Introduction

Voltage-gated sodium channels are responsible for action potential initiation and propagation in excitable cells, including nerve, muscle, and neuroendocrine cell types. They are also expressed at low levels in nonexcitable cells, where their physiological role is unclear. Sodium channels are the founding members of the ion channel superfamily in terms of their discovery as a protein and determination of their amino acid sequence. This article presents an introduction to their biochemical, molecular, and genetic properties, physiological roles, and pharmacological significance.

Sodium Channel Subunits

Sodium channels consist of a highly processed α subunit, which is approximately 260 kDa, associated with auxiliary β subunits (Catterall, 2000). Sodium channels in the adult central nervous system and heart contain β₁ through β₄ subunits, whereas sodium channels in adult skeletal muscle have only the β₁ subunit (Isom, 2001). The pore-forming α subunit is sufficient for functional expression, but the kinetics and voltage dependence of channel gating are modified by the β subunits, and these auxiliary subunits are involved in channel localization and interaction with cell adhesion molecules, extracellular matrix, and intracellular cytoskeleton. The α subunits are organized in four homologous domains (I-IV), each of which contain six transmembrane α helices (S1-S6) and an additional pore loop located between the S5 and S6 segments (Fig. 1). The pore loops line the outer, narrow entry to the pore, whereas the S5 and S6 segments line the inner, wider exit from the pore. The S4 segments in each domain contain positively charged amino acid residues at every third position. These residues serve as gating charges and move across the membrane to initiate channel activation in response to depolarization of the membrane. The short intracellular loop connecting homologous domains III and IV serves as the inactivation gate, folding into the channel structure and blocking the pore from the inside during sustained depolarization of the membrane.

Sodium Channel Classification and Nomenclature

A variety of different sodium channels has been identified by electrophysiological recording, biochemical purification, and cloning (Goldin, 2001). The sodium channels are members of the superfamily of ion channels that includes voltage-gated potassium and calcium channels (Yu and Catterall, 2004); however, unlike the different classes of potassium and calcium channels, the functional properties of the known sodium channels are relatively similar. Despite their similarity of function, the sodium channels were originally named in many different ways, with no consistent nomenclature for the various isoforms. To eliminate confusion resulting from the multiplicity of names, a standardized nomenclature was developed for voltage-gated sodium channels (Goldin et al., 2000). This nomenclature is based on that for voltage-gated potassium channels (Chandy and Gutman, 1993). It uses a numerical system to define subfamilies and subtypes based on similarities between the amino acid sequences of the channels. A comparable nomenclature has also been adopted for voltage-gated calcium channels (Ertel et al., 2000; Catterall et al., 2005). In this nomenclature system, the name of an individual channel consists of the chemical symbol of the principal permeating ion (Na) with the principal physiological regulator (voltage) indicated as a subscript (Na_V). The number following the subscript indicates the gene subfamily (currently only Na_V1), and the number following the full point identifies the specific channel isoform (e.g., Na_V1.1). This last number has been assigned according to the approximate order in which each gene was identified. Splice variants of each family member are identified by lowercase letters following the numbers (e.g., Na_V1.1a).

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Fig. 1.

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. 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, sites of probable N-linked glycosylation; P, sites of demonstrated protein phosphorylation by protein kinase A (circles) and protein kinase C (diamonds); shaded, pore-lining S5-P-S6 segments; white circles, the outer (EEDD) and inner (DEKA) rings of amino residues that form the ion selectivity filter and tetrodotoxin binding site; ++, S4 voltage sensors; h in shaded circle, inactivation particle in the inactivation gate loop; open shaded circles, sites implicated in forming the inactivation gate receptor. Sites of binding of α- and β-scorpion toxins and a site of interaction between α and β1 subunits are also shown.

