Electrical Activity에 관한 공부 (Cardiac Electrophysiology) 1) Generation and Conduction of Action Potential Generation of ECG: - conduction of depolarization wave - electrical heterogeneity of AP 3) Ionic Basis of Action Potentials: How to understand the generation of electrical signal (V) from the characteristics of ion channels and currents (I): I vs V 4) Abnormal electrical activity: Abnormal AP//Abnormal conduction --- Arrhythmias

Ion current through ion channels electrical signal

Since, Iion = Iinward + Ioutward , Equivalent Circuit Model Im = Iion + Cm*dV/dt = 0 dVm = -∫ Iion dt / Cm Since, Iion = Iinward + Ioutward , Iinward > Ioutward depolarization Ioutward > Iinward hyperpolarization

Ionic Basis of Action Potentials of a single cell: Specific form of AP can be understood by Biophysical characteristics of individual ion channels Distribution of various channels specific to the cell type General principle: dV/dt = −Iion/Cm Outward Current ; Cause repolarization or hyperpolarization Cause depolarization Inward Current: I(Ca) I(Na/Ca) I(back) I(Na) I(K) I(to) I(pump)

Ion Channels Selectivity for a specific ion: Na channel, Ca channel, K channel, Non-selective cationic channel, Cl channel. Gating by a specific signal: Voltage-gated, Ligand-gated, Ca2+-activated, ATP-dependent...

Selectivity of ion channels determines the direction of the current flow. Channels selective for Na+ or Ca2+, or non-selective to cations produce inward currents, thus cause depolarization. Channels selective for K+ produce outward currents, thus cause repolarization or hyperpolarization. Current direction depends on electrochemical gradient

If membrane is selectively permeable to a specific ion, X; Na+ Ca2+ K+ Membrane potential approaches to EX

X+ moves outward when the membrane potential, Em > Ex X+ moves inward when the membrane potential , Em < Ex K+ Na+ If gNa=gK, net current flow = 0 at (ENa+EK)/2 Outward Inward

Resting membrane potential is the potential where inward current equals to outward current. K+ Na+ RMP Since gK>>gNa at resting state, RMP is close to EK.

If conductance changes to make gNa>>gK, Em moves toward ENa.

Without current injections, membrane potential changes in the range of reversal potentials for major ions: EK < Em (RMP< AP) < ENa

Ion channel conductance is changed by Gating mechanisms Gating signals Voltage-gated: depolarization/hyperpolarization Receptor-gated Ca2+-activated ATP-sensitive Stretch-activated H+, O2, other ligands Temperature: heat or cold Gating kinetics Time dependence: transient, slowly activating, rapidly activating Time-independent

Analysis of current recordings to understand ion channel properties Dissection of an individual channel: Pharmacological methods Ion substitution: external solution/pipette solution Electrophysiological methods: various pulse protocol Analysis of channel properties: Current-voltage relationship (I-V curve): Erev, slope Activation curve/Inactivation curve: V1/2, slope Time constant

Dissection of membrane currents using electrophysiological methods Pipette solution Pulse protocol K+ pipette Cs+ pipette +50 mV -40 mV 40 mV -80 mV -50 mV -80 -70 6000 IK 5000 4000 3000 2000 1000 200 400 600 800 1000 1200 1400 -1000 ICa,L ms -2000 -3000 INa -4000 pA -5000 -6000

Current-voltage relationship Can be drawn by measuring: Instantaneous current Peak current Steady state current Analysis parameter: Reversal potential --- represents ionic selectivity Conductance: slope/chord Shape: linear/rectification/bell

Current-voltage relationship for K+ current Inward Rectification Outward Rectification Inward rectifing K+ EK Voltage-gated K+ EK Background K EK Linear

Current-voltage relationship for Na+ or Ca2+currents Bell shape Voltage-gated Ca2+, low threshold Voltage-gated Ca2+, high threshold Voltage-gated Na+

Current-voltage relationship for nonselective cation currents Reversial Potential (Erev) = gNa*ENa/(gNa+gK) + gK*EK/(gNa+gK) Linear: Background Hyperpolarization-activated

a(V) Open Close n b(V) 1-n Gating analysis for voltage-gated channel a(V) Open Close n b(V) 1-n Open 1 I V a b Close 0 mV V1/2 1. Kinetic analysis time constant =1/(a+b) 2. Steady state analysis: Activation curve n = a/(a+b) = 1/(1+exp((V-V1/2)/dx)) (Boltzmann function) fast activation / slow activation voltage-dependence of activation

Function of Ion Channels can be understood… Biophysical properties --- simulation Diseases Drugs Knock-out of specific genes

Ventricular myocyte SA node cell

Cardiac Ion Channels  Electrical properties (Resting Membrane Potential, Action Potential)를 결정지을 뿐 아니라, 수축의 발생 및 조절과도 밀접한 관계.  Pathophysiology of Diseases, 또는 side effect of drug 와 관련됨.  Target of therapeutics: Ion channel blocker, Ion channel opener들이 부정맥, 고혈압의 치료제로 쓰임.

depolarization-activated hyperpolarization-activated -40 mV 40 mV -80 mV -50 mV B-1 -40 -50 -90 -2 200 ms nA ICa,L Ih INa B-2 A-2 5 -50 50 mV -100 500 pA -1000 -2000 -50 50 100 mV -5 ICa,L INa -10 -15 nA

