Electrical Characteristics of Channelopathies Involving Skeletal Muscle

1. Electrical Characteristics of Channelopathies Involving Skeletal Muscle Bob Ruff, M.D., Ph.D. Chief, SCI Service Louis Stokes Cleveland VAMC Barbara E. Shapiro, M.D., Ph.D. Case Western Reserve Univ. Jacob Levitt, M.D. Albert Einstein College of Medicine

2. Objectives • To understand factors regulating membrane excitability in skeletal muscle • To understand how impaired Na+ channel inactivation can produce myotonia • To appreciate how persistent depolarization produces paralysis (Myotonia vs HyperKPP) • To learn different ways to produce a persistent depolarization (HyperKPP vs HypoKPP)

3. Roles of Na+, K+ and Cl- Channels in Membrane Excitability • Kir sets resting membrane potential • Kv (delayed rectifier) repolarizes after AP • Cl- channel stabilizes membrane potential • INa drives AP

4. Potassium Sets Membrane Resting Potential • K+ conductance 20% of membrane conductance - Inward or anomalous rectifier K+ channel (KIR) • AP Termination - Delay Rectifier K+ Channel

5. Inward (Anomolous) Rectifier

6. Sodium channel gating properties • Depolarization activates Na channels - changes from a closed to an open state • The declining portion of INa - transition of open channels to a non-conducting fast inactivated state

8. Two Types of Skeletal Muscle Sodium Channel Inactivation • Fast inactivation – msec, Slow inactivation - seconds  • Fast inactivation helps to terminate the AP • Slow inactivation operates at more negative potentials - regulates the number of excitable sodium channels as a function of the membrane potential

9. Action Potential

10. Factors Determining Action Potential Threshold • Number of excitable Na+ channels (# of channels and fraction that are excitable) • Voltage dependence ofNa+ channel opening • Amount of Cl- conductance • Inward rectifier K+ conductance with depolarization

11. Periodic Paralysis • Results from persistent membrane depolarization  inactivation of normal Na+ channels  membrane inexcitability • HyperKPP – Na+ channelopathy – depolarization due to abnormal persistent INa • HypoKPP – • Type I - Indirect Ca2+ Channelopathy • Type 2 - Na+ channelopathy – loss of function

12. Hyperkalemic Periodic Paralysis (HyperPP) - AD • episodic attacks of flaccid weakness • myotonia is often present (vs HypoK-PP) • paralysis caused by membrane depolarization  Na+ channel inactivation • Overlap: Na+ Ch myotonias, paramyotonia Lehmann-Horn, Rudel, Ricker

13. Impaired fast inactivation can produce myotonia 1 msec Note: Loss of inactivation in a small % of channels →myotonia Myotonia stopped in part due to accumulated slow inactivation

14. Key to Paralysis vs Myotonia is Persistent Depolarization Impairment of Slow Inactivation will facilitate persistent opening of mutant channels

15. Hypokalemic Periodic Paralysis (HypoKPP) - AD • Episodic attacks of flaccid paralysis • Myotonia never present (vs HyperKPP) • Insulin paralytic attack without  K+ • Membrane excitability impaired – low conduction velocity: Drs. Haenen, Links, Oosterhuis, Stegeman, van der Hoevan, van Weerden & Zwarts

16. Depolarization not blocked by TTXInsulin Enhances Depolarization Lehmann-Horn, Rudel, Ricker

17. Paralysis parallels drop in K+

18. In HypoKPP Weakness Parallels Depolarization & Reduction in EMG Amplitude

19. Skeletal Muscle Membrane Excitability Is Impaired in HypoKPP (Type1) • Muscle fibers very susceptible to depolarization-induced inexcitable • Small depolarizations (10 mV) make HypoKPP fibers unexcitable • Slow conduction velocity (Zwarts’ lab) suggests impaired Na+ channel function in HypoKPP

20. Two Genotypes - Similar Phenotype • Type 1 HypoKPP is linked to 1Q31-32 • Defective gene (CACNL1A3) encodes a skeletal muscle dihydropyridine (DHP) sensitive or L-type calcium channel • Mutations - segment 4 of domain 2 (R528H) and segment 4 of domain 4 (R1239H, R1239G) of the -subunit of the skeletal muscle L-type Ca+2 channel

