Voltage-Gated Sodium Channels and Electrical Excitability of the Heart

Introduction

Voltage-gated sodium channels initiate action potentials in nerves, skeletal muscles, cardiac myocytes, and other excitable cells, ^, and they are responsible for the propagation of action potentials through the atria, conduction system, and ventricles of the heart (see Chapter 9 ). As shown in Fig. 1.1 , action potentials in atrial and ventricular muscle fibers rise very rapidly from a resting potential near –80 mV and reach their peak within one millisecond. During this brief interval, cardiac sodium channels respond to the change in pacemaker potential as it reaches threshold, and they open to allow for rapid Na ⁺ entry. Sodium channels begin to inactivate as soon as they open and inactivate to 98% or 99% completion within a few milliseconds. The plateau phase of the cardiac action potential is generated by the opening of voltage-gated calcium channels (see Chapter 2 ), and the cell is finally repolarized by the slower opening of voltage-gated potassium channels (see Chapter 3 ). The rate of conduction of the action potential through the cardiac tissue depends directly on the rate of rise of the cardiac action potential and therefore on the density of the active sodium channels and their rate of activation.

Much is now known about the molecular mechanisms of activation, inactivation, and ion conduction by the sodium channel protein, as summarized in this chapter. Multiple genes encode sodium channel subunits, and the distinct sodium channel subtypes have subtle differences in functional properties and differential distribution in subcellular compartments of cardiac myocytes. These differences in function and localization may contribute to the specialized functional roles of sodium channels in cardiac physiology and pharmacology.

Subunit Structure of Sodium Channels

Sodium channel proteins purified from excitable cells are complexes composed of around a 260-kDa α-subunit in association with one or two auxiliary β-subunits of around 33 to 39 kDa in size. Purified sodium channel complexes of α- and β-subunits are sufficient for voltage-dependent gating and ion conduction in artificial lipid membranes, and expression of the α-subunit alone is sufficient for physiologic function in recipient nonexcitable cells, indicating that this subunit has all of the necessary structural elements for voltage-dependent gating and ion conduction. The primary sequence predicts that the sodium channel α-subunit will fold into four internally repeated domains (I–IV), each of which will contain six α-helical transmembrane segments (S1-S6; Fig. 1.2 ). ^, In each domain, the S1 through S4 segments serve as the voltage-sensing module, and the S5 and S6 segments and the reentrant P loop between them serve as the pore-forming module. One large extracellular loop connects either the S5 or S6 transmembrane segment to the P loop in each domain, whereas the other extracellular loops are small. Large intracellular loops link the four homologous domains, and the large N-terminal and C-terminal domains also contribute substantially to the mass of the intracellular face of sodium channels. This view of sodium channel architecture, originally derived from a hydrophobicity analysis of the amino acid sequence, ^, has been largely confirmed by biochemical, electrophysiologic, and structural experiments. ^,

The auxiliary β1- and β2-subunits were identified in the initial purification studies of sodium channels. These subunits have a single transmembrane segment, a large N-terminal extracellular domain, and a short C-terminal intracellular segment (see Fig. 1.2 ). ^, The β-subunits interact with α-subunit extracellular domains, modulate α-subunit function, and enhance their cell surface expression. They also serve as cell adhesion molecules by interacting with extracellular matrix proteins, cell adhesion molecules, signaling proteins, and cytoskeletal linker proteins. These interactions are thought to localize and stabilize sodium channels in specific subcellular compartments and to bring crucial signaling molecules to the sodium channel to regulate it. Deletion of the genes encoding β-subunits causes alterations in sodium channel function, reduced action potential conduction and abnormal development of myelin folds in axons, hyperexcitability and epilepsy in the brain, and arrhythmias in the heart.

Sodium Channel Genes

Sodium channels are the founding members of the ion channel superfamily, which includes voltage-gated calcium channels; transient receptor potential (TRP) channels; voltage-gated, inward rectifying, and two-pore-domain potassium channels; and cyclic nucleotide-gated (CNG) and hyperpolarization activated cyclic nucleotide-gated (HCN) channels. Many of these ion channels are important for generating the action potential of the heart, including voltage-gated calcium channels (see Chapter 10, Chapter 2 ) and voltage-gated potassium channels (see Chapter 20, Chapter 3 ). In evolution, the four-domain sodium channel was last among the voltage-gated ion channels to appear, and it is only found in complex single-celled eukaryotes and in multicellular organisms. It is thought that sodium channels evolved through two rounds of gene duplication from ancestral single-domain bacterial sodium channels. Voltage-gated sodium channel genes are present in a variety of metazoan species, including the fly, leech, squid, and jellyfish. The biophysical properties, pharmacology, gene organization, and even intron-splice sites of these invertebrate sodium channels are largely similar to the mammalian sodium channels.

