Structure and Physiology

The cardiac Na⁺ channel complex is composed of a primary α and multiple ancillary β subunits. The approximately 2000-amino-acid α subunit contains the channel’s ion-conducting pore and controls the channel selectivity for Na⁺ ions and voltage-dependent gating machinery. This subunit contains all the drug and toxin interaction sites identified to date. The α subunit (Na_v1.5), encoded by the SCN5A gene, consists of four internally homologous domains (I to IV) that are connected to each other by cytoplasmic linkers (Fig. 2-1). Each domain consists of six membrane-spanning segments (S1 to S6), connected to each other by alternating intracellular and extracellular peptide loops. The four domains are arranged in a fourfold circular symmetry to form the channel. The extracellular loops between S5 and S6 (termed the P segments) have a unique primary structure in each domain (Fig. 2-2). The P segments curve back into the membrane to form an ion-conducting central pore whose structural constituents determine the selectivity and conductance properties of the Na⁺ channel.²

FIGURE 2-1 The sodium channel macromolecular complex. See text for discussion.

(From Boussy T, Paparella G, de Asmundis C, et al: Genetic basis of ventricular arrhythmias. Heart Fail Clin 6:249-266, 2010.)

FIGURE 2-2 Transmembrane organization of sodium channel subunits. The primary structures of the subunits of the voltage-gated ion channels are illustrated as transmembrane folding diagrams. Cylinders represent probable α-helical segments: S1 to S3, blue; S4, green; S5, orange; S6, purple; outer pore loop, shaded orange area. Bold lines represent the polypeptide chains of each subunit, with length approximately proportional to the number of amino acid residues in the brain sodium channel subtypes. The extracellular domains of the β₁ and β₂ subunits are shown as immunoglobulin-like folds. Ψ shows sites of probable N-linked glycosylation. P represents sites of demonstrated protein phosphorylation by protein kinase A (red circles) and protein kinase C (red diamonds); h in the blue circle signifies an inactivation particle in the inactivation gate loop; the empty blue circles represent sites implicated in forming the inactivation gate receptor. The structure of the extracellular domain of the β subunits is illustrated as an immunoglobulin-like fold based on amino acid sequence homology to the myelin P0 protein. Sites of binding of α and β scorpion toxins (α-ScTx, β-ScTx) and a site of interaction between α and β₁ subunits also are shown. H₃N and NH₃ = ammonia.

(From Caterall WA, Maier SK: Voltage-gated sodium channels and electrical excitability of the heart. In Zipes DP, Jalife J, editors: Cardiac electrophysiology: from cell to bedside, ed 5, Philadelphia, 2009, Saunders, pp 9-17.)

Four auxiliary β subunits (Na_vβ1 to Na_vβ4, encoded by the genes SCN1B to SCN4B, respectively) have been identified; each is a glycoprotein with a single membrane-spanning segment. The β₁ subunit likely plays a role in modulation of the gating properties and level of expression of the Na⁺ channel.²

Na⁺ channels are the typical example of voltage-gated ion channels. Na⁺ channels switch among three functional states: deactivated (closed), activated (open), and inactivated (closed), depending on the membrane potential (E_m). These channel states control Na⁺ ion permeability through the channel into the cardiomyocyte. Na⁺ channel activation allows Na⁺ ion influx into the cell, and inactivation blocks entry of Na⁺ ions.²

On excitation of the cardiomyocyte by electrical stimuli from adjacent cells, its resting E_m (approximately −85 mV) depolarizes. The positively charged S4 segment of each domain of the α subunit functions as the sensor of the transmembrane voltage; these segments are believed to undergo rapid structural conformational changes in response to membrane depolarization, thus leading to channel opening (activation) from its resting (closed) state and enabling a large and rapid influx of Na⁺ (inward Na⁺ current [I_Na]) during the rapid upstroke (phase 0) of the action potential in atrial, ventricular, and Purkinje cardiomyocytes.³

Normally, activation of Na⁺ channels is transient; fast inactivation (closing of the pore) starts simultaneously with activation, but because inactivation is slightly delayed relative to activation, the channels remain transiently open to conduct I_Na during phase 0 of the action potential before it closes. Each Na⁺ channel opens very briefly (less than 1 millisecond) during phase 0 of the action potential; collectively, activation of the channel lasts a few milliseconds and is followed by fast inactivation.

