at the plasma membrane of kidney epithelial cells and participate in transepithelial transport. The importance of ClC- K/barttin channels is illustrated by human genetic diseases and by transgenic mouse models. Mutations in the gene encoding barttin affect both ClC-Ka and ClC-Kb and cause type III Bartter’s syndrome in kidney and sensoneuronal hearing loss, whereas mutations in ClC-Kb cause type IV Bartter’s syndrome without sensoneuronal hearing loss since the ClC-Ka expressed in inner ear is still functional. ClC-Ka is primarily expressed in the medulla (the thin ascending limb of the loop of Henle) and ClC-Kb in the renal cortex (the thick ascending limb of the loop of Henle). The differential localization of ClC-Ka and ClC-Kb gives rise to two different renal phenotypes in knock-out mouse models, only one of which has been confirmed in humans. In mice the inactivation of ClC-K1 leads to nephrogenic diabetes insipidus. (Jentsch et al. 2005, Veizis & Cotton 2007.)

2.2.2 Cystic fibrosis transmembrane conductance regulator (CFTR)

Cystic fibrosis (CF), the most lethal genetic disease among Caucasians, is complicated by abnormal epithelial solute and fluid transport due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Anion flow through this channel is needed for normal function of epithelia. Without anion flow, water movement slows, and dehydrated mucus clogs ducts and collects in

36 the lungs where it ultimately fosters lethal bacterial infections. (Gadsby et al. 2006.) CFTR is a protein kinase A regulated plasma membrane chloride channel that belongs to the ATP-binding cassette (ABC) transporter superfamily. CFTR is the only member of this superfamily that functions as an ion channel. CFTR contains 12 predicted transmembrane helices and five cytoplasmic domains consisting of two nucleotide binding domains (NBD1 and NBD2) and a regulatory (R) domain containing numerous consensus phosphorylation sequences, six of which are used in vivo. It also contains a glycosylation site in an extracellular loop. (Nilius & Droogmans 2003.) The regulatory (R) region of the CFTR is intrinsically disordered and must be phosphorylated at multiple sites for full CFTR channel activity, with no one specific phosphorylation site required. In addition, nucleotide binding and hydrolysis at the NBDs of CFTR are required for channel gating. (Baker et al. 2007.) The human NBD1 structures solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces (Lewis et al. 2005). There are four main trafficking pathways in the life of the CFTR: the standard route from the ER to the plasma membrane after synthesis, endocytic retrieval from the plasma membrane to the early or sorting endosomes, recycling of endocytosed CFTR back to the cell surface and targeting the endocytosed CFTR for degradation (Ameen et al. 2007). The di-acidic exit code in the cytosolic tail of CFTR is used in COPII-dependent export of the CFTR from the ER to the Golgi elements (Wang et al. 2004). The CFTR is also regulated by interactions with various other proteins (e.g. Rab GTPases (Saxena & Kaur 2006)). Both the C-terminus and N-terminus provide an important regulatory site for interaction with proteins. In addition the CFTR plays a role in regulating other membrane channels, transporters and cellular functions. (Nilius & Droogmans 2003.) A ΔF508 mutation in the CFTR gene is the most common of more than 1000 mutations causing cystic fibrosis. It accounts for 70% of CF chromosomes in the United States. (Davis 2006.) Crystal structures of the channel show minimal changes in protein conformation between normal and the ΔF508 mutation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. (Lewis et al. 2005.) A ΔF508 variant fails to fold properly in the ER, is rapidly degraded by the ER quality control machinery and never reaches the cell surface (Ameen et al. 2007).

37 2.2.3 Volume regulated chloride channels (VRAC)

Cells need to regulate their volume in the face of several external and internal challenges. To regulate their volume, cells are endowed with various ion and organic osmolyte transport proteins that activate upon cell swelling or cell shrinkage. (Jentsch et al. 2002.) The volume regulated chloride channels (VRACs) can be activated not only by cell swelling, induced by dialyzing the cell with an intracellular solution of increased osmolarity or by challenging the cells with an extracellular hypotonic solution, but also under isoosmotic, isovolumic conditions by reducing intracellular ionic strength, by intracellular GTPγS, or by application of shear stress in endothelial cells. The mechanism by which the cell senses “changes in volume” or “ionic strength” and transduces this physical stimulus into the opening of VRAC is poorly understood. Several molecular candidates have been proposed for VRAC (including P-glycoprotein and ClC-3), but have been discarded, so the molecular identification of VRAC is still to come. (Nilius & Droogmans 2003.)

2.2.4 Calcium-activated chloride channels (CaCC)

The permeability of the plasma membrane for Cl- is modulated in many cell types by changes in the intracellular concentration of free Ca2+. Non-excitable cells, such as epithelial, endothelial and also blood cells, express calcium-activated chloride channels (CaCCs). They are also found in many excitable cells, including neurons, cardiac and smooth muscle cells. CaCCs participate in many different physiological processes such as transepithelial transport, excitability of neurons and muscle cells, and oocyte fertilization. Within the airways, CaCCs contribute to epithelial fluid secretion. The salient features of CaCCs are very similar in various cell types. They are activated in a voltage-dependent way by an increase in Ca2+ concentration, probably due to binding of Ca2+ to the channel. The CaCCs are characterized by a small single channel conductance and a permeation profile of SCN- > I- > Cl- > gluconate, which is different from that of ClCs. The kinetics of CaCCs are Ca2+ and voltage dependent, but the molecular nature of CaCCs is not yet resolved. Putative candidates are some recently cloned and related membrane proteins, including the endothelial adhesion proteins Lu-ECAM-1 (bCLCA2), the bovine

38 bCLCA1,2, murine mCLCA1-4, pig pCLCA1 and human hCLCA1-4 proteins. (Nilius & Droogmans 2003, Eggermont 2004, Hartzell et al. 2005.)

