Michael K Pugsley, PhD, FBPharmacolS, DSP Scientific Director, Janssen Research Fellow, Global Safety Pharmacology, Preclinical Development & Safety

The Biophysics of Cardiac Ion Channels

Electrical Properties of Cell Membranes Electrical properties defined as ‘idealized’ circuit (conductors, capacitors, voltage-

generators (batteries) Heart is considered a ‘volume’ conductor – a 3-D network of linked resistors Cardiac cell membranes behave like resistor-capacitor (RC) circuits:

Membrane is a capacitor (~1.0µF/cm2) Ion Channel is a resistor

But ion channels allow permeation = Vr results from the Equilibrium Potential (Veq) for each permeant ion

Vm is a function of K, Na, Ca & (Cl) concentration gradients and membrane permeability….

Resting membrane potential Vm is ~ -80 to -90 mV…

Use the Goldman-Hodgkin-Katz (GHK) equation to determine the reversal potential

across a cell's membrane….accounts for permeant ions…

Vr = (RT/F)⋅ln{(PK[K+]o+PNa[Na+]o+PCl[Cl-]i)/(PK[K+]i+PNa[Na+]i+PCl[Cl-]o)}

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Electrical Properties of Cell Membranes The cell membrane potential determines the ability of the myocyte to transmit

electrical activity

Changes in myocyte cell membrane permeability lead to changes in cell membrane potential

Nerbonne, 2000

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Introduction to Ion Channels Ion channels:

Pore-forming integral membrane proteins: Gate ion flow across cell membranes Establish Vm Shape (cardiac) action potential

High rate of transport (106/sec) Only pass ions of certain size/charge

= selective permeability Many ion channel families

- Voltage-gated, Ligand-gated etc….

Voltage-gated ion channels: Activated by changes in electrical Vm potential Composed of subunits forming a central pore Has a ‘voltage-sensor’ & ‘selectivity filter’ Multiple properties:

• I-V relationship • Rectification ‘preferred direction of ion’ • Gating ‘voltage-sensor’

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Properties of Voltage-Gated Ion Channels Current-Voltage Relationship

For a given level of current flow due to the opening of an ion channel there is a change in membrane potential

Voltage = V and Current (I) = flow of Na through channel The current is determined by the conductance (gx) of the channel

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Ion channel Activation/Inactivation (e.g., sodium channel)

Properties of Voltage-Gated Ion Channels

Hodgkin-Huxley (1952)

- gINa in squid giant axon - Steep volt-dep. activation of INa - Membrane ‘gates’ = change Vm - ‘m’ – activation gate; ‘h’ inactivation gate - Proposed ‘voltage-sensor’ (gating current) - ‘Activation’ independent of ‘Inactivation’ - gNa described by gNa = GNa•m3•h - gK described by gK = GK•n4

Voltage-gated Na+ channels exist in 3 distinct states: deactivated (closed) activated (open) inactivated (closed)

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Properties of Voltage-Gated Ion Channels Hodgkin-Huxley (H-H) Model Predicts Action Potentials

The Hodgkin–Huxley model is a “conductance-based model” that accounts for ion fluxes and permeability changes in excitable membranes

It is an empirical kinetic description of the electrical characteristics (AP shape and conduction velocity) of excitable cells - neurons and cardiac myocytes

Total current through membrane:

Action potential and underlying Na and K channel opening calculated from H-H model

Loligo vulgaris

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The Cardiac (Nav1.5) Sodium Channel

- Consists of an α (~2000 aa) + β subunit(s) - α-subunit = repeat domains (DI-DIV), 6 TM segments (S1-S6); S4 voltage sensor - S6 regions form the pore region of the channel - The P loops from each domain comprise the ‘selectivity’ filter - Bell-shaped with 47% of the protein structure cytoplasmic - Toxins (TTX) block by binding the extracellular pore - AA drugs (Lidocaine) block the channel intracellularly - Drug block occurs in domains IS6, IIIS6, IVS6

INa Peak

INa Late

Savio-Galimberti et al., Front Pharmacol. 2012

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- VGCC (Ca2+) (L-type) transduce Vm changes into intracellular Ca2+ transients

- At resting Vm VGCCs are closed; activated (i.e., opened) at depolarized Vm

- Cardiac E-C coupling & pacemaker conduction

- Blockers slow contractility, reduce conduction, antiarrhythmic

- L-type CC possess high affinity, stereoselective-binding domains for channel blocking drugs

The Cardiac (CaV1.2) Calcium Channel

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The Cardiac Potassium Channel(s) - All K+ currents have a similar primary aa sequence - highly conserved structural regions - Activation repolarizes cardiac cell membrane - Constitutive activation (Voltage-independent) maintains cell resting Vm - Many drugs block K channels…

Current Activation Inactivation

Ito,f Fast Fast

Ito,s Fast Slow

IKr Fast Fast

IKs Very Slow N/A

IKur Very Fast N/A

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INa

ICa,L Cav1.2

Dep

olar

izin

g

IK1

Ito,1

Ito,2

IKr

IKs KV7.1 (KvLQT1)

Kv11.1 (hERG)

Kv4.x/KChIP2

Kir2.x

Rep

olar

izin

g

0

Nav1.5

Implications: Ion Channel Heterogeneity

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Suggested Reading

Catterall, W.A. (2012). Voltage-gated sodium channels at 60: structure, function and pathophysiology. J Physiol. Jun 1;590(Pt 11):2577-89. Hille, B. (1976). Gating in sodium channels of nerve. Ann. Rev. Physiol. 38, 139-152. Hille, B. 1992. “Ionic Channels of Excitable Membranes.” Sinauer Associates Inc, Sunderland. Hodgkin, A.L., and Huxley, A.F. (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. (London) 116, 500-544. Nerbonne, J.M. (2000). Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium. J Physiol 525 Pt 2, 285-298. Pugsley, M.K. and Quastel, D.M.J. 1998. “Basic Cardiac Electrophysiology. In “Methods in Cardiac Electrophysiology” (M.J.A. Walker and M.K. Pugsley, Eds.), pp. 1-24. CRC Press, Boca Raton. Striessnig, J. et al. (2014). L-type Ca2+ channels in heart and brain. Wiley Interdiscip Rev Membr Transp Signal. 2014 Mar 1;3(2):15-38.

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