1. Neurological Channelopathies- mechanisms Stephen M. Smith Division of Pulmonary & Critical Care Medicine, Department of Physiology & Pharmacology, OHSU.

2. Channelopathies Definition: A disease caused by mutations of ion channels. Increasingly recognized as important cause of disease (>30 diseases). Numerous mutation sites may cause similar channelopathy e.g. cystic fibrosis where >1000 different mutations of CFTR described

3. Channel properties In nervous system, coordinated activation of channels generate action potentials, postsynaptic potentials, pacemaker potentials. In addition, mediate epithelial transport, transduce chemical signaling and regulate intracellular ion concentrations. Channels mediate ion movement down electrochemical gradients. Activation of channel permeable to ion X shifts membrane potential towards E X.

4. Channel Function Ion channels are not open continuously but open and close in a stochastic or random fashion. Ion channel function may be decreased by decreasing the open time (o), increasing the closed time (c), decreasing the single channel current amplitude (i) or decreasing the number of channels (n).

5. I. Production II. Processing III. Conduction IV. Gating Molecular Mechanisms of Channel Disruption Channelopathies

6. Copyright restrictions may apply. Human Channelopathies Ptacek, L. J. et al. Arch Neurol 2004;61:1665-1668.

7. Channelopathies But new additions: Absence epilepsyGABA A  2 subunitGABA channel Febrile convulsions Painful neuropathyNa v 1.7Na channel Furthermore: Numerous diseases seen in ko animals that mimic human diseases Eg Deafess L-type VACC channel Disease states recognized as a result of GPCR opathy often involve ion channels. Eg Autosomal dominant hypocalcemiaCaR

8. Channelopathies- general characteristics Although mutation is continuous the disease may be episodic such as periodic paralysis or progressive like spinocerebellar ataxia. Abnormalities in same channel may present with different disease states. Lesions in different channels may lead to same disease eg periodic paralysis.

9. Copyright restrictions may apply. Ptacek, L. J. et al. Arch Neurol 2004;61:1665-1668. The targets: voltage-gated ion channel alpha subunit schema 4 domains, 6 membrane- spanning segments

10. Periodic Paralysis ● AD inherited ● PP manifests as paroxysmal weakness affecting all or some muscles ● Classified by blood [K] during episode ● Hyperkalemic (High K), normokalemic or hypokalemic (low K)

11. Hyperkalemic Periodic Paralysis ● Most common PP ● Short period of weakness (1-2 hr), no progressive permanent weakness, young age of onset (>10 yr) ● Triggers- post-exercise rest, cold, stress, K ingestion, fasting ● Myotonia- sustained involuntary muscle contraction ● Most commonly caused by gain of function Na channel mutations

12. Impaired inactivation of Na channels underlies HyperPP Cannon,1991 Normal NaCh open and close rapidly due to inactivation. NaCh from hyperPP close more slowly and reopen. Mutant myotube (Met 1592 Val)

13. Hayward, L. J. et al. Neurology 1999;52:1447 Recovery from inactivation is hastened for I693T channels WT and mutant whole cell Na currents Impaired inactivation of Na channels underlies HyperPP

14. Hayward, L. J. et al. Neurology 1999;52:1447 Recovery from slow or fast inactivation is accelerated by mutations associated with hyperkalemic periodic paralysis

15. Cannon,1991 Impaired inactivation of Na channels causes the Myotonia seen in HyperPP

16. Cannon,1991 Level of basal Na channel activity determines firing pattern in model muscle cell

17. Hypokalemic Periodic Paralysis ● Longer period of weakness, possible progressive permanent weakness, age of onset (>20 yr) ● Triggers- post carbohydrate meal, or Na-rich food ● No Myotonia ● Most commonly caused by loss of function Ca channel mutations or less often by altered voltage sensor of Na v 1.4 channels

18. Skeletal Muscle Functions of Cav 1.1 L-type channel or dihydropyridine receptor. Present in skeletal muscle. Found at triads and connect T-tubules with SR. Essential for excitation- contraction coupling Cav1.1 ko fails to contract but is rescued by the channel. From Hille, 2001 Fine structure skeletal muscle (transverse and longitudinal). Arrows show RyR-Cav1.1 complexes

19. Skeletal Muscle Functions of Cav 1.1 Mediate voltage sensing for the RyR (Ca channels in SR) to initiate rise in [Ca] i and contraction. Ca-entry unimportant as: muscle contracts in Ca- free Ringers and current activation too slow over 1-2 sec. From Hille, 2001

20. Cav1.1 mutants have decreased VACC current in Hypokalemic Periodic Paralysis- ● Unclear how this decreased activity causes HypoPP. Morrill and Cannon,1999

21. In some cases of HypoPP Na current inactivation is left- shifted and activation is right-shifted Sokolov et al, 2007 But this inhibitory effect does not explain dominant inheritance pattern of loss of positively charged arginine on voltage sensor (S4) WT vs R666G

22. Sokolov et al, 2007 HypoPP Na channel gating pore current is increased at negative potentials WT vs R666G

23. Gating pore Na current depolarizes membrane potential and increases [Na] i to reduce muscle contraction in HypoPP In R666G and other HypoPP mutants gating pore excludes Ca but permeable to monovalents

24. Voltage sensor mutation of Na v 1.4 channels: a less common cause of Hypokalemic Periodic Paralysis ● Voltage sensors (blue) encircle central pore in Na v 1.4 channels ● Loss of positively charged arginine leads to gating pore current at RMP ● Explains no sustained contraction with Hypo PP ● Gain of function explains dominant inheritance ● Could increased basal energy requirements cause chronic myopathy? ● Other channelopathies have voltage sensor mutations Sokolov et al, 2007

25. VACC-opathies Found in nerve, muscle, and endocrine cells. Trigger transmitter release, plasticity and gene expression in neurons, muscle contraction, hormone release. Target for dihydropyridines, diltiazem, verapamil, novel analgesics (SNX-111).

