The VGL-Chanome: A Protein Superfamily Specialized for Electrical Signaling and Ionic Homeostasis

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Science's STKE  05 Oct 2004:
Vol. 2004, Issue 253, pp. re15
DOI: 10.1126/stke.2532004re15


Complex multicellular organisms require rapid and accurate transmission of information among cells and tissues and tight coordination of distant functions. Electrical signals and resulting intracellular calcium transients, in vertebrates, control contraction of muscle, secretion of hormones, sensation of the environment, processing of information in the brain, and output from the brain to peripheral tissues. In nonexcitable cells, calcium transients signal many key cellular events, including secretion, gene expression, and cell division. In epithelial cells, huge ion fluxes are conducted across tissue boundaries. All of these physiological processes are mediated in part by members of the voltage-gated ion channel protein superfamily. This protein superfamily of 143 members is one of the largest groups of signal transduction proteins, ranking third after the G protein–coupled receptors and the protein kinases in number. Each member of this superfamily contains a similar pore structure, usually covalently attached to regulatory domains that respond to changes in membrane voltage, intracellular signaling molecules, or both. Eight families are included in this protein superfamily—voltage-gated sodium, calcium, and potassium channels; calcium-activated potassium channels; cyclic nucleotide–modulated ion channels; transient receptor potential (TRP) channels; inwardly rectifying potassium channels; and two-pore potassium channels. This article identifies all of the members of this protein superfamily in the human genome, reviews the molecular and evolutionary relations among these ion channels, and describes their functional roles in cell physiology.


Complex multicellular organisms require rapid and accurate transmission of information among cells and tissues and tight coordination of distant functions. Electrical signals and the resulting intracellular calcium transients, in vertebrates, control contraction of muscle, secretion of hormones, sensation of the environment, processing of information in the brain, and output from the brain to peripheral tissues. In nonexcitable cells, calcium transients signal many key cellular events, including secretion, gene expression, and cell division. In epithelial cells, huge ion fluxes are conducted across tissue boundaries. All of these physiological processes are mediated in part by members of the voltage-gated ion channel protein superfamily [figure 1 in (1)]. This protein superfamily of 143 members is one of the largest groups of signal transduction proteins, ranking third after the G protein–coupled receptors and the protein kinases in number. In analogy to the term "kinome" introduced for the protein kinase superfamily (2), we propose the term "chanome" for the universe of ion channel proteins and the VGL-chanome for the superfamily of voltage-gated and related cation channels: the voltage-gated–like ion channels. Other large groups of ion channels, such as the ligand-gated ion channels and chloride channels, might be referred to by similar shorthand names such as LG-chanome and Cl-chanome. Here we review the molecular and evolutionary relations among the members of the VGL-chanome and describe their functional roles in cell physiology.

Structural and Functional Motifs

The architectures of the ion channel families consist of four variations built on a common pore-forming structural theme. The founding members of this superfamily are the voltage-gated sodium channels (Fig. 1, NaV) (36). Their principal α subunits are composed of four homologous domains (I to IV) that form the common structural motif for this family (7, 8) (Fig. 2A). Each of these domains contains six regions that are thought to form membrane-spanning α helices (termed segments S1 to S6) and a membrane-reentrant loop between the S5 and S6 segments (9). Structural analysis by high-resolution EM and image reconstruction shows that the four homologous domains surround a central pore and suggests laterally oriented entry ports in each domain for ion transit toward the central pore (10) (Fig. 2A). Voltage-gated calcium (CaV) channels have a similar structure (see below). Voltage-gated potassium channels, first identified as the product of the gene encoding the Shaker mutation in the fruit fly Drosophila, exemplify the second architectural theme in the ion channel superfamily. They are composed of tetramers of α subunits that each resemble one homologous domain of sodium and calcium channels (Fig. 1, KV) (1113). Several other families of ion channels also have this tetrameric architecture (Fig. 1; see below). The inwardly rectifying potassium channels constitute the simplest structural motif in the ion channel protein superfamily. These channels are complexes of four subunits that each have only two transmembrane segments, termed M1 and M2, which are analogous in structure and function to the S5 and S6 segments of voltage-gated sodium, calcium, and potassium channels (Fig. 1, Kir) (14, 15). Two of these pore motifs are linked together to generate the fourth structural architecture, the two-pore potassium channels (Fig. 1, K2P) (16, 17).

Fig. 1.