Ten related sodium channel genes are found in vertebrates, and nine encode voltage-gated sodium channels ( Fig. 1.3 ). ^, More than 20 exons comprise each of the sodium channel α-subunit genes in mammals. Genes encoding sodium channels Na _V 1.1, Na _V 1.2, Na _V 1.3, and Na _V 1.7 are localized on chromosome 2 in humans, and these channels share similarities in sequence, biophysical characteristics, block by nanomolar concentrations of tetrodotoxin, and broad expression in neurons. A second cluster of genes encoding Na _V 1.5, Na _V 1.8, and Na _V 1.9 channels is localized to human chromosome 3p21-24. Although they are more than 75% identical in amino acid sequence to the group of channels on chromosome 2, these sodium channels all contain amino acid substitutions that confer varying degrees of resistance to the pore blocker tetrodotoxin. In Na _V 1.5, the principal cardiac isoform encoded by the SCN5A gene, a single amino acid change from phenylalanine to cysteine in the pore region of domain I is responsible for a 200-fold reduction in tetrodotoxin sensitivity compared with those channels on chromosome 2. At the identical position in Na _V 1.8 and Na _V 1.9, the amino acid residue is serine, and this change results in even greater resistance to tetrodotoxin. These two channels are primarily expressed in peripheral sensory neurons. Compared with the sodium channels on chromosomes 2 and 3, Na _V 1.4, which is expressed in skeletal muscle, and Na _V 1.6, which is highly abundant in the central nervous system (CNS), have greater than 85% sequence identity and similar functional properties, including tetrodotoxin sensitivity in the nanomolar concentration range. A tenth sodium channel, Na _x , whose gene is located near the sodium channels of chromosome 2, is evolutionarily more distant. It has key differences in functionally important regions of voltage sensors and inactivation gates. It is likely that this unusual sodium channel responds to extracellular sodium concentration and is involved in the regulation of plasma salt levels.

The Na _V β-subunits are encoded by four distinct genes in mammals. ^{, , ,} The gene encoding β1 maps to human chromosome 19q13, whereas β2 and β4 are located on chromosome 11q22-23 and β3 is located nearby on chromosome 11q24. The β1- and β3-subunits associate noncovalently with the α-subunits, whereas β2 and β4 are covalently linked by a disulfide bond. These distinct modes of association and the corresponding similarities in amino acid sequence suggest that the similar pairs of β-subunits (β1 and β3 vs. β2 and β4) may be able to substitute for each other when interacting with sodium channel α-subunits. All four of the β-subunits are expressed in the heart. ^,

Three-Dimensional Structure of Sodium Channels

Sodium channel architecture was revealed in three dimensions by determining the crystal structure of the bacterial sodium channel Na _V Ab at high resolution (2.7Å; see Fig. 1.3 ). This ancient sodium channel has a very simple structure—four identical subunits where each is similar to one homologous domain of a mammalian sodium channel, without the large intracellular and extracellular loops of the mammalian protein ( Fig. 1.4A–C ). This structure has revealed a wealth of new information about the molecular basis for voltage-dependent gating, sodium selectivity and conductance, and blockage of the channel by therapeutically important drugs. More recently, the structure of the larger and more complicated mammalian cardiac sodium channel has been determined by cryogenic electron microscopy (cryo-EM; see Fig. 1.4D–F ). Because the basics of sodium channel structure and function are easier to visualize in the simpler bacterial sodium channel, we begin with consideration of that structure.

As viewed from the top, Na _V Ab has a central pore surrounded by four pore-forming modules composed of S5 and S6 segments and the intervening pore loop (see Fig. 1.4B , blue ). Four voltage-sensing modules composed of S1 to S4 segments are symmetrically located around the outer rim of the pore module ( Fig. 1.4B , red, green ). The transmembrane architecture of Na _V Ab shows that the adjacent subunits have swapped their functional domains such that each voltage-sensing module is most closely associated with the pore-forming module of its neighbor (see Fig. 1.4B–C ). It is likely that this domain-swapped arrangement enforces concerted gating of the four subunits or domains of sodium channels.

The mammalian cardiac sodium channel Na _V 1.5 has a similar core structure of transmembrane segments and pore, but they are embedded in a much larger and more complex overall structure (see Fig. 1.4E–F ). The four domains of Na _V 1.5 are positioned in a pseudosymmetric, nearly square array rather than in a strictly symmetrical square array as in the case of Na _V Ab, but they are organized in a domain-swapped manner as for the bacterial sodium channels. The structures of the transmembrane cores of Na _V Ab and Na _V 1.5 can be overlaid with less than 3 Å difference, essentially at the resolution of the structures ; therefore, it is likely that the core functions of sodium channels have a similar structural basis from bacteria to man. Nevertheless, there are important adaptations of the mammalian cardiac sodium channels that are conferred by changes in amino acid side chains in the context of the similar core structures, as described in the next section on structure and function.

Comparisons of the primary structures of the isoforms of auxiliary β-subunits to those of other proteins revealed a close structural relationship to the family of proteins that contain immunoglobulin-like folds, which include many cell-adhesion molecules. ^{, , ,} The extracellular domains of these type I single-membrane-spanning proteins were predicted to fold in a similar manner to myelin protein P0, whose immunoglobin-like fold is formed by a sandwich of two β sheets held together by hydrophobic interactions (see Fig. 1.2 ). Three-dimensional structures of the extracellular domains of the β3 and β4 subunits ^, and both the complete Na _V β1 and Na _V β2-subunits, bound to the intact sodium channels from nerve and skeletal muscle, ^, confirmed the predictions from the homology analysis. Like typical cell adhesion molecules, Na _V β-subunits interact with extracellular matrix molecules, other cell adhesion molecules, and intracellular cytoskeletal proteins and signaling proteins. In addition, homophilic and heterophilic interactions of β-subunits have been demonstrated at both the cellular and structural levels. ^, Nevertheless, in contrast to the neuronal and skeletal muscle sodium channels, the structure of the cardiac sodium channel Na _V 1.5 did not reveal associated Na _V β-subunits. Differences in amino acid residues at the key points of interaction of the β-subunits suggest that their binding to Na _V 1.5 may have lower affinity than for sodium channels in nerve and muscle.