26. VACC are heterologous multimers Each alpha1subunit has 4 homologous repeat domains, each comprised of 6 transmembrane segments alpha1 modulated by other subunits

27. Channelopathies of Cav2.1

28. Three distinct diseases from distinct mutations of P/Q channel. Familial Hemiplegic Migraine (FHM) Intermittent headache with aura and paraesthesia, hemiplegia, hemianopia or dysphasia. Episodic Ataxia type 2 (EA2) Intermittent cerebellar disturbance incl, vertigo, diplopia and nystagmus. Mild progressive cerebellar atrophy. Rarely associated with absence seizures. Spinocerebellar Ataxia type 6 (SCA6) Progressive cerebellar degeneration.

29. Comparison of alpha 1A mutant diseases. FHMEA2SCA6 MutationmissensePremature stop codon & splice site mutation CAG repeat ProteinPoint mutationTruncated channel Altered C- terminus & Polyglutamine Pattern of inheritance Dominant Impact on function Altered I Ca density. Shift I-V to left Non-functional channel Reduced P/Q surface expression and altered in/activation. Is polyglutamine toxic? Mechanism of disease unclear Null mutant mouse may facilitate understanding.

30. An animal model of these channelopathies. The cerebellum develops in absence of  1A subunit. WT  1A -/- Cerebellar vermis PN How does loss of the channel affect function?

31. Copyright ©2004 Society for Neuroscience Song, I. et al. J. Neurosci. 2004;24:5249-5257 Absence seizures in alpha1A-/- mice EEG recordings from the cortex of freely moving mice Absence seizures seen in humans with episodic ataxia type 2 Co-deletion of T-type channel leads to absence of EEG changes and seizures

32. Is loss of coordination a result of absent P/Q VACC currents in alpha1A-/- mice Purkinje neurons

33. Total current density in PN is decreased due to the elimination of P-type currents but L- and N-type current densities are increased.

34. Copyright ©1999 by the National Academy of Sciences Jun, Kisun et al. (1999) Proc. Natl. Acad. Sci. USA 96, 15245-15250 Synaptic transmission intact but altered in hippocampus from alpha1A-/- mice Mechanism of disease still unclear as VACC density and type altered in many parts of nervous system

35. Null Mutant similar to EA2. P/Q nullFHMEA2SCA6 MutationNull mutantmissensePremature stop codon & splice site mutation CAG repeat ProteinAbsentPoint mutationTruncated channel Altered C- terminus & Polyglutamine Pattern of inheritance RecessiveDominant Impact on function Absent P/Q currents Altered I Ca density. Shift I-V to left Non-functional channel Reduced P/Q surface expression and altered in/activation. Is polyglutamine toxic?

36. Mechanism still unclear. How do alpha 1A mutations lead to disease states? Is poor coordination the result of impaired synaptic transmission or reduced current density in the soma?. Currents from rat, mouse, rabbit and human mutations are affected differently. Need to better understand neuronal function e.g. Purkinje cell firing pattern (see Walter and Khodakhah).

37. How do alpha1A mutations lead to disease states? Introduction: Purkinje neuron AP represents cerebellar output Only P/Q channels activate Ca-activated K channels in P-cells Pacemaker rate set by afterhyperpolarization Hypothesis: Endogenous pacemaker rate set by Ca- activated K channels. APs activates P/Q channels, permits Ca entry which activates the Ca-activated K channels. Thus subsequent hyperpolarization and rate will depend on normal P/Q channel function. Uses ataxic mice with a variety of alpha 1a mutations to argue modulation of Ca-activated K channels is key to maintaining intrinsic pacemaker activity in P-cells.

38. Multiple measures confirm Purkinje cell firing is irregular in ataxic mice with P/Q mutations Walter et al., 2006

39. Activation of SK with EBIO improves Purkinje cell precision Walter et al., 2006

40. Activation of SK with EBIO improves motor performance of Ducky mice Walter et al., 2006 RotarodBalance beam

41. P/Q VAC Channelopathies KO mouse similar to Episodic Ataxia T2. But recessive vs dominant- “slots” taken by truncated channel Compensation by other VACC insufficient. How does reduced P/Q current cause ataxia- P cell output irregular as loss of afterhyperpolarization.

42. Summary. Channel mutations are an increasingly recognized cause of disease. Many channelopathies episodic despite persistently abnormal channel. Triggers recognized for some diseases. Abnormalities in same channel may present with different disease states. Lesions in different channels may lead to same disease eg periodic paralysis. Disease mechanism often unclear despite identification of mutation.