The nine mammalian sodium channel isoforms that have been identified and functionally expressed are all greater than 50% identical in amino acid sequence in the transmembrane and extracellular domains, where the amino acid sequence is similar enough for clear alignment (Fig. 2A). For potassium channels and calcium channels, all members of distinct subfamilies are less than 50% identical to those of other families, and there is much closer sequence similarity within families (Chandy and Gutman, 1993; Ertel et al., 2000). The sodium channel sequences vary more continuously, without defining separate families. By this criterion, all of the nine sodium channel isoforms may be considered members of one family.

Sodium Channel Genes

To test this hypothesis more critically, the nine sodium channel amino acid sequences were aligned and compared for relatedness using a maximum parsimony procedure that measured their evolutionary distance by calculating the number of nucleotide changes required for the change in codon at each position (Fig. 2B). The resulting phylogenetic tree is consistent with designation of these sodium channels as a single family. Na_V1.1, Na_V1.2, Na_V1.3, and Na_V1.7 are the most closely related group by this analysis. All four of these sodium channels are highly tetrodotoxin-sensitive and are broadly expressed in neurones. Their genes are all located on human chromosome 2q23-24, consistent with a common evolutionary origin. Na_V1.5, Na_V1.8, and Na_V1.9 are also closely related (Fig. 2B), and their amino acid sequences are greater than 64% identical to those of the four sodium channels encoded on chromosome 2. These sodium channels are tetrodotoxin-resistant to varying degrees due to changes in amino acid sequence at a single position in domain I, and they are highly expressed in heart and dorsal root ganglion neurons (Fozzard and Hanck, 1996; Catterall, 2000). Their genes are located on human chromosome 3p21-24, consistent with a common evolutionary origin. The isoforms Na_V1.4, expressed primarily in skeletal muscle, and Na_V1.6, expressed primarily in the central nervous system, are set apart from these other two closely related groups of sodium channel genes (Fig. 2B). Although their amino acid sequences are greater than 84% identical to the group of sodium channels whose genes are located on chromosome 2 (Fig. 2A), their phylogenetic relationship is much more distant when analyzed by parsimony comparison (Fig. 2B). This distant evolutionary relationship is consistent with the location of the genes encoding these two sodium channels on chromosomes 17q23-25 and 12q13, respectively. The chromosome segments carrying the sodium channel genes are paralogous segments that contain many sets of related genes, including the homeobox gene clusters. These segments were generated by whole genome duplication events during early vertebrate evolution (Plummer and Meisler, 1999). The comparisons of amino acid sequence identity and phylogenetic and chromosomal relationships lead to the conclusion that all nine members of the sodium channel family that have been functionally expressed are members of a single family of proteins and have arisen from gene duplications and chromosomal rearrangements relatively recently in evolution. These results contrast with those for potassium channels and calcium channels, for which distinct gene families have arisen earlier in evolution and have been maintained as separate families to the present (Chandy and Gutman, 1993; Ertel et al., 2000).

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Fig. 2.

Amino acid sequence similarity and phylogenetic relationships of voltage-gated sodium channel α subunits. Phylogenetic relationships by maximum parsimony analysis of rat sodium channel sequences Na_v1.1-Na_v1.9 and Na_x. To perform the analysis, the amino acid sequences for all isoforms were aligned using Clustal W. The amino acid sequences in the alignments were then replaced with the published nucleotide sequences, and the nucleotide sequence alignments were subjected to analysis using the program PAUP^*. Divergent portions of the terminal regions and the cytoplasmic loops between domains I-II and II-III were excluded from the PAUP^* analysis. The tree was rooted by including the invertebrate sodium channel sequences during the generation of the tree, although these sequences are not shown in the figure.

