Molecular genetics and electrophysiological studies of hypokalemic periodic paralysis (HypoPP) [Elektronische Ressource] / Chao Hang

Caohang

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Department of Applied Physiology University of Ulm Head: Prof. Dr. F. Lehmann-Horn Molecular genetics and electrophysiological studies of hypokalemic periodic paralysis (HypoPP) Dissertation Applying for Degree of Doctor Human Biology Faculty of Medicine, University of Ulm Presented by Chao Hang Yangzhou, P. R. China 2002, Ulm Amtierender Dekan: Prof. Dr. Reihard Marre 1. Berihterstatter: HD. Dr. Karin Jurkat-Rott 2. Berihterstatter: Prof. Dr. Reihardt Rüdel Tag der Promotion: 19. 04. 2002 Content Abbreviations 1. Introduction 1.1 Hypokalemic periodic paralysis (HypoPP) 1 1.2 HypoPP-1 and calcium channel gene (CACNA1S) 1 1.3 Phenotype and genotype studies of HypoPP-1 2 1.4 Electrophysiological studies of HypoPP-1 3 1.5 HypoPP and sodium channel Gene (SCN4A)? 5 1.6 Aims of this study 5 2. Patients, materials and methods 2.

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• Biologie

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• Publié par: universitat_ulm
• Publié le: 01 janvier 2002
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

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  Department of Applied Physiology  University of Ulm  Head: Prof. Dr. F. Lehmann-Horn      Molecular genetics and electrophysiological  studies of hypokalemic periodic paralysis  (HypoPP)   D  Faculty of Medicine, University of Ulm     Presented by  Chao Hang  Yangzhou, P. R. China    2002, Ulm  

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Amtierender Dekan: Prof. Dr. Reihard Marre

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1.Berihterstatter: HD. Dr. Karin Jurkat-Rott

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2.Berihterstatter: Prof. Dr. Reihardt Rüdel

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Tag der Promotion: 19. 04. 2002

Content Abbreviations  1.  Introduction 1.1   1Hypokalemic periodic paralysis (HypoPP) 1.2  HypoPP-1 and calcium channel gene (CACNA1S) 1 1.3   2Phenotype and genotype studies of HypoPP-1 1.4  Electrophysiological studies of HypoPP-1 3 1.5  HypoPP and sodium channel Gene (SCN4A)? 5 1.6   5 study thisAims of  2.  Patients, materials and methods 2.1  Patients and families 7 2.2  PCR and allele specific PCR 7 2.3  Single strand conformation analysis 8 2.4   9Sequencing of PCR products 2.5   11Genetic linkage verification 2.6  In vitro site-directed mutagenesis 12 2.7   15Cell culture 2.8  Transfection 15 2.9  Whole-cell recordings 16  3.  Results 3.1  Mutation screening results ofCACNA1S 21 3.2  Mutation screening results ofSCN4A 23 3.3  Linkage verification of HypoPP-2 26 3.4  Allele specific PCR as mutation screening method 30 3.5   30Statistical comparison of clinical symptoms of HypoPP-1 and -2 3.6   34Whole-cell recording results of HypoPP-1

 4.  Discussion 4.1  HypoPP-1 mutations and voltage sensor segments inCACN1AS 52 4.2  SCN4A, a new HypoPP gene 52 4.3   53Phenotype and genotype studies of HypoPP-1 and -2 4.4   54Electrophysiological consequences of HypoPP-1 4.5  Pathogenesis theory of HypoPP 57  5.   6.   7.   8. 

References60

Summary

Acknowledgements

CV

Abbreviations  Biological abbreviations: ASPCR Allele specific polymerase chain reaction CACNA1SSkeletal muscle voltage-gated calcium channelα1 subunit cDNA Complementary DNA cM centiMorgan DHPR Dihydropyridine receptor HG Mutant with double mutation, R650H+R1362G HH Mutant with double mutation, R650H+R1362H HypoPP Hypokalemic periodic paralysis HyperPP Hyperkalemic periodic paralysis kb kilobase (pair) mdg muscular dysgenesis mouse (CACNA1Sknockout) LOD Logarithm of the odds PCR Polymerase chain reaction SSCA Single strand conformation analysis SCN4ASkeletal muscle voltage-gated sodium channelαsubunit WT Wild type   Chemical abbreviations: Acr/Bis Acrylamide/Bis-acrylamide APS Ammonium persulfate (+) Bay K8644 1,4 Dihydro-2,6-dimethyl-5-nitro-4-[trifluoromethyl)-  phenyl]-3-pyridine carboxylic acid methyl ester Bis-acrylamide N, N -methylene-Bis-acrylamide  EDTA Ethylenediamine tetraacetic acid tetrasodium