2.2.5 Intracellular chloride channels (CLIC)

The CLICs have been identified during the last few years. A striking feature of the CLIC family of ion channels is that they can exist in a water-soluble as well as membrane-bound state. The CLICs have a broad tissue distribution. Besides being found in plasma membranes they have been located in organelle membranes such as mitochondria, nuclear membranes, large dense core vesicles, trans-Golgi vesicles, secretory vesicles and the endoplasmic reticulum membrane. The CLICs are involved in a wide variety of fundamental cellular events including regulated secretion, cell division and apoptosis. (Cromer et al. 2002, Ashley 2003.) Seven members of the CLIC family have been identified so far: CLIC1 (a putative nuclear membrane channel), CLIC2, CLIC3 (MAP kinase-associated channel, CLIC4 (exo/endocytosis and apoptosis), CLIC5, p64 and parchorin. The p64 seems to be a splice variant of CLIC5, based on an analysis of human genome sequence databases. A link between the CLIC family and the glutathione S-transferase (GST) superfamily has been found. An obvious difference between CLIC1 and GSTs is that the former is a monomer whereas the latter are dimers. (Cromer et al. 2002.) The first crystal structure of a CLIC family member, CLIC1, has been determined. It is monomeric and folds into two domains: an N-terminal domain processing a thioredoxin fold and an all-helical C-terminal domain. (Cromer et al. 2002.) Coexpression of CLIC1 with CFTR in Xenopus oocytes leads to an increased cAMP-activated plasma membrane conductance compared to expression of CFTR alone. Based on this it can be hypothesized that CFTR regulates CLIC1 in a cAMP-dependent manner, leading to membrane insertion and plasma membrane channel activity of this protein. The regulation of CLIC1 by CFTR suggests that some aspects of the cystic fibrosis disease process may be due to a failure to activate CLIC channel activity. However, until the roles of CLIC1 in normal physiology are known, there is little basis for speculation as to what part of the cystic fibrosis phenotype may be attributable to dysregulation of CLIC1. (Edwards 2006.)

39 2.2.6 Ligand gated chloride channels

The ligand-gated ion channels permit cells to respond rapidly to changes in their external environment. They are particularly well known for mediating fast neurotransmission in the nervous system. Fast inhibitory neurotransmission in the mammalian central nervous system is mediated primarily by the neurotransmitters GABA and glycine. The glycine receptor (GlyR), the nicotinic acetylcholine receptor cation channel (nAChR) and the anion-permeable GABA type A and C receptors GABAAR and GABACR belong to this group. Members of this group have a common structure in which five subunits form an ion channel. (Jentsch et al. 2002.)

2.3 Myotonia congenita

Myotonia is a neuromuscular disorder characterized by the slow relaxation of the muscles after voluntary contraction or electrical stimulation. Myotonia is a common symptom in patients with dystrofia myotonica and in a group of disorders called channelopathies. Channelopathies are caused by mutations in the sodium, potassium or chloride channels that affect the muscle membrane. Functional defects in ClC-1 result in reduced chloride conductance and electrical hyperexcitability (spontaneous, repetitive action potential firing), which in turn impairs muscle relaxation and leads to myotonia. Stiffness, however, is, temporary and is most pronounced when muscle activity is initiated after a long period of rest. Repeated activity then relieves the situation. This warm-up effect is a conspicuous and so far unexplained feature of myotonia congenita. The warm- up effect is short-lived, wearing off after 5 minutes rest. There is considerable variation between the symptoms even of patients with identical mutations. These may be explained by other genetic factors, differential allelic expression and the influence of environmental factors. The only statistically significant effect on severity of symptoms is seen with gender; symptoms may be more pronounced in men with a recessive form of myotonia congenita. In humans myotonia congenita can be inherited both in an autosomal recessive form (Becker type) and in an autosomal dominant form (Thomsen type). Sometimes the inheritance is not clear. Over 80 mutations in this gene have been described, including point mutations, frame shifts, splice site mutations, deletions and premature transcription terminations. None of the mutations appears to have particularly elevated frequency compared to other mutations in the general population. Many more

40 recessive than dominant mutations have been found and these are quite evenly scattered throughout the protein. The phenomenon that the same pathogenic mutation may be found in dominant and recessive pedigrees is a peculiar and almost unique feature of myotonia congenita. The truncation mutation R894X is one of those found both in recessive and dominant pedigrees. Myotonia due to mutation in this muscle ClC-1 chloride channel gene is also found in goats, mice and other animals. Myotonia in goats is autosomal dominant whereas in mice it is autosomal recessive. (Pusch 2002, Colding-Jorgensen 2005, McKay et al. 2006, Kuo et al. 2006.)