Analysis of Na+ current -80 to +40 mV -120 mV activation inactivation g=I/(Vm-ENa) Activation curve I-V curve

TTX h-gate

Analysis for Na+ channel inactivation Inactivation curve Inactivation time constant

Contribution of INa : Simulation study Ventricle SA node

Na channel inactivation을 느리게 하는 약물

LQT3 Syndrome Brugada Syndrome Conduction disturbance

Na channel toxins Tetrodotoxin, saxitoxin, -conotoxin: outer pore occlusion Veratridine, aconitine: persistent activation due to negative shift of m() Local anesthetics binding to inner surface of IV-S6 (IV)  resting and use-dependent block

Specific tissue localization of Na+-channel  subunits NaV1.1 – 1.3: CNS, PNS, embryonic(1.3) NaV1.4: skeletal m. NaV1.5 (SCN5A): heart m. NaV1.6: CNS, PNS, node of Ranvier, glia NaV1.7: PNS Schwann cell NaV 1.8 - 1.9: PNS Nax: heart, uterus, smooth m., glia (TTX-resistant: underlined)

Cardiac Na Channel  활성화(activation)되면 전기화학적 경사에 의해 Na 이온이 세포내로 유입되어 내향전류가 발생.  심실근 활동전압의 빠른 upstroke (수십 V/s)는 Na 통로의 활성화에 기인: fast action potential fast conduction velocity  Na 통로는 수 ms내에 곧 비활성화(inactivation)되므로 지속적으로 내향전류를 발생하지는 않음.  비활성화(inactivation)의 장애 --재분극 지연 -- APD 증가로 인한 long QT syndrome의 원인.  Na channel blocker: - TTX (high concentration), local anesthetics (lidocain, quinidine등은 Na 통로의 비활성화를 negative로 shift -- 부정맥 치료에 쓰임)

Ca channels L-type Ca current recording Present in heart T-type L-type -35 mV 40 mV -80 mV -50 mV -60 -40 -20 20 40 -2500 -2000 -1500 -1000 -500 pA mV T-type L-type Present in heart

A. SA-Node B. Ventricle

Effect of Ca channel blocker

Ca2+ currents  활성화되었을 때 Ca 이온이 세포내로 유입되며 내향전류가 발생한다.  Na 통로에 비해 activation, inactivation이 느림  심실근, 심방근에서의 Ca 전류는 활동전압의 plateau유지에 기여  동방결절이나 방실결절 같이 안정막전압이 낮아서 Na channel은 비활성인 세포에서는 활동전압의 upstroke에 기여: upstroke dV/dt 느림 --- slow AP --- slow conduction  유입된 Ca은 흥분-수축 연결에서 작용 : 수축의 유발, 수축 크기 결정에 기여. L-type Ca channel blocker - inorganic blocker: Mn, Co, Ni - organic blocker: verapamil, D-600, diltiazem, nifedipine 등. - 부정맥, 고혈압 치료에 쓰임.

Excitation-secretion gene name Pre-synaptic terminal Excitation-secretion coupling present in heart

L-type (Long lasting) Ca2+ channel Subunits: α1C, α1D, α1F, or α1S, α2δ, β3a Blockade Sensitive to dihydropyridine (DHP) agonists and antagonists Also blocked by phenylalkylamines (verapamil), benzothiazepines (diltiazem) & calciseptine Activation: Strong depolarization Inactivation by depolarization: Little Localization Skeletal muscle: α1S Brain (Neuronal soma & proximal dendrites): α1D Cardiac muscle: α1C Neuroendocrine: α1D Retina: α1F General function in muscle: Excitation-contraction coupling - Cav 1.1 (A1S): Skeletal muscle; Also acts as voltage sensor Cav 1.2 (A1C): Heart & Smooth muscle

T-type (Transient) Ca2+ channel - Activation By depolarization near resting potential Low voltage activation (LVA) threshhold - Inactivation Rapid Steady-state inactivation occurs over a similar voltage range as activation Window current: Small range of voltages where T-type channels can open, but do not inactivate completely Rapid deinactivation - Reactivation: Requires strong hyperpolarization - Blockade Nickel ions: Especially Cav 3.2 Mibefradil Do not bind dihydropyridines - Tissue localization: Cardiac & vascular smooth muscle; Nervous system - Function Rhythmic action (pacemaker) potentials in cardiac muscle & neurons Burst firing mode of action potentials Regulate intracellular Ca++ concentrations

SA node cell Pacemaker current (If, Ih) : IK ICa Pacemaker current (If, Ih) : hyperpolarization-activated inward currents

Functional role of hyperpolarization-activated inward current in cardiac pacemaker activity. SA node Purkinje fiber

HCN channels (Pacemaker channels) Hyperpolarization-activated, Cyclic nucleotide-gated (PK+/PNa+ = 5), Erev = –30 mV Generation of repetitive spontaneous electrical activity Found in pacemaking cells in the heart and the brain Cyclic nucleotide binding domain Regulated by neurotransmitters: faster activation by sympathetic (cAMP) slower activation by parasympathetic (cGMP)

Hyperpolarization-activated inward current is regulated by neurotransmitters

Species difference in the density of Hyperpolarization-activated inward current

Hyperpolarization-activated inward current Cyclic nucleotide binding domain HCN isoforms

Ventricular AP Sinoatrial AP INa ICa,L Ih , ICa,T MDP -65 mV RMP Repolarization INa ICa,L MDP -65 mV RMP -90 mV Ih , ICa,T