1. Cardiac Ion Channels Ling-Ping Lai, MD, PhD National Taiwan University Taipei, Taiwan

2. Recent Advances in Basic EP §Molecular cloning of ion channels §Intracellular recordings §Voltage clamp and patch clamp

3. What is an ion channel? §Ion channels are proteins §Ion channels are membrane bound §Ion channels are pores §Ion channels have ion selectivity §Ion channels have gating properties

4. Cardiac Ion Channels §Physiology l Action potential in excitable cells §Pharmacology l AAD classification

5. Ion channels in action potential

6. Ion channels in the heart §Depolarizing current l Sodium channels l Calcium channels §Repolarizing current l Potassium channels l Chloride channels

7. Classification of AADs §Class I: Na channel blockers l Ia: prolong APD l Ib: shorten APD l Ic: No change of APD §Class II: beta-blockers §Class III: K channel blockers §Class IV: Ca channel blockers

8. Basic Concepts §Electrico-chemical gradient §Resting membrane potential and action potential §Voltage-gated ion channels §Voltage-clamp and patch-clamp

10. Basic Physics §Ernst equation l Ec=(RT/F)*ln([C o ]/[C i ]) §Resting membrane potential l Vm=g*Ec 1 +g*Ec 2 +....

11. Gating of Ion Channels §Voltage gated §Ligand gated §Stretch activated §Others

12. Clamp §Nobel Prize(1963): Eccles,Hodgkin,Huxley l Voltage clamp §Nobel prize(1991): Sakmann,Neher l Patch clamp for single channel study

13. Advantage of Mol Biol §High sensitivity and specificity §Analytic and preparative §Versatile

14. Common Molecular Techniques §PCR §Plasmid and E. coli §Restriction enzyme digestion §Gel elctrophoresis §Northern and southern blotting §DNA sequencing

15. Cloning of Ion channels §Discovery of new channels §Functional studies §Site directed mutagenesis §Measurement of amount of channels

16. Cloning of channels §Reverse genetics §Expression screening §Homology screening

17. Molecular biology §Shaker mutant of fruit fly §Other mutant of fruit fly §Homology screening: l mammal and human counterpart §Reverse genetics

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19. Reverse genetics §Shaker family of potassium channel §Congenital long QT syndrome §Brugada syndrome

20. Congenital long QT syndrome §LQT1: K channel KvLQT1 §LQT2: K channel HERG §LQT3: Na channel §LQT4: Ankyrin B §LQT5: K channel minK §LQT6: K channel MirP

21. Screening Cloning §Expression screening: minK §Homology screening: l Shab, Shaw, Shal l Funny current channel

22. Functional Expression §PCR cloning of cDNA §Mass production by plasmid and E coli §Transcription: l Transfection l In vitro transcription §Protein synthesis §Clamp study

27. In Vitro Cloning and Expression of I Ks

28. Ion channels in action potential

29. Cardiac sodium channel §Molecular identity l NaV1.5 (SCN5A) l Exclusively expressed in the heart §Electrophysiology l Open when depolarization l Rapid inactivation l Upstroke of AP l TTX in-sensitive

34. Recovery from block   recovery time constant §Fast: lidocaine (use-dependent) §Intermediate §Slow: flecainide (tonic blockade)

35. Calcium Channel §Molecular identities l L-type: CaCNA1C (CaV1.2) l T-type: CaCNA1G,H,I (CaV3.1, 3.2, 3.3) §Electrophysiological properties l Open when depolarization l Inactivation l Similar to I Na but slower kinetic l Plateau of AP

37. Cardiac potassium channels §The most diverse class of channels l I K1 : inward rectifier in resting MP l I to : transient outward in early AP l I K : delayed rectifier in late AP I Kur, I Kr and I Ks :

38. Inward rectifier §Electrophysiological properties l Activated by hyperpolarization l Inward current only l Maintaining RMP §Molecular identities l Kir2.1

41. Transient outward current §Electrophysiological properties l Open when depolarization l Rapid inactivation l Spike and dome of AP §Molecular identities: l KV4.3 l KV4.2

43. Delayed rectifiers §Molecular identities l I Kr : KCNH1 (HERG) + MirP (KCNE2) l I Ks : KCNQ1 (KvLQT1) + minK (KCNE1) l I Kur : KCNA5 (Kv1.5) §Electrophysiological properties l Open when depolarization l Different kinetics

44. I Ks

45. I Kur I Kr I Ks