Cardiac Ion Channels

Ling-Ping Lai, MD, PhD

National Taiwan University

Taipei, Taiwan

Recent Advances in Basic EP

Molecular cloning of ion channelsIntracellular recordingsVoltage clamp and patch clamp

What is an ion channel?

Ion channels are proteinsIon channels are membrane boundIon channels are poresIon channels have ion selectivityIon channels have gating properties

Cardiac Ion Channels

Physiology Action potential in excitable cells

Pharmacology AAD classification

Ion channels in action potential

Ion channels in the heart

Depolarizing current Sodium channels Calcium channels

Repolarizing current Potassium channels Chloride channels

Classification of AADsClassification of AADs

Class I: Na channel blockers Ia: prolong APD Ib: shorten APD Ic: No change of APD

Class II: beta-blockersClass III: K channel blockersClass IV: Ca channel blockers

Basic Concepts

Electrico-chemical gradientResting membrane potential and action

potentialVoltage-gated ion channelsVoltage-clamp and patch-clamp

Basic PhysicsBasic Physics

Ernst equation Ec=(RT/F)*ln([Co]/[Ci])

Resting membrane potential Vm=g*Ec1+g*Ec2+....

Gating of Ion ChannelsGating of Ion Channels

Voltage gatedLigand gatedStretch activatedOthers

ClampClamp

Nobel Prize(1963): Eccles,Hodgkin,Huxley Voltage clamp

Nobel prize(1991): Sakmann,Neher Patch clamp for single channel study

Advantage of Mol Biol

High sensitivity and specificityAnalytic and preparativeVersatile

Common Molecular Techniques

PCRPlasmid and E. coliRestriction enzyme digestionGel elctrophoresisNorthern and southern blottingDNA sequencing

Cloning of Ion channels

Discovery of new channelsFunctional studiesSite directed mutagenesisMeasurement of amount of channels

Cloning of channels

Reverse geneticsExpression screeningHomology screening

Molecular biologyMolecular biology

Shaker mutant of fruit flyOther mutant of fruit flyHomology screening:

mammal and human counterpart

Reverse genetics

Reverse genetics

Shaker family of potassium channelCongenital long QT syndromeBrugada syndrome

Congenital long QT syndromeCongenital long QT syndrome

LQT1: K channel KvLQT1LQT2: K channel HERGLQT3: Na channelLQT4: Ankyrin BLQT5: K channel minKLQT6: K channel MirP

Screening Cloning

Expression screening: minKHomology screening:

Shab, Shaw, Shal Funny current channel

Functional Expression

PCR cloning of cDNAMass production by plasmid and E coliTranscription:

Transfection In vitro transcription

Protein synthesisClamp study

In Vitro Cloning and Expression of IKs

d-H2O

Time in ms

0 3000 6000 9000 12000

Cu

rre

nt

in n

A

0

20

40

60minK mRNA

Time in ms

0 3000 6000 9000 12000

Cu

rre

nt

in n

A

0

20

40

60

Ion channels in action potential

Cardiac sodium channel

Molecular identity NaV1.5 (SCN5A) Exclusively expressed in the heart

Electrophysiology Open when depolarization Rapid inactivation Upstroke of AP TTX in-sensitive

Recovery from block

recovery time constantFast: lidocaine (use-dependent) IntermediateSlow: flecainide (tonic blockade)

Calcium Channel

Molecular identities L-type: CaCNA1C (CaV1.2) T-type: CaCNA1G,H,I (CaV3.1, 3.2, 3.3)

Electrophysiological properties Open when depolarization Inactivation Similar to INa but slower kinetic Plateau of AP

Cardiac potassium channels

The most diverse class of channels IK1: inward rectifier in resting MP

Ito: transient outward in early AP

IK: delayed rectifier in late AP

• IKur, IKr and IKs:

Inward rectifier

Electrophysiological properties Activated by hyperpolarization Inward current only Maintaining RMP

Molecular identities Kir2.1

Transient outward current

Electrophysiological properties Open when depolarization Rapid inactivation Spike and dome of AP

Molecular identities: KV4.3 KV4.2

Delayed rectifiers

Molecular identities IKr: KCNH1 (HERG) + MirP (KCNE2)

IKs: KCNQ1 (KvLQT1) + minK (KCNE1)

IKur: KCNA5 (Kv1.5)

Electrophysiological properties Open when depolarization Different kinetics

IKs

IKur IKr

IKs