2.3.1 Autosomal dominant form

A dominant form of myotonia congenita was described as early as 1876 (Thomsen 1876). The prevalence of the dominant disorder in Germany is 0.2 per 100 000 (Becker 1977). Conductivity measurements have shown a lack of function for several recessive ClC-1 mutants and all dominant mutants. Therefore, the dominant Thomsen disease is thought to result froma dominant negative effect upon dimer formation. Nearly all mutations that result in a dominant type of myotonia congenita shift the voltage dependence of ClC-1 common gating to more positive potentials. Dominant mutations cluster at the interface of the ClC-1 channel monomers (Duffield et al. 2003) and more precisely in exon 8 (Fialho et al. 2007).

2.3.2 Autosomal recessive form

The existence of a recessive form of myotonia congenita was convincingly proved by Dr. Becker (Becker 1966). The worldwide prevalence is about 1 per 100 000, but a 10-fold higher prevalence has been reported in Northern Scandinavia (Baumann et al. 1998, Sun et al. 2001). A proportion of myotonia congenita patients have been shown to be compound heterozygotes (Meyer- Kleine et al. 1995, Sun et al. 2001). Transient weakness is a clinical symptom that has been reported in some recessive patients, whereas it has not been reported in patients with dominant myotonia (Deymeer et al. 1998). Recessive mutations are generally assumed to result in mutant channels that are nonfunctional or at least have reduced function in such a way that the normal wild-type pore in a mutant:wt heterodimer complex is minimally affected. Surprisingly, expression of some mutations yields functional channels with biophysical properties only

41 slightly (V165G, F167L, F413C and V236L) or not at all (Y150C, Y261C and V327U) different from those of wild-type channels. (Pusch 2002, Colding- Jorgensen 2005.)

42 3 Aims of the study

The dominant form of myotonia congenita was first described as early as 1876 and the recessive form 90 years later. In the beginning of the 1990’s the ClC-1 protein and gene were characterized, linkage between the gene and the disease was established and the first mutations were described. Since then over 80 different mutations have been identified throughout the protein-coding region of the muscle specific chloride channel. Less is known about the regulation and cellular events of this protein and the mechanism by which the different mutations cause myotonic symptoms in muscle cells. The specific aims of this study were: 1. To search for mutations in the CLCN1 gene causing myotonia congenita in Northern Finland and to evaluate the genotype-phenotype correlations. 2. To clarify the localization of the normal ClC-1 mRNA and protein in muscle cells. 3. To examine if the mutations in ClC-1 affect mRNA or protein localization.

44 4 Materials and methods

A summary of materials and methods are given in tables 2, 3 and 4, and they have been described in detail in the original publications. Some pivotal methods are also shortly described.

Table 2. Experimental methods used in the thesis.

Method Used in Cell Culture Myoblast cell culture II, III Myotube cell culture II, III Myofiber cell culture II, III Viral infections II, III Electric stimulation II Staurosporine treatment II Molecular biology PCR I,III Sequencing I Southern blotting I Site directed mutagenesis II, IIII RNA in situ hybridization III Protein biochemistry Immunoprecipitation II, III SDS-PAGE II, III Pulse-chase labeling II, III EndoH digestion II Western blotting II, III Protein analyses Immunocytochemistry II, III Confocal laser scanning microscopy II, III

Table 3. ClC-1 constructs and viruses used in the thesis.

Construct Corresponding virus Used in pSFV1-myc-ClC-1 wt recSFV-myc-ClC-1 II, III pSFV1-myc-ClC-1 F413C recSFV-myc-ClC-1 F413C III pSFV1-myc-ClC-1 A531V recSFV-myc-ClC-1 A531V III pSFV1-myc-ClC-1 R894X recSFV-myc-ClC-1 R894X III

45 Table 4. List of antibodies and fluorescence markers used in the thesis.

Antibody Source/reference Used in ClC-1 rabbit pAb II II, III Myc mouse mAb Santa Cruz II, III DHPR mouse mAb Affinity Bioreagents II β-dystroglycan mouse mAb Novocastra II BODIBY-α-bungarotoxin Molecular Probes II GM130 mouse mAb BD BIosciences II GM130 rabbit pAb (Nakamura et al. 1995) III COPII rabbit pAb Affinity Bioreagents III DIG mouse mAb Roche III AlexaFluor488, goat anti mouseIgG Molecular Probes II, III AlexaFluor568, goat anti mouseIgG Molecular Probes II, III AlexaFluor488, goat anti rabbitIgG Molecular Probes II, III AlexaFluor568, goat anti rabbitIgG Molecular Probes II, III mAb monoclonal antibody, pAb polyclonal antibody

4.1 Patients

Twenty-four families originating from Northern Finland were included in this study, and 46 patients and 16 unaffected relatives were analyzed. The diagnoses were based on careful neurologic and electromyographic (EMG) examinations. Genomic DNA was isolated from the patients’ blood cells and further analyzed.

4.2 Myofiber cell culture

Myofibers were isolated from female Sprague-Dawley rat flexor digitorum brevis (FDB) muscles. The muscle was excised and treated with collagenase followed by trituration with a pipette. The isolated myofibers were cultured on dishes coated with Matrigel, in Dulbecco’s MEM containing 5% horse serum, in an atmosphere of 5% CO2. Each dish contained approximately 50-100 muscle fibers. Myofibers were used for experiments after overnight cultivation.