Na⁺ channel inactivation comprises different conformational states, including fast, intermediate, and slow inactivation. Fast inactivation is at least partly mediated by rapid occlusion of the inner mouth of the pore by the cytoplasmic interdomain linker between domains III and IV of the α subunit, which has a triplet of hydrophobic residues that likely functions as a hinged “latch” that limits or restricts Na⁺ ion pass through the pore. The carboxyl terminus (C-terminus) also plays an important role in the control of Na⁺ channel inactivation and stabilizing the channels in the inactivated state by interacting with the loop linking domains III and IV. Importantly, although most Na⁺ channels open before inactivating, some actually inactivate without ever opening (a process known as closed-state inactivation).^1,4

Once inactivated, Na⁺ channels do not conduct any more current and cannot be reactivated (reopened) until after recovery from inactivation. The recovery of the Na⁺ channel to reopen is voltage dependent. Channel inactivation is removed when the E_m of the cell repolarizes during phase 4 of the action potential. Membrane repolarization is facilitated by the fast inactivation of the Na⁺ channels (limiting the inward current) and is augmented by activation of voltage-gated K⁺ channels (allowing the outward current). The recovery of channels from inactivation is also time dependent; Na⁺ channels typically activate within 0.2 to 0.3 milliseconds and inactivate completely within 2 to 5 milliseconds.

Following recovery, Na⁺ channels enter a closed state that represents a nonconducting conformation, which allows the channels to be activated again during the next action potential. The fraction of channels available for opening varies from almost 100% at −90 mV and 50% at −75 mV to almost 0% at +40 mV. Consequently, highly polarized (−80 to −90 mV) cell membranes can be depolarized rapidly by stimuli because more Na⁺ channels reopen, whereas partially depolarized cells with potentials close to threshold −70 mV generate a much slower upstroke because of the inactivation of a proportion of Na⁺ channels. Given that Na⁺ channels are major determinants of conduction velocity, this velocity generally slows at a reduced E_m.

Na⁺ channel activation, inactivation, and recovery from inactivation occur within a few milliseconds. At the end of phase 1 of the action potential, more than 99% of Na⁺ channels transit from an open (activated) state to an inactivated state. However, a very few Na⁺ channels are not inactivated and may reactivate (reopen) during action potential phase 3. The small current produced by these channels (less than 1% of the peak I_Na) is called the “window” current because it arises when the sarcolemma reaches a potential that is depolarized sufficiently to reactivate some channels, but not enough to cause complete inactivation. The voltage range for the window current is very restricted and narrow in healthy hearts, thus granting it a small role during the cardiac action potential.

In addition to these rapid gating transitions, Na⁺ channels are also susceptible to slower inactivating processes (slow inactivation) if the membrane remains depolarized for a longer time. These slower events can contribute to the availability of active channels under various physiological conditions. Whereas fast-inactivated Na⁺ channels recover rapidly (within 10 milliseconds) during the hyperpolarized interval between stimuli, slow inactivation requires much longer recovery times (ranging from hundreds of milliseconds to many seconds). The molecular movements leading to slow inactivation are less well understood. The P segments seem to play a key role in slow inactivation.⁴

Some Na⁺ channels occasionally show alternative gating modes consisting of isolated brief openings occurring after variable and prolonged latencies and bursts of openings during which the channel opens repetitively for hundreds of milliseconds. The isolated brief openings are the result of the occasional return from the inactivated state. The bursts of openings are the result of occasional failure of inactivation.¹ Prolonged opening or reopening of some Na⁺ channels during phases 2 and 3 can result in a small late I_Na (I_NaL). Despite its minor contribution in healthy hearts, I_NaL can potentially play an important role in diseased hearts.³

Function

Na⁺ channels play a pivotal role in the initiation, propagation, and maintenance of the normal cardiac rhythm. The I_Na determines excitability and conduction in atrial, His-Purkinje system (HPS), and ventricular myocardium. On membrane depolarization, the voltage-gated Na⁺ channels respond within a millisecond by opening, thus leading to the very rapid depolarization of the cardiac cell membrane (phase 0 of the action potential), reflected by the fast (within tenths of a microsecond) subsequent opening of Na⁺ channels triggering the excitation-contraction coupling. Na⁺ entry during phase 0 of the action potential also modulates intracellular Na⁺ levels and, through Na⁺-Ca²⁺ exchange, intracellular Ca²⁺ concentration and cell contraction.