21. Two Genotypes - Similar Phenotype • Type 2 HypoK-PP has a similar phenotype to type 1 HypoK-PP • Associated with point mutations in the Na+ channel gene (SCN4A) • Surface membrane INa is reduced to about 50% of normal (reduced expression and increased resting inactivation)

22. Type 1 HypoKPP – Altered Inward (Anomolous) Rectifier

23. Insulin  outward current component of KIR in HypoKPP Circle – no insulin Square - insulin Unfilled – HypoKPP Filled – Control

24. Insulin Reduces K+ Conductance Even When [K+]o is High Circle – no insulin Square - insulin Unfilled – HypoKPP Filled – Control

25. Summary of Alterations of Inward Rectifier K+ Channel in HypoKPP • Baseline Inward Rectifier Conductance Including KATP Channels is Reduced • Insulin selectively reduces the K+ conductance for outward currents • Lowering [K+]o causes depolarization due to TTX- and DHP-insensitive depolarizing current (low Kir conductance for outward current facilitates depolarization) • Note: Andersen-Tawil Syndrome due to Kir mutation

26. Why do Type I and Type II HypoKPP have similar phenotypes? • The effects of the Na+ channel mutations in Type II HypoKPP are to reduce membrane channel density and to increase the amount of resting inactivation - both lead to  INa • Susceptibility of Type I HypoKPP fibers to depolarization-induced inactivation and lower AP conduction velocities suggest reduced INa in HypoKPP (Zwarts’ lab)

27. Small Depolarizations Produce Paralysis in HypoKPP

28. Comparison of Na+ Channel Properties and Action Potential (AP) Thresholds in Fast Twitch, Type IIb, Skeletal Muscle Fibers from Five Patients with HypoKPP and Seven Controls. Controls HypoKPP Na+ Channel Properties Max INa (mA/cm2) 23.7 15.4 ±1.3 ±1.9 (p<0.001) Action Potential (AP) Thresholds AP Threshold (mV) -58.7 -53.4 ±1.5 ±1.1 (p<0.001)

29. Which Membrane Change Correlates Best with Paralytic Attacks in Type 1 HypoKPP? • INa correlated inversely with frequency of paralytic attacks (Pearson’s correlation coefficient, r = -0.996) • AP threshold correlated with the frequency of paralytic attacks (r=-0.921) • Peak outward K+ conductance of the inward rectifier K+ channel correlated weakly with the frequency of paralytic attacks (r = -0.121).

30.  Na+ current correlated with the frequency of paralytic attacks  K+ current did not have a strong correlation Patients 1 2 3 4 5 Peak INa Max INa,max 11.9 12.2 16.9 17.7 18.2 (mA/cm2) ±1.8 ±2.0 ±1.8 ±1.7 ±1.9 Action Potential (AP) Thresholds AP Thresh -50.6 -51.0 -54.9 -55.1 -55.4 (mV) ±1.9 ±1.7 ±1.7 ± 1.8 ±1.8 Peak Outward IK in 80 mM K+ with 12mU/ml Insulin Conductance 260 271 279 268 251 (µS/cm2) ±30 ±29 ±39 ±42 ±36 Number of Paralytic Attacks (lasting >1 hour) in one year 15 13 3 2 1

31. How Can Ca2+ Channel Mutations Alter Na+ & K+ Channel Properties? • The Ca2+ channel mutations may disturb intracellular [Ca2+] • Intracellular Ca2+ is known to regulate Na+ channel expression and can alter the expression and properties of other channels

32. Intracellular [Ca2+] is increased in HypoKPP Fibers • Intracellular [Ca2+] Determined with a Calcium Sensitive Electrode in Type I, IIa and IIb Control and HypoKPP Human Intercostal Muscle Fibers • Intracellular [Ca2+](µM) According to Fiber Type • Type IType IIaType IIb • Controls 0.113±0.005 0.094±0.005 0.081±0.003 • n=27 n=22 n=58 • HypoPP 0.129±0.009 0.112±0.008 0.100±0.006 • n=11 n=12 n=16 • p<0.05 p<0.05 p<0.01

33. Indirect Channelopathy -Intracellular [Ca2+] may Down Regulate Na+ and KIR (incl. KATP) Channels Ca2+ mutations in HypoKPP may reduce Na+ channel density (and perhaps alter Inward Rectifier K+ Channel Function) by elevating intracellular [Ca2+], which reduces the level of the Na+ channel a-subunit mRNA (and perhaps reduces expression of KATP Channels)

34. Thyrotoxic Periodic Paralysis: the brother of Hypokalemic Periodic Paralysis Bob Ruff, M.D., Ph.D. Chief, SCI Service Louis Stokes Cleveland VAMC Director Rehabilitation Research & Development Department of Veterans Affairs.