4.3 Confocal microscopy

Cells were fixed, permeabilized, blocked and stained with the appropriate antibodies. In the control samples, cells were left uninfected or the primary antibody was omitted. Stained samples were embedded in Mowiol 4-88 (Hoechst;

46 Frankfurt, Germany) with 2.5% 1,4-diazobicyclooctane as a fading inhibitor. Samples were examined with a Zeiss LSM510 confocal microscope equipped with argon and helium-neon lasers (Carl Zeiss Inc; Göttingen, Germany). A 100- fold oil immersion objective was used. Images were processed with Adobe Photoshop®.

48 5 Results

5.1 Mutation analyses of the patients (I)

Since myotonia congenita is relatively common in northern Finland (Baumann et al. 1998) we decided to analyze the CLCN1 gene of these patients. Southern analyses using total genomic DNA isolated from some of the patients and unaffected relatives, and ClC-1 cDNA probes did not detect large deletions, insertions or rearrangements in the CLCN1 gene. All 23 exons and the nearby flanking regions of the exons in the CLCN1 gene from the patients representing at least one patient from 24 families were sequenced. In the 46 patients only three different mutations were found. In exon 11 of the gene one nucleotide substitution, T to G, changed a codon of phenylalanine (TTC) to a codon of cysteine (TGC) at position 413 (F413C). In exon 15 a codon for alanine (GCG) changed to a codon for valine (GTG) at position 531 (A531V). Another C to T transition generated a premature translational stop codon from the codon for arginine at position 894 in exon 23 (R894X), causing truncation of the polypeptide by 95 amino acids. The three mutated alleles identified here were found in all possible genotypic combinations in the patients (Table 5). Consequently a high number of compound heterozygotes were found in the population. No statistically significant correlation between the clinical symptoms and DNA findings were found, but a clear male preponderance was observed among the patients.

Table 5. The combinations of mutations found in Northern Finnish patients.

Type of mutation Number of patients Relative frequency F413C/F413C 9 20 R894X/R894X 5 11 A531V/A531V 1 2 F413C/R894X 11 24 F413C/A531V 3 7 A531V/R894X 7 15 R894X/- 4 9 A531V/- 2 4 No mutation found 4 9

49 Seven additional sequence variants were detected, none of which segregated with the myotonia congenita disease. Four were silent mutations, i.e. third base changes of codons that did not cause substitutions of amino acids. The three other variants were A437T, P697L and P727L and were classified as polymorphisms based on previous reports (for review see (Pusch 2002).

5.2 ClC-1 mRNA localization (III)

The in situ hybridization method was used to verify that the viral expression system did not change the localization patterns of ClC-1 mRNAs (Fig. 5). The endogenous ClC-1 mRNA was localized around the nuclei and beneath the sarcolemma on frozen FDB muscle sections and in single isolated myofibers. When the wild-type or mutated ClC-1 transcript was expressed from the recombinant virus, the intensity of the staining was slightly higher and the perinuclear staining was less prominent than with the endogenous ClC-1. These findings indicate that the viral expression and the point mutations did not reduce the amount of the transcripts or change the normal localization suggesting that mRNA anomalies are not the cause of myotonia congenita, at least not in the case of these three mutations. Therefore the amount of mRNA and its localization should not affect the localization pattern of the respective proteins. These staining patterns were abolished when the anti-sense probe was replaced with a sense probe, indicating the specificity of the staining.

50 Fig. 5. The localization of mRNAs encoding ClC-1 protein in FDB muscle.

51 5.3 ClC-1 protein (II, III)

5.3.1 ClC-1 peptide antibody

Gurnett et al. (1995) have reported production of functional anti-ClC-1 antibodies. However, these antibodies are not available, and at the time we initiated these studies, no commercial antibodies against ClC-1 were available. Therefore we prepared anti-ClC-1 antibodies. At present some commercial antibodies are also available, however they have not proven reliable in our myofiber experiments. Antibodies against a synthetic peptide corresponding to the 15 last amino acids of the carboxyterminal sequence of ClC-1 were raised in two rabbits. Both of the immunized rabbits produced anti-ClC-1 antibodies. In western blotting the affinity-purified antibodies recognized a polypeptide of 130 kDa (Fig. 6A), corresponding to the size of ClC-1 (Gurnett et al. 1995). Cell lysates of EDL that represent the fast twitch type muscle showed a considerably higher intensity 130 kDa band than did lysates of the slow twitch type muscle Sol. No staining was seen when muscle cryosections from the myotonic ADR mouse (EDL, Sol) were analyzed, whereas normal mouse muscle exhibited prominent sarcolemmal staining (Fig. 6B).

52 Fig. 6. The specificity of the ClC-1 peptide antibody. A) Western blot indicating that purified anti-ClC-1 antibodies recognize a 130 kDa band from the lysates of EDL (fast twitch) and Sol (slow twitch) muscle. Molecular weight markers are indicated on the left. B) In normal mouse muscle cryosections the ClC-1 localizes to the sarcolemma of EDL and Sol muscle (left panel). In ADR-mouse no sarcolemmal staining was seen (right panel). Scale bar 10 μm.

5.3.2 ClC-1 protein in myoblasts

The established rat muscle cell line, L6, was used as a myoblast model. Immunofluorescence studies of recSFV-mycClC-1-infected L6 muscle cells with the anti-myc and anti-ClC-1 antibodies revealed a uniform infection of the cells. Confocal microscopic studies surprisingly indicated that ClC-1 was localized to the intracellular membranes in L6 myoblasts. No plasma membrane staining was observed.

