cardiac ion channels ling-ping lai, md, phd national taiwan university taipei, taiwan
Post on 21-Dec-2015
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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
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+....
ClampClamp
Nobel Prize(1963): Eccles,Hodgkin,Huxley Voltage clamp
Nobel prize(1991): Sakmann,Neher Patch clamp for single channel study
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
Molecular biologyMolecular biology
Shaker mutant of fruit flyOther mutant of fruit flyHomology screening:
mammal and human counterpart
Reverse genetics
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
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
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