The cardiac Na⁺ channel also plays a crucial role in the propagation of action potentials throughout the atrium, HPS, and ventricles. The opening of Na⁺ channels in the atria underlies the P wave on the ECG, and in the ventricles I_Na underlies the QRS complex and enables a synchronous ventricular contraction. Because the upstroke of the electrical potential primarily determines the speed of conduction between adjacent cells, Na⁺ channels are present in abundance in tissues where speed is of importance. Cardiac Purkinje cells contain up to 1 million Na⁺ channels, a finding that illustrates the importance of rapid conductance in the heart.²

Na⁺ channels also make a contribution in the plateau phase (phase 2) and help determine the duration of the action potential. After phase 0 of the action potential, I_Na decreases to less than 1% of its peak value over the next several milliseconds because of voltage-dependent inactivation. This persistent or “late” inward I_Na (I_NaL), along with the L-type Ca²⁺ current (I_CaL), helps maintain the action potential plateau.⁵

Furthermore, Na⁺ channel inactivation is very important as it prevents cells from being prematurely reexcited because of the unavailability of the voltage-gated Na⁺ channels. With repolarization, the Na⁺ channel normally recovers rapidly from inactivation (within 10 milliseconds) and is ready to open again. Hence, Na⁺ channels help to determine the frequency of action potential firing. To a lesser extent, cardiac Na⁺ channels are also present in the sinus node and the atrioventricular node (AVN), where they contribute to pacemaker activity.

Regulation

The regulatory proteins interacting with Na_v1.5 may be classified as follows: (1) anchoring-adaptor proteins (e.g., ankyrin-G, syntrophin proteins, multicopy suppressor of gsp1 [MOG1]), which play roles only in trafficking and targeting the channel protein in specific membrane compartments; (2) enzymes interacting with and modifying the channel structure (post-translational modifications), such as protein kinases or ubiquitin ligases; and (3) proteins modulating the biophysical properties of Na_v1.5 on binding (e.g., caveolin-3, calmodulin, glycerol 3-phosphate dehydrogenase 1–like [G3PD1L], telethonin, Plakophilin-2).⁵ Coexpression of Na_v1.5 with its β subunits induces acceleration in the recovery from inactivation and enhancement of I_Na amplitude.

The cardiac Na⁺ channels are subject to phosphorylation and dephosphorylation by kinases or phosphatases. The intracellular linker between domains I and II contains eight consensus sites for cyclic adenosine monophosphate (cAMP)–dependent protein kinase A (PKA) phosphorylation. cAMP-dependent PKA and G protein stimulatory α subunit (Gsα) modulate the function of expressed cardiac Na⁺ channels on β-adrenergic stimulation and enhance I_Na.¹

In contrast, activation of α-adrenergic stimulating protein kinase C (PKC) results in the reduction of I_Na. The effect of PKC is largely attributable to phosphorylation of a highly conserved serine in the linker between domains III and IV. PKC reduces the maximal conductance of the channels and alters gating. Na⁺ channels exhibit a hyperpolarizing shift in the steady-state availability curve, suggesting an enhancement of inactivation from closed states.

All subunits of the Na⁺ channel are modified by glycosylation. The β₁ and β₂ subunits are heavily glycosylated, with up to 40% of the mass being carbohydrate. In contrast, the cardiac α subunit is only 5% sugar by weight. Sialic acid is a prominent component of the N-linked carbohydrate of the Na⁺ channel. The addition of such a highly charged carbohydrate has predictable effects on the voltage dependence of gating through alteration of the surface charge of the channel protein.¹

Pharmacology

Na⁺ channels are the targets for the action of class I antiarrhythmic drugs. Na⁺ channel blockers bind to a specific receptor within the channel’s pore. The binding blocks ion movement through the pore and stabilizes the inactivated state of Na⁺ channels. Blockade of Na⁺ channels tends to decrease tissue excitability and conduction velocity (by attenuating peak I_Na) and can shorten action potential duration (by attenuating late I_Na).^1,6