53 5.3.3 ClC-1 protein in myotubes

Mononucleated L6 muscle cells can be induced to differentiate into multinucleated myotubes. Myotubes are more mature than myoblasts, but not fully differentiated and do not express endogenous ClC-1. Myotubes infected with a recombinant virus encoding wild type ClC-1 or each of the mutants were used in experiments where metabolic labeling was needed (pulse chase, endoH), since these methods are not sensitive enough to produce results with myofibers. In both myoblasts and myotubes confocal microscopic studies indicated that the myc-tagged ClC-1 was localized to the intracellular membranes. No plasma membrane staining was observed. The mutant proteins showed similar intracellular localization. (Fig. 7) According to the ClC-1 amino acid sequence there is one potential N-linked carbohydrate attachment site. Digestion with EndoH resulted in a slightly faster moving band when compared to the non-digested control sample, indicating that ClC-1 contains N-linked sugars. A subsequent 3h chase period did not result in EndoH resistance. (Fig. 8A) Each recombinant virus produced a single band when analyzed with SDS- PAGE and autoradiography, the wild type ClC-1, the F413C and the A531V viruses at 130 kDa, and the R894X viruses at 110 kDa as expected. It can be seen that the stability of the F413C mutant protein does not differ markedly from that of the wild type, but the mutant A531V and R894X proteins show instability during the 4h chase period. (Fig. 8B)

Fig. 7. The intracellular localization of ClC-1 in myotubes. Myotubes were infected with a recombinant virus encoding wild type ClC-1 or each of the mutants. Confocal sections are shown. Scale bars, 10 μm.

54 Fig. 8. The effects of endoH treatment (A) and chase period (B) on the ClC-1 protein. A) EndoH digestion results in a change of mobility indicating that ClC-1 is glycosylated. B) Two mutations (A531V and R894X) cause reduced stability of the ClC-1 protein. Mean intensities of duplicate bands are shown in arbitrary units. Change in intensity indicates change of the mean intensities between pulse and chase.

5.3.4 ClC-1 protein in myofibers

Myofibers are mature muscle cells that can be isolated and maintained in culture for few days. While in muscle cryosections ClC-1 was seen in the sarcolemma (Fig 6B) it surprisingly disappeared from the sarcolemma upon isolation of the fibers from their tissue environment (Fig. 9, left panel). After an overnight incubation with the protein kinase C (PKC) inhibitor, staurosporine, surface staining with the ClC-1 antibodies was clearly detectable in the isolated myofibers (Fig. 9, middle panel). Staurosporine had a similar effect on both noninfected and infected myofibers. Another PKC inhibitor, H7, had a similar but less profound effect. The sarcolemmal staining was also detectable after electrical stimulation of the myofiber cultures (Fig. 9, right panel). Electrical stimulation of infected fibers was not possible, since the myofiber cultures could not bear the stress of two exhausting treatments without severe damage.

55 Fig. 9. The results of stimulation experiments on myofibers. Both staurosporine and electric stimulation restores the sarcolemmal ClC-1 component. Confocal sections are shown. Scale bars, 10 μm.

It has been previously documented that sarcolemmal components are unevenly distributed, eg. β-dystroglycan is known to distribute on the sarcolemma as cross- striations and longitudinal stripes (Williams & Bloch 1999) and leave transverse tubule openings clear (Rahkila et al. 2001). Accordingly the sarcolemma comprises a mosaic structure. Upon recruiting ClC-1 to the sarcolemma, it was found that the distribution of ClC-1 was not uniform but comprised patches that were arranged in a cross-striated fashion and overlapped considerably with that of β-dystroglycan (Fig. 10A). When neuromuscular junctions were localized with α- bungarotoxin (BTX), it was seen that ClC-1 staining was more intense in neuromuscular junctions both in muscle cryosections and in isolated myofibers (Fig. 10B). When ClC-1 staining was compared with the staining of various marker proteins no colocalization was seen with the dihydropyridine receptor (DHPR) that marks the transverse tubules (Fig. 10C). In addition to the cross- striated staining pattern, the ClC-1 protein was seen as spots representing a typical Golgi staining pattern in myofibers (Rahkila et al. 1997). Double staining with the Golgi marker GM130 confirmed this (Fig. 10D). These findings indicate

56 that ClC-1 in cultivated myofibers is able to leave the ER, nevertheless it does not arrive at the sarcolemma unless stimulated electrically or chemically with staurosporine.

Fig. 10. Colocalization of ClC-1 with various markers. A) Sarcolemmal distribution of ClC-1 colocalizes with β-dystroglycan considerably. B) The intensity of ClC-1 staining is stronger in NMJ. C) Intracellular staining shows very little colocalization with the T- tubule marker DHPR. D) ClC-1 is transported from the ER to the Golgi elements, which are visualized with the Golgi marker GM130. Confocal sections are shown. Scale bars, 10 μm.

57 When compared with the wild type ClC-1 staining pattern, the F413C and A531V mutant proteins showed only the cross-striations over the I bands together with the perinuclear components. This pattern represents the ER of myofibers showing that the transport from the ER to the Golgi elements was severely reduced (Fig. 11). COPII which has been shown to colocalize with proteins blocked in the ER by treatment with Brefeldin A, was used as an ER-marker (Bannykh et al. 1996). It is notable that COPII also marks the Golgi elements, as reported earlier (Ralston et al. 2001). There was a faint Golgi staining present in a minor fraction of the fibers infected with the F413C mutation-encoding virus, but the difference between the mutant and wild type was striking. The A531V never labeled the Golgi elements. On the other hand, the R894X mutant, that lacks a C- terminal segment of the cytoplasmic portion, labeled the Golgi elements efficiently (Fig. 11) like the wild type ClC-1 indicating efficient trafficking from the ER to the Golgi elements.