35. Objectives • To understand distinguishing features of Thyrotoxic Periodic Paralysis (TPP) • To compare channel defects in TPP with HypoKPP • To consider how thyrotoxicosis contributes to the pathogenesis of TPP

36. Clinical: TPP vs HypoKPP

37. Periodic Paralysis • Results from persistent membrane depolarization  inactivation of normal Na+ channels  membrane inexcitability • HyperKPP – Na+ channelopathy – depolarization due to abnormal persistent INa • HypoKPP – • Type I - Indirect Ca2+ Channelopathy • Type 2 - Na+ channelopathy – loss of function • TPP – Not Associated with HypoKPP channel defects

38. Common Features of TPP & HypoKPP • Episodic attacks of flaccid paralysis • Myotonia never present (vs HyperKPP) • Insulin paralytic attack without  K+ • Membrane excitability impaired – low conduction velocity, low CMAP amplitude, CMAP reduction with exercise

39. Genetics of TPP • Familial cases increasingly recognized • HypoKPP Na channel mutations not found • HypoKPP Ca channel mutations not found • Reports of selective single nucleotide polymorphisms (SNP) in regulatory region of Ca channel gene – region of thyroid hormone binding sites

40. Methods - Patient with TPP • 32 yo man with TPP in the T-toxic state and 4 months later when euthyroid & asymptomatic • Measured INa with a loose patch voltage clamp, inward rectifier IK with a 3-electrode voltage clamp, action potential (AP) threshold with a 2 electrode clamp and intracellular [Ca2+] using Ca2+-sensitive electrodes • Intercostal type IIb muscle fibers from patient with TPP, 5 patients with Type I HypoKPP (R528H mutation) and 7 controls(C).

41. Summary of Alterations of Inward Rectifier K+ Channel in HypoKPP • Baseline Inward Rectifier Conductance Including KATP Channels is Reduced • Insulin selectively reduces the K+ conductance for outward currents

42. KIR in TPP (nA/mm2)

43. Max INa (mA/cm2)

44. AP Threshold (mV)

45. Intracellular [Ca2+] (nM) in TPP & HypoKPP

46. TPP & HypoKPP- Indirect Channelopathies -[Ca2+] may Down Regulate Na+ and KIR Channels • Ca2+ mutations in HypoKPP may reduce Na+ channel density and alter KIR function by elevating intracellular [Ca2+] • In TPP - SNPs at the thyroid hormone responsive element may affect the binding affinity of the thyroid hormone responsive element and modulate the stimulation of thyroid hormone on the Ca(v)1.1 gene

47. Summary – HyperKPP • Paralysis produced by prolonged membrane depolarization • Difference between mutations that produce myotonia vs paralysis is probably that paralysis is associated with prolonged pathological INa • Impairment of slow inactivation will facilitate prolonged pathological INa • Mutations that impair slow inactivation associated with paralysis

48. Summary – HypoKPP • INa is reduced in both types of HypoKPP • Inward Rectifier K+ conductance is altered in Type I HypoKPP and Andersen-Tawil Syndrome • Type I HypoKPP - Frequency of paralytic attacks correlates with decrease of INa • Type I HypoKPP – indirect Channelopathy - alteration of Na+ and K+ channel function may be mediated by  intracellular [Ca2+]

49. Supported by the Clinical Research and Development Service, Office of Research and Development, Department of Veterans Affairs

50. Rx of HyperKPP • REDUCE PARALYTIC ATTACK FREQUENCY • 1) Eat regular meals high in carbohydrates and low in K • 2) Avoid strenuous exercise followed by rest, emotional stress and cold