One important component in the action of antiarrhythmic drugs is a voltage-dependent change in the affinity of the drug-binding site (i.e., the channel is a modulated receptor). Additionally, restricted access to binding sites can contribute to drug action, a phenomenon that has been called the guarded receptor model. Open and inactivated channels are more susceptible to block than resting channels, likely because of a difference in binding affinity or state-dependent access to the binding site. Consequently, binding of antiarrhythmic drug occurs primarily during the action potential (known as use-dependent block), and the block dissipates after repolarization (i.e., in the interval between action potentials). When the time interval between depolarizations is insufficient for block to recover before the next depolarization occurs (secondary to either abbreviation of the interval between action potentials during fast heart rates or slow kinetics of the unbinding of the Na⁺ channel blocker), block of Na⁺ channels accumulates (resulting in an increased number of blocked channels and enhanced blockade).⁶ A drug with rapid kinetics produces less channel block with the subsequent depolarization than does a drug with slower recovery. Use-dependent block is important for the action of antiarrhythmic drugs because it allows strong drug effects during fast heart rates associated with tachyarrhythmias but limits Na⁺ channel block during normal heart rates. Importantly, drug recovery kinetics can potentially be slowed by pathophysiological conditions such as membrane depolarization, ischemia, and acidosis.¹ This property is known as use-dependence and is seen most frequently with the class IC agents, less frequently with the class IA drugs, and rarely with the class IB agents.

Class I antiarrhythmic drugs can be classified into three groups according to rates of drug binding to and dissociation from the channel receptor. Class IC drugs (flecainide and propafenone) block both the open and inactivated state (which is induced by depolarization) Na⁺ channels and have the slowest kinetics of unbinding during diastole. The results are prolongation of conduction at normal heart rates and a further increase in the effect at more rapid rate (use-dependence).

The class IB agents (lidocaine, mexiletine, and tocainide) block both open and inactivated Na⁺ channels and dissociate from the channel more rapidly than do other class I drugs. As a consequence, class IB drugs exhibit minimal or no effects on the Na⁺ channels in normal tissue but cause significant conduction slowing in depolarized tissue, especially at faster depolarization rates. Furthermore, class IB drugs are less effective in the atrium, where the action potential duration is so short that the Na⁺ channel is in the inactivated state only briefly compared with the relatively long diastolic recovery times; thus, accumulation of block is less likely to result from the rapid recovery of block.

Class IA drugs (quinidine, procainamide, and disopyramide) exhibit open state block, have intermediate effects on Na⁺ channels, and generally only cause significant prolongation of conduction in cardiac tissue at rapid heart rates. Because the open state block is dominant and recovery from block is slow, these drugs are effective in both the atrium (where action potential duration is short) and the ventricle (where the action potential duration is long).

The late I_Na (I_NaL) also can be a target for blockade. Several drugs exhibit relative selectivity for block of late I_NaL over peak I_Na, including mexiletine, flecainide, lidocaine, amiodarone, and ranolazine.

Importantly, class IA drugs also have moderate K⁺ channel blocking activity (which tends to slow the rate of repolarization and prolong the action potential duration) and anticholinergic activity, and they tend to depress myocardial contractility. At slower heart rates, when use-dependent blockade of I_Na is not significant, K⁺ channel blockade becomes predominant (reverse use-dependence), leading to prolongation of the action potential duration and QT interval and increased automaticity. Flecainide and propafenone also have K⁺ channel blocking activity and can increase the action potential duration in ventricular myocytes. Propafenone has significant β-adrenergic blocking activity.

Inherited Channelopathies

Mutations in genes that encode various subunits of the cardiac Na⁺ channel or proteins involved in regulation of the inward I_Na have been linked to several types of electrical disorders (Table 2-1). Depending on the mutation, the consequence is either a gain of channel function (with consequent prolongation of action potential duration because more positive ions accumulate in the cell) or an overall loss of channel function that influences the initial depolarizing phase of the action potential (with consequent decrease in cardiac excitability and electrical conduction velocity). It is noteworthy that a single mutation can cause different phenotypes or combinations thereof.²