HEPEs N-(2-hydroxyethyl) piperazine N-2-ethanesulfonic acid SDS Sodium dodecyl sulfate TBE Tris borate EDTA buffer TEMED N, N, N, N-tetramethyl-ethylenediamine Triton X-100 Octylphenol-polyethyleneglycol ether  Physical abbreviations: kHz kilohertz ml milliliter mM millimolar ms millisecond mV millivolt mΩ microohm pA picoampere pF picofarad µM micromolar  

1. Introduction  1.1  Hypokalemic periodic paralysis (HypoPP)   Hypokalemic periodic paralysis (HypoPP, MIM 170400) is characterized by transient attacks of generalized flaccid muscle weakness accompanied by a decrease in serum potassium level. It is inherited as an autosomal dominant disorder. Incomplete penetrance is observed more often in women than in men. The estimated prevalence is about 1:100,000. Clinical onset is usually before the age of 20. Provoking factors include carbohydrate-rich food, rest after excise, exposure to cold, infection and mental stress. Episodic muscle weakness usually involves all four limbs and lasts from hours to days. The frequency is variable from once during a lifetime to several attacks per week. After the age of 40, attacks become less frequent, and some patients develop progressive myopathy. As a diagnostic test, glucose (2 g/kg) and insulin (0.1 U/kg) may be infused under strict medical supervision to provoke hypokalemia and subsequent muscle weakness. Medical treatment during attacks is potassium salt administered orally. Regular acetazolamide intake is an effective preventive measure. (for overview see (Lehmann-Horn et al. 1994)).  1.2  HypoPP-1 and the calcium channel gene (CACNA1S)   By linkage analysis HypoPP was assigned to chromosome 1q31-32 and cosegregated with an intragenic marker forCACNA1S (= HypoPP-1 locus) (Fontaine et al. 1994).CACNA1S the gene encoding the skeletal muscle is voltage-gated calcium channelα1 subunit, which is the pore-forming subunit of the so-called dihydropyridine receptor (DHPR). The DHPR consists of five subunits,α1,α2/δ, βandγ (Figure 3.1), and serves dually as

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voltage sensor for intracellular calcium release initiating contraction and as a voltage-gated calcium channel (see review (Hofmann et al. 1994)). Theα1 subunit belongs to the super-family of voltage-gated cation channelα  subunits. It is composed of four homologous domains (DI to DIV), each having sixα-helical transmembrane segments (S1-S6) (Figure 3.1). The S4 segments contain a high density of positively charged amino acids, with every third being lysine or arginine. They serve as the voltage sensor in voltage-gated ion channel gating (for review see (Lehmann-Horn and Jurkat- Rott 1999)).  Screening inCACN1ASyielded three causative HypoPP mutations encoding R528H, R1239H, and R1239G (Jurkat-Rott et al. 1994, Ptacek et al. 1994). These mutations are all located in the voltage-sensor segments of calcium channel, and all reduce the positive charge density. One mutation replaces the outermost positively charged arginine at the position 528 in segment S4 of domain II by a weakly positive histidine (R528H). The other two mutations affect the second outermost arginine at the position 1239 in the S4 of domain IV and replace it by either histidine or uncharged glycine residue, R1239H and R1239G.  1.3  Phenotype and genotype studies on HypoPP-1  HypoPP-1 mutations were present in various ethnic groups from different regions: America, Britain, Europe, Japan, and Korea (Davies et al. 2001, Elbaz et al. 1995, Fouad et al. 1997, Grosson et al. 1996, Sillen et al. 1997, Wada et al. 2000). R1239H was the most frequent form in American (Fouad et al. 1997) whereas R1239G was rarely present. A founder effect of R528H was found in some Danish (Sillen et al. 1997), but not in American families (Grosson et al. 1996). Occurrence of a de novo R1239G mutation was