Fig. 11. Colocalization of mutated ClC-1 protein with ER-exit site marker COPII. The missense mutations (F413C and A531V) cause transport block and the protein stays in the ER, whereas the nonsense mutation (R894X) does not affect the protein transport from the ER to the Golgi elements. Confocal sections are shown. Scale bars, 10 μm.

58 6 Discussion

6.1 Tools used in the thesis

A variety of mutations have been shown to cause the myotonia disease world wide. In this study we identified the mutations causing the myotonia disease in Northern Finland. We combined the Southern method and sequencing to delineate the mutations causing myotonia congenita in Northern Finland. Southern analysis was expected to reveal large deletions or rearrangements and direct sequencing is so far the most accurate method to detect small sequence variations. Sequencing of the whole gene was not feasible, so we decided to sequence all the exons and nearby exon flanking regions of the gene. Using this strategy we found three significant point mutations. In order to study how the point mutations are reflected at the mRNA and protein levels we used immunohistochemical localization studies as a key method. Immunochemistry is a valuable tool to study protein expression, but it has to be optimized and well controlled. We characterized the anti ClC-1 antibody very carefully (I) using peptide competition and a myotonic ADR-mouse as negative controls. We have also used two antibodies for detection of ClC-1. A myc-tag attached to the N-terminus of the ClC-1 also made it possible to use antibodies against myc for detection of ClC-1. In the case of the R894X mutated ClC-1 protein this myc-tag was the only possibility to detect mutated protein, since this mutation causes the truncation of the protein by 95 amino acids and our peptide antibody recognizes the last 15 amino acids of the polypeptide. Immunohistochemistry is not a quantitative method, thus comparison of the intensity of the immunosignal between samples is only semiquantitative. When infecting myofibers, there are always some cells that are not infected and these provide a reliable basis to evaluate background. These noninfected myofibers also serve as internal control cells and allow us to compare the intensity of the native ClC-1 protein and virally expressed protein when using anti-peptide antibodies. When antibody against the myc-tag was used, these internal controls were very useful to determine if the particular fiber was infected or not. In this study we have used L6 muscle cell line as a muscle model. Myoblasts of the rat L6 cell line are easy to cultivate and can be induced to differentiate into multinucleated myotubes. Although, in general, they do not fully mature (Neville et al. 1998). Since differentiation in these cell lines is not complete, the situation

59 in myotubes cannot totally correspond to that in mature myofibers. In experiments where metabolic labeling was needed (pulse chase, endoH) myotubes were used, since these methods are not sensitive enough to produce results with myofibers. The expression of developmentally mature proteins and the absence of fetal proteins, in addition to the maintenance of normal calcium regulation, highlights the FDB culture system as a more mature and more relevant culture system for the study of adult skeletal muscle function (Ravenscroft et al. 2007). However, the behavior and survival of multinucleated skeletal muscle cells is dependent on innervation and muscle activity (Conte-Camerino et al. 1985, Chen & Jockusch 1999), and this is the point were myofiber cultures differ from the intact muscle. Lueck et al. (2007b) concluded that the functional properties of ClC-1 channels are remarkably similar in Hek293 cells, myotubes and native FDB fibers. All cell models seem to be useful when studying molecular function, however, our results indicate that normal externalization of ClC-1 only occurs in the intact muscle where innervation and the basement membrane are present. Thus the advantages and disadvantages of each model and method used have to be considered before drawing final conclusions.

6.2 Myotonia congenita in Northern Finland

The present results elucidate the biological basis for the high prevalence of myotonia congenita in Northern Finland. Only three different mutations were identified in the ClC-1 in the study area. This can be explained with the population structure of this sparsely populated region with founding ancestors and a subsequent clustering of the mutations due to regional isolation. This is consistent with other known diseases of the Finnish disease heritage. It was later found that the same three mutations also dominated in patient populations from Northern Sweden and Northern Norway (Sun et al. 2001). The observed male preponderance of the disease may be partly due to chance occurrence, to a relatively high number of men with both alleles affected, or to yet unknown sex-related modifying factors. Some of the patients diagnosed with myotonia congenita did not have mutations in CLCN1. It is possible that the mutations of these patients are in regions of CLCN1 not analyzed in this study, such as the promoter region or intronic regions, or are other mutations not detectable by our Southern blot analysis. It is also possible that the A531V and R894X mutations can be expressed in a heterozygous form in some patients (i.e., as a dominant allele), as

60 has already been observed with the R894X mutation in other populations. So far there are no functional studies on the A531V mutation that would clarify the nature of this mutation.