Acquired Diseases

In heart failure, peak I_Na is reduced (likely secondary to reduced SCN5A expression), whereas I_NaL is increased (likely because of increased phosphorylation of Na⁺ channels). Na_v1.5 expression is reduced in the surviving myocytes in the border zone of the myocardial infarct (MI). Importantly, Na⁺ channel blockers can increase the risk for SCD in patients with ischemic heart disease, possibly by facilitating the initiation of reentrant excitation waves. Additionally, I_NaL increases during myocardial ischemia, explaining why I_NaL inhibition may be an effective therapy for chronic stable angina. Na_v1.5 expression is reduced in response to persistent atrial tachyarrhythmias as part of the “electrical remodeling” process, leading to attenuation of I_Na.³

Furthermore, mutations in SCN5A can predispose affected individuals to acquired LQTS induced by a variety of drugs such as antihistamines or antibiotics. These mutations result in changes in channel activity that exert a significant impact on action potential duration only when combined with drug-induced alteration of other channels.

Structure and Physiology

Cardiac K⁺ channels are membrane-spanning proteins that allow the passive movement of K⁺ ions across the cell membrane along its electrochemical gradient. The ion-conducting or pore-forming subunit is generally referred to as the α subunit. The tripeptide sequence glycine-tyrosine-glycine GYG is common to the pore of all K⁺ channels and constitutes the signature motif for determining K⁺ ion selectivity. A gating mechanism controls switching between open-conducting and closed-nonconducting states.^1,15

K⁺ channels represent the most diverse class of cardiac ion channels (Fig. 2-3). Cardiac K⁺ currents can be categorized as voltage-gated (K_v) and ligand-gated channels. In K_v channels, pore opening is coupled to the movement of a voltage sensor within the membrane electric field, and they include the rapidly activating and inactivating transient outward current (I_to); the ultrarapid (I_Kur), rapid (I_Kr), and slow (I_Ks) components of the delayed rectifier current; and the inward rectifier current (I_K1). In contrast, pore opening in ligand-gated channels is coupled to the binding of an organic molecule, including channels activated by a decrease in the intracellular concentration of adenosine triphosphate (K_ATP) or activated by acetylcholine (K_ACh). Other classes of K⁺ channels respond to different stimuli, including changes in intracellular Ca²⁺ concentration and G proteins.¹⁵

FIGURE 2-3 Molecular compositions of cardiac potassium (K⁺) channels. Amino termini (N) and carboxyl termini (C) are indicated. A, Voltage-gated (K_v), inward rectifier (Kir), and two-pore α subunits are integral membrane proteins with six, two, and four membrane-spanning domains, respectively. B, These α subunits assemble as tetramers or dimers to form K⁺-selective pores.

(Modified from Oudit GY, Backx PH: Voltage-regulated potassium channels. In Zipes DP, Jalife J, editors: Cardiac electrophysiology: from cell to bedside, ed 5, Philadelphia, 2009, Saunders, pp 29-42.)

On the basis of the primary amino acid sequence of the α subunit, K⁺ channels have been classified into three major families (Table 2-2):

Each voltage-gated K⁺ channel (K_v family) is formed by the coassembly of 4 identical (homotetramers) or a combination of 4 different (from the same subfamily, heterotetramers) α subunits. A total of 38 genes has been cloned and assigned to 12 subfamilies of voltage-gated K channels (K_v1 to K_v12) on the basis of sequence similarities. Each α subunit contains one domain consisting of 6 membrane-spanning segments (S1 to S6), connected to each other by alternating intracellular and extracellular peptide loops (similar to 1 of the 4 domains of voltage-gated Na⁺ and Ca²⁺ channels), with both the amino terminus (N-terminus) and the C-terminus located on the intracellular side of the membrane. The central ion-conducting pore region is formed by the S5 and S6 segments and the S5-S6 linker (P segment); the S5-S6 linker is responsible for K⁺ ion selectivity. The S4 segment serves as the voltage sensor.^1,15

The α subunits of K_v channels can generate voltage dependent K⁺ current when expressed in heterologous systems. However, the assembly of a functional tetramer can occur only in the presence of multiple auxiliary units (see Table 2-2). In many cases, auxiliary subunits coassociate with the α subunits and likely modulate cell surface expression, gating kinetics, and drug sensitivity of the α subunit complex. Most K⁺ channel β subunits assemble with α subunits and give rise to an α₄β₄ complex. K⁺ channel β subunits represent a diverse molecular group, which includes cytoplasmic proteins (K_vβ1 to K_vβ3, KChIP, and KChAP) that interact with the intracellular domains of K_v channels; single transmembrane spanning proteins (e.g., minK and minK-related proteins [MiRPs]) encoded by the KCNE gene family; and large ATP-binding cassette (ABC) transport-related proteins (e.g., the sulfonylurea receptors [SURs]).^1,16

The diversity of K⁺ currents in native tissues exceeds the number of K⁺ channel genes identified. The explanations for this diversity include alternative splicing of gene products, post-translational modification, and heterologous assembly of β subunits within the same family and assembly with accessory β subunits that modulate channel properties.