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reported in two cases (Elbaz et al. 1995, Ptacek et al. 1994). Incomplete penetrance was described mainly for R528H primarily in females (Elbaz et al. 1995), but later also in males (Sillen et al. 1997). Among the carriers of the same mutation, men often suffered more severe clinical features than women did. Among the three HypoPP-1 mutations, slight differences were found in clinical features, age of onset, hypokalemia, duration and frequency of attacks. R528H was found to be less severe, while R1239G was the most severe (Fouad et al. 1997).  1.4  Pathogenesis of HypoPP-1  Byin vitrostudies on native muscle fibers from HypoPP patients, prolonged depolarization and reduced action potentials of muscle cells were found to be the direct reasons for the attacks of paralysis. And hypokalemia further depolarized muscle cells and also contributed to the attacks (Lehmann-Horn et al. 1987, Rüdel et al. 1984).  In order to understand the functional defects of mutated calcium channel resulting HypoPP, electrophysiological studies were performed on muscle fibers and myotubes (polynuclear muscle cells) of HypoPP patients and also on different cell lines heterologously expressing mutant channels.  Voltage gated calcium is characterized by a simplified three states model, which contains a resting closed state, open state, and inactivated close state (for review see (Lehmann-Horn and Jurkat-Rott 1999)). Resting closed state is when a cell is held in polarized potential around 90 mV. With the depolarization S4 segments are supposed to move outwards, and then the channel opens and enters into open state. This process is called activation and the reverse called deactivation. With the depolarization, the channel

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from resting closed or open states can enter into inactivated closed state, in which the channel will be unable to open regardless of the cell membrane depolarization. This process is called inactivation and the reverse called recovery.  Electrophysiological recordings of calcium currents were performed on myotubes from HypoPP-1 patients with the two main mutations, R528H and R1239H (Jurkat-Rott et al. 1998, Lehmann-Horn et al. 1995, Morrill et al. 1998, Sipos et al. 1995). Both mutations showed a reduced current density compared to wild type, R528H by 30% and R1239H by 70% (Morrill et al. 1998, Sipos et al. 1995). Contradictory results on R528H were reported. Voltage dependent activation and inactivation of R528H showed a shift to more negative potential by 6 mV and no change on the kinetics of activation (Jurkat-Rott et al. 1998), whereas another research group observed significant slowing of the rate of activation (Morrill et al. 1998).  To explore the functional consequence of HypoPP-1 mutations, calcium channel heterologous cellular expression systems were developed. HypoPP- 1 mutations were introduced by in vitro mutagenesis into the rabbit CACNA1S which are highly homologous with their human cDNAs, counterpart. Mutated cDNAs were transfected into different cell lines, mouse L-cells (Lapie et al. 1997), muscular dysgenesis mouse (CACNA1S  knockout mouse) cell line (Jurkat-Rott et al. 1998) and Xenopus oocytes (Morrill and Cannon 1999). Reduced current density for R528H, R1239H and R1239G were confirmed. But no significant changes were found in the kinetics of channel activation, inactivation and recovery.  Contradictory results existed in the electrophysiological measurements of HypoPP-1 mutations. Secondary effects such as the disturbed sodium or

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potassium channel function was suggested for the pathogenesis of HypoPP- 1.  1.5  HypoPP and the sodium channel gene (SCN4A)?  The geneSCN4A the voltage-gated sodium channel encodesα subunit (SkM1), which shares a highly homologous structure to the calcium channel α1 subunit (Figure 3.2). Depolarization of the muscle fiber membrane activates the sodium channel into a positive feed back mechanism. This produces an action potential, which rises to a peak of approximately 10-20 mV within about 1 ms. The channels inactivation occurs within a few milliseconds. The skeletal muscle voltage-gated sodium channel plays an important role in generating the action potential owning to its intrinsic fast activation and inactivation kinetics (Lehmann-Horn and Jurkat-Rott 1999).  Missense mutations in theαsubunit of the skeletal muscle sodium channel (SkM1) cause hyperkalemic periodic paralysis (HyperPP), which is also characterized by muscle weakness as HypoPP but accompanied by the increased serum potassium levels. The medication for HypoPP, intake of potassium, is often the triggering effect for HyperPP. A mutation in the sodium channel was suggested to cause HypoPP in a small family only with 4 affected members (Bulman et al. 1999). The possibility of misjudgment of HyperPP by HypoPP could not be excluded.  1.6  Aims of this study  1. Previous mutation screening in part ofCACNA1Sencoding intracellular loop produced negative results (Boerman et al. 1995). We planed to screen all the transmembrane segments to verify that the voltage sensor segments in

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