In addition to these nine sodium channels that have been functionally expressed, closely related sodium channel-like proteins have been cloned from mouse, rat, and human but have not yet been functionally expressed (Na_x). They are approximately 50% identical to the Na_V1 subfamily of channels but more than 80% identical to each other. They have significant amino acid sequence differences in the voltage sensors, inactivation gate, and pore region that are critical for channel function and have previously been proposed as a distinct subfamily (George et al., 1992). These atypical sodium channel-like proteins are expressed in heart, uterus, smooth muscle, astrocytes, and neurones in the hypothalamus and peripheral nervous system. Because of their sequence differences, it is possible that these channels are not highly sodium-selective or voltage-gated. Although these proteins have striking differences in amino acid sequence in highly conserved regions of sodium channels, their amino acid sequence is greater than 50% identical to other sodium channels. They are closely related phylogenetically to the group of sodium channels on human chromosome 2q23-24, where their gene is also located (Goldin et al., 2000). Successful functional expression of these atypical sodium channel-like proteins and identification of additionally related sodium channels may provide evidence for a second sodium channel subfamily.

Four auxiliary subunits of sodium channels have been defined thus far: Na_Vβ₁, Na_Vβ₂, Na_Vβ₃, and Na_Vβ₄ (Catterall, 2000; Isom, 2001; Yu et al., 2004). In the event that additional subunits are identified, we propose that the nomenclature should be comparable to that for the auxiliary subunits of calcium channels (Ertel et al., 2000).

Sodium Channel Molecular Pharmacology

All of the pharmacological agents that act on sodium channels have receptor sites on the α subunits. At least six distinct receptor sites for neurotoxins and one receptor site for local anesthetics and related drugs have been identified (Cestèle and Catterall, 2000; Table 1). Neurotoxin receptor site 1 binds the nonpeptide pore blockers tetrodotoxin and saxitoxin and the peptide pore blocker μ-conotoxin (Fozzard and Hanck, 1996; Terlau and Stühmer, 1998; Catterall, 2000). The receptor sites for these toxins are formed by amino acid residues in the pore loops and immediately on the extracellular side of the pore loops at the outer end of the pore. Neurotoxin receptor site 2 binds a family of lipid-soluble toxins, including batrachotoxin, veratridine, aconitine, and grayanotoxin, which enhance activation of sodium channels. Photoaffinity labeling and mutagenesis studies implicate transmembrane segments IS6 and IVS6 in the receptor site for batrachotoxin (Cestèle and Catterall, 2000). Neurotoxin receptor site 3 binds the α-scorpion toxins and sea anemone toxins, which slow the coupling of sodium channel activation to inactivation. These peptide toxins bind to a complex receptor site that includes the S3-S4 loop at the outer end of the S4 segment in domain IV (Cestèle and Catterall, 2000). Neurotoxin receptor site 4 binds the β-scorpion toxins, which enhance activation of the channels. The receptor site for the β-scorpion toxins includes the S3-S4 loop at the extracellular end of the voltage-sensing S4 segments in domain II (Cestèle and Catterall, 2000). Neurotoxin receptor site 5 binds the complex polyether toxins brevetoxin and ciguatoxin, which are made by dinoflagellates and cause toxic red tides in warm ocean waters (Cestèle and Catterall, 2000). Transmembrane segments IS6 and IVS5 are implicated in brevetoxin binding from photoaffinity labeling studies (Cestèle and Catterall, 2000). Neurotoxin receptor site 6 binds δ-conotoxins, which slow the rate of inactivation like the α-scorpion toxins. The location of neurotoxin receptor site 6 is unknown. Finally, the local anesthetics and related antiepileptic and antiarrhythmic drugs bind to overlapping receptor sites located in the inner cavity of the pore of the sodium channel (Catterall, 2000). Amino acid residues in the S6 segments from at least three of the four domains contribute to this complex drug receptor site, with the IVS6 segment playing the dominant role.

Tables 2, 3, 4, 5, 6, 7, 8, 9, 10 summarize the major molecular, physiological, and pharmacological properties for each of the nine sodium channels that have been functionally expressed. Quantitative data are included for voltage dependence of activation and inactivation, single-channel conductance, and binding of drugs and neurotoxins, focusing on those agents that are widely used and diagnostic of channel identity and function.