6.3 ClC-1 mRNA in myofibers

The endogenous ClC-1 mRNA was localized around the nuclei and beneath the sarcolemma on frozen FDB muscle sections and in single isolated myofibers. This is typical for mRNA species encoding membrane proteins (Mitsui et al. 1997, Awad et al. 2001, Nissinen et al. 2005). Both the wild type as well as the mutated mRNAs also showed a perinuclear and subsarcolemmal localization when a Semliki forest virus expression system was used. The intensity of the staining was markedly higher, as expected for the recombinant virus, probably due to the fact that the perinuclear staining was less prominent than with the endogenous ClC-1 mRNA. It is known that when the hemagglutinin (HA) protein of the influenza virus is expressed from the recombinant vesicular stomatis virus encoding HA, the mRNA is produced in the cytoplasm and influenza virus mRNAs, including the one encoding the HA glycoprotein are produced in the nucleus. These two different mRNA localizations also affect the HA protein localization in myofibers (Kaakinen et al. 2007). The viral vector, SFV, which was used in this study did not distort the mRNA localization pattern of the wild type ClC-1. Therefore the localization pattern of the expressed ClC-1 protein should not be affected either. No anomalies in the localization of the mutated mRNAs were seen when compared to the wild type species.

6.4 Sarcolemmal ClC-1

There is some controversy about the localization of ClC-1. Physiological experiments on skeletal muscle have localized Cl- conductance to the T-tubular system (Dulhunty 1979, Coonan & Lamb 1998). Both previous (Gurnett et al. 1995) and our immunohistochemical results localizes ClC-1 in the sarcolemmal membrane. Interestingly, our data indicate that ClC-1 in intact muscle was not uniformly distributed on the sarcolemma but rather appeared as a mosaic. The distribution pattern followed that of β-dystroglycan that typically leaves the areas clear where T-tubules protrude. It is possible that glycerol treatment, which is used to seal the T-tubular system to eliminate their role in the measurements, also disrupts sarcolemmal Cl- conductance by facilitating ClC-1 internalization. In

61 skinned fibers that have lost the sarcolemma, the ClC-1 apparently internalized during the isolation procedure and was possibly incorporated into the T-tubular membrane after the fiber has been electrically stimulated. Another possibility is that ClC-1 is, in fact, present in T-tubules of intact skeletal muscle as well as in the sarcolemma, but is not visible with the antibody that has been used. The antibody binds to the residues in the carboxyl tail of ClC-1 and it is possible that when ClC-1 is localized in T-tubules this part of the carboxyl tail of ClC-1 is unavailable for antibody binding. One scenario is that ClC-1 has a T-tubule variant lacking the recognition segment of our antibody. ClC-3 has been reported to have two isoforms that result from splicing variations (Nilius & Droogmans 2003) and non-functional splicing variants of ClC-1 have been found in embryonic samples and in DM patients where tandem repeats in a different gene cause also missplicing of ClC-1 mRNA (Kanadia et al. 2006, Lueck et al. 2007a). As seen from our data, ClC-1 staining was also more intense at the neuromuscular junction than at other areas of the sarcolemma. Based on the findings from other members of the ClC family, several hypotheses can be drawn as to how muscle cells may maintain an uneven sarcolemmal distribution. In addition to splicing variants ClC-3 and ClC-5 also have tissue specific glycoforms (Schmieder et al. 2001, Schmieder et al. 2007) and in this respect it is interesting that ClC-1 is glycosylated. This glycosylation might differ in specific membrane compartments. ClC-6 and also ClC-2 are associated with lipid rafts (Hinzpeter et al. 2007, Ignoul et al. 2007). ClC-1 did not float (our unpublished results) suggesting it is a non-raft protein, leaving space for a raft associated chloride channel in muscle. ClC-Ks and ClC-7 channels require a β-subunit for proper function (Estevez et al. 2001, Lange et al. 2006), and so far these two β-subunits are the only ones known in the ClC protein family. It remains possible that ClC-1 has one or several β-subunits affecting trafficking to the plasma membrane compartment or stability or some other function of the protein. The mechanisms that maintain the uneven distribution of ClC-1 on the sarcolemma remain to be resolved. The behavior and survival of multinuclear skeletal muscle cells is dependent on innervation and muscle activity. In denervated fibers, ClC-1 current is drastically reduced (Conte-Camerino et al. 1985, Chen & Jockusch 1999). In agreement with these data electrophysiological studies have indicated that ClC-1 current at the plasma membrane of isolated myofibers is down-regulated when compared to freshly isolated intact muscles (Chen & Jockusch 1999), and it has been proposed that phosphorylation was involved in the process. Our present

62 study reveals that ClC-1 disappears from the sarcolemma upon isolation of the fibers, and this phenomenon obviously results in the downregulation of current. Our data with staurosporine indicate that ClC-1 targeting to the plasma membrane is dependent on phosphorylation-dephosphorylation signaling. However, Lueck and co-workers (2007b) were able to measure robust ClC-1 channel activity in both freshly dissociated FDB fibers and in primary mouse skeletal myotubes after nuclear injection of mClC-1 cDNA. Since these experiments provide no measure of ClC-1 channel activity before fiber isolation, significant ClC-1 internalization during the isolation procedure remains possible. The antibodies used in our analysis bind to residues in the cytoplasmic tails of ClC-1 and it is possible that this part of ClC-1 interacts with intracellular proteins, rendering ClC-1 unavailable for antibody binding. If this interaction is somehow phosphorylation dependent the staurosporine treatment is able to make the recognition site again available for antibody binding. It is also possible that changes of the extracellular matrix during the myofiber isolation process may stimulate pathways leading to internalization of the channel.