As with voltage-dependent Na⁺ and Ca²⁺ channels, K_v channels typically fluctuate among distinct conformational states because of molecular movements in response to voltage changes across the cell membrane (voltage-dependent gating). The K_v channel activates (opens) on membrane depolarization, thus allowing the rapid passage of K⁺ ions across the sarcolemma. After opening, the channel undergoes conformational transition in a time-dependent manner to a stable nonconducting (inactivated) state. Inactivated channels are incapable of reopening, even if the transmembrane voltage is favorable, unless they “recover” from inactivation (i.e., enter the closed state) on membrane repolarization. Closed channels are nonconducting but can be activated on membrane depolarization.¹⁷

Three mechanistically distinct types of K_v channel inactivation that are associated with distinct molecular domains have been identified: N-type, C-type, and U-type. N-type (“ball and chain”) inactivation involves physical occlusion of the intracellular mouth of the channel pore through binding of a small group of amino acids (“inactivation ball tethered to a chain”) at the extreme N-terminus. In contrast, C-type inactivation involves conformational changes in the external mouth of the pore. C-type inactivation exists in almost all K⁺ channels and may reflect a slow constriction of the pore. This inactivation process is thought to be voltage independent, coupled to channel opening, and is usually slower than N-type inactivation. Recovery from C-type inactivation is relatively slow and weakly voltage dependent. Importantly, the rate of C-type inactivation and recovery can be strongly influenced by other factors, such as N-type inactivation, drug binding, and changes in extracellular K⁺ concentration. These interactions render C-type inactivation an important biophysical process in regulating repetitive electrical activity and determining certain physiological properties such as refractoriness, drug binding, and sensitivity to extracellular K⁺.¹⁷

In addition to N-type and C-type inactivation, some K_v channels also show another type of inactivation (U-type), which exhibits a U-shaped voltage dependence with prolonged stimulation rates. Those channels appear to exhibit preferential inactivation from partially activated closed states, rapid and strongly voltage-dependent recovery from inactivation and, in some channel types, accelerated inactivation with elevation of extracellular K⁺. The exact conformational changes underlying U-type inactivation remain unclear. Importantly, there is extreme diversity in the kinetic and potentially molecular properties of K_v channel inactivation, particularly of C-type inactivation.¹⁷

Function

K⁺ channels are a diverse and ubiquitous group of membrane proteins that regulate K⁺ ion flow across the cell membrane on the electrochemical gradient and regulate the resting E_m, the frequency of pacemaker cells, and the shape and duration of the cardiac action potential. Because the concentration of K⁺ ions outside the cell membrane is approximately 25-fold lower than that in the intracellular fluid, on activation, the opening of K⁺ channels generates an outward current resulting from the efflux of positively charged ions that offers a mechanism to counteract, dampen, or restrict the depolarization front (phases 1 through 4 of the action potential) triggered by an influx of cations (Na⁺ and Ca²⁺).^1,3,15,16

The variation in the level of expression of K⁺ channels that participate in the genesis of the cardiac action potential explains the regional differences of the configuration and duration of cardiac action potentials from sinus node and atrial to ventricular myocytes and across the myocardial wall (endocardium, midmyocardium, and epicardium). Moreover, the expression and properties of K⁺ channels are not static; heart rate, neurohumoral state, pharmacological agents, cardiovascular diseases (cardiac hypertrophy and failure, MI) and arrhythmias (e.g., AF) can influence those properties, and they underlie the change in action potential configuration in response to variation in heart rate and various physiological and pathological conditions.^1,15,16

Transient Outward Potassium Current (I_to)

Ultrarapidly Activating Delayed Outward Rectifying Current (I_Kur)