6.5 ClC-1 degradation in myotubes

In order to determine whether there are differences in the stability of mutant proteins we performed pulse chase experiment in L6 myotubes. During a 4h chase period the stability of the F413C mutant protein did not differ markedly from the wild type, but the mutant A531V and R894X proteins showed instability during the chase period. Previous findings indicate that in Xenopus oocytes the R894X mutant protein showed dramatically reduced expression levels when compared to the wild type ClC-1 (Macias et al. 2007). This can be explained by degradation. The results obtained with L6 myotubes are problematic because no ClC-1 proteins were exported. Functional surface expression has been shown in primary myotubes (Lueck et al. 2007b), but the amount of intracellular ClC-1 in this experiment is not known. It is possible that our methodology was not sensitive enough to reveal the surface expression due to more pronounced intracellular expression. Lueck et al. (2007b) also concluded that the functional properties of the ClC-1 channel are remarkably similar in Hek293 cells, myotubes and native FDB fibers, but they did not perform localization studies. The deactivation rate was slower in FDB fibers as would be expected. The protein degradation we have shown in myotubes is also likely to occur in myofibers, but at a slower rate.

63 6.6 Transport of ClC-1 from the ER to the Golgi elements

The worldwide F413C mutation, also common in Northern Finland, has been found to yield only a small shift (20 mV) in chloride conductance in a human embryonic kidney (Hek) cell line (Zhang et al. 2000). The finding indicates that, in spite of the mutation, the protein appeared at the plasma membrane in these mononucleated cells. It seems that mononucleated cells are able to transport the mutant proteins or that very inefficient transport to the plasma membrane is enough to show chloride currents in these cells. In the adult rat muscle cells the F413C mutation caused inefficient protein transport from the ER to the Golgi elements. The A531V mutated ClC-1 was also found to be blocked in the ER in our study, and the block was associated with decreased stability. It is not known whether the A531V mutant protein is functional if exported, since its conductivity properties have not been measured. A similar transport defect has been observed for the most common form of cystic fibrosis (∆F508) where a nucleotide triplet encoding phenylalanine is deleted in the CFTR gene. As a result, a di-acidic cytoplasmic ER exit code of the CFTR protein is masked (Wang et al. 2004) and the protein is not exported. The missense point mutations investigated in this study (F413C and A531V) localize into a membrane-spanning segment of the ClC-1 protein and are not expected to mask a possible cytoplasmic exit code. A more likely reason for the retardation in the ER is that the ClC-1 mutant did not fold correctly in the muscle cells and remained bound to chaperones. Investigations are in progress with pharmacological chaperones and their ability to repair folding defects e.g. in the case of cystic fibrosis transmembrane conductance regulator (CFTR). It will be important to determine whether pharmacological chaperones that can repair certain types of folding defects (Yang et al. 2003, Loo et al. 2005, Wang et al. 2006) can enhance transport of the F413C and A531V mutant proteins to the sarcolemma. The nonsense mutation R894X did not influence the transport from the ER to the Golgi elements, since the Golgi elements were intensively stained when this mutant protein was expressed in myofibers. However, staurosporine treatment did not have as strong an effect on the mutated proteins as it had with the wild-type ClC-1 suggesting differences in phosphorylation.

64 7 Conclusions

The presence of only few mutations in the Northern Finnish population is due to a founder effect and makes it possible to use DNA diagnostics to provide better genetic counseling for the patients with myotonia congenita in this area. The sarcolemmal localization of the ClC-1 channel is regulated in many ways; previous studies have shown regulation by extracellular and intracellular anion binding sites, extracellular and intracellular pH, and intracellular adenosine nucleotides. Here we have shown that phosphorylation and electrical activity are also important regulators. ClC-1 targeting to the plasma membrane is dependent on the physiological environment of the cell and this prerequisite is not fulfilled in isolated myofibers under culture conditions. We found a method to restore the physiological requirements for ClC-1 targeting by using staurosporine or electrical stimulation. This indicates that phosphorylation and dephosphorylation pathways are associated with targeting of the ClC-1 protein. Two of the three mutations (F413C and A531V) examined here affected the transport from the ER. Previously, it has been demonstrated that the F413C mutant protein yields nearly normal current in Hek cells, meaning that in mononucleated cells it is transported at least to some extent to the plasma membrane. The transport defect in muscle cells could explain the myotonic symptoms. On the contrary, truncated ClC-1 (R894X) was efficiently transported to the Golgi elements, but experiments with myotubes indicated reduced stability. Functional expression of the R894X mutation of the ClC-1 protein has indicated a large reduction though not a total loss of chloride conductance in Xenopus oocytes (Meyer-Kleine et al. 1995) and showed dramatically reduced expression levels when compared to the wild type ClC-1 (Macias et al. 2007), and these also could be explained with degradation. Both retardation and degradation are likely to result in too few channels present at the plasma membrane to maintain normal physiological function. Chloride channels have been relegated to the sidelines of ion channel research for many years, but have recently become one of the most exciting topics in channel research. The ClC family has offered many surprises during the past years: First, the phenomenon that the same pathogenic mutation may be found in dominant and recessive pedigrees is peculiar and almost a unique feature of the ClC-1 channel disease myotonia congenita. Second, the two subunits form a channel consisting of not just one pore, but two pores, one in each subunit. Third, the ClC proteins are the first family where channels and transporters coexist with

65 a very similar and conserved structural core. Probably the next surprise is waiting just around the corner.

66 References

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