Representation of the amino acid sequence relations of the minimal pore regions of the voltage-gated ion channel superfamily. This global view of the 143 members of the structurally related ion channel genes highlights seven groups of ion channel families and their membrane topologies. Four-domain channels (CaV and NaV) are shown as blue branches, potassium selective channels are shown as red branches, cyclic nucleotide–gated channels are shown as magenta branches, and transient receptor potential (TRP) and related channels are shown as green branches. Background colors separate the ion channel proteins into related groups: light blue, CaV and NaV ; light green, TRP channels; light red, potassium channels, except KV10–12, which have a cyclic nucleotide–binding domain and are more closely related to CNG and HCN channels; light orange, KV10–12 channels and cyclic nucleotide–modulated CNG and HCN channels. Minimal pore regions bounded by the transmembrane segments M1 or S5 and M2 or S6 were aligned by Clustal X (221) and refined manually for the 143 ion channel members (Appendix); amino acid sequence positions with gaps in the alignment were omitted from the analyses. The pore regions of the fourth homologous domain of NaV and CaV channels, the second domain of TPC, and the first pore regions of the K2P channels were used to assemble the alignment. This unrooted consensus tree was built by minimum evolution analysis using PAUP v. 4.0b10 (222) software; it summarizes 214 bootstrapped replicates of heuristic searches with random stepwise additions and "tree-bisection-reconnection" (TBR) branch swapping settings. Bootstrap values from 1000 replicates of neighbor-joining analysis are shown by the colors of the branches of the figure that represent each ion channel family: colored lines, bootstrap values >50%; black lines, bootstrap values <50%. The scale bar represents the tree distance corresponding to 0.05 substitutions per site in the sequence. The bootstrap values indicate the significance of the order of joining of the branches of the tree. To confirm the significance of the relations among the families that constitute the VGL ion channel superfamily, we tested the significance of the amino acid sequence relations using the HMM searching procedure. HMM searches of the complete RefSeq database revealed that each ion channel family profile identified another family of voltage-gated–like ion channels as the nearest relative in amino acid sequence of its pore. For example, for the KV channel profile, the nearest neighbor was CNGA1 (HMM E value of 2.6 × 10−3); for the cyclic nucleotide–modulated channel profile, KV11.2 (HMM E value of 1.5 × 10−6; for the TRP channel profile, CaV3.1 (HMM E value of 1.8 × 10−3); and for the NaV/CaV profile, CatSper3 (HMM E value of 9.4 × 10−6). The E value is a measure of the number of hits from HMM searches that would be expected by chance; values less than 1.0 indicate a highly significant amino acid sequence relation to the probe profile.

Fig. 2.

Structure of voltage-gated sodium channels and the potassium channel minimal pore region. (A) Left, Schematic representation of the sodium channel α subunit. Roman numerals indicate the homologous domains. The α-helical transmembrane segments S5 and S6 and the pore loop region between them are highlighted in green. The positively charged S4 segments are highlighted in red. The inactivation gate and the IFMT motif that is crucial for inactivation are highlighted in yellow. The probable N-linked glycosylation sites are indicated by the symbol, Ψ. The circles in the reentrant loops in each domain represent the amino acids that form the ion selectivity filter (the outer rings have the sequence EEDD and inner rings DEKA) (19, 65, 222). Right, The three-dimensional structure of the NaV channel α subunit at 20 Å resolution, compiled from electron micrograph reconstructions. [Adapted from Sato et al. (10)] (B) Open and closed conformations of the potassium channel pore structures. Two subunits of the bacterial K+ channels of KcsA (28), representing a "closed" conformation, and MthK (32), representing an "open" conformation, are shown in the figure. The selectivity filter is orange, and the outer helix (M1) is depicted adjacent to the lipid bilayer. The inner helix (M2) is marked with a space-filling model of the conserved glycine residue (red), which is thought to be critical for the bending of M2 helix in the open conformation.

The functional elements of the ion channel family of proteins can be divided into three complementary aspects: ion conductance, pore gating, and regulation. The ion-conducting pore and selectivity filter (18) of the six-transmembrane–segment(6-TM) channels (for example, the NaV, CaV, and KV families) are formed by their S5 and S6 segments and the membrane-reentrant pore loop (P) between them (1927) (Fig. 2A). The analogous M1 and M2 segments and pore loop form the complete transmembrane structure of the 2-TM potassium channels (14, 15). X-ray crystallographic analysis of the three-dimensional structure of a 2-TM bacterial potassium channel (KcsA), analogous in overall topology to the inwardly rectifying potassium channels, reveals an "inverted teepee" arrangement of the M1 and M2 segments in a square array around a central pore (Fig. 2B) (28). The narrow outer mouth of the pore is formed by the intervening membrane-reentrant pore loop. This structure is cradled in a cone formed by the tilted M1 and M2 transmembrane segments, with the M2 segments lining most of the inner pore, surrounded by the M1 segments. A cavity in the center of the structure is water-filled and contains permeating potassium ions. The pore appears to be constricted at the intracellular end by crossing of the M2α helices.

Insight into gating of the pore has come from structural and functional experiments. Biophysical studies revealed gated access of substituted amine blockers to the pore from the intracellular side of voltage-gated sodium and potassium channels (2931). In the three-dimensional structure of a bacterial 2-TM calcium-activated potassium channel (MthK) (Fig. 2B) analyzed in its calcium-bound, presumably activated form, the M2α helices are bent at a highly conserved glycine residue (32, 33). This bend appears to open the intracellular mouth of the pore sufficiently to allow permeation of ions. Substitution of proline for this glycine, which should greatly favor the bent conformation, dramatically enhances activation and slows pore closure of a bacterial sodium channel, which provides functional evidence for bending at this position as a key step in opening the pore (34).

Addition of the S1 to S4 segments to the pore structure in the NaV, CaV, and KV channels confers voltage-dependent pore opening. Although the mechanism of voltage-dependent gating is not known in detail, an overall view of the process has emerged from a combination of biophysical, mutagenesis, and structural studies. Movement of charged amino acid residues associated with NaV and KV channel gating [gating currents (35)] are consistent with the outward translocation of about 12 positive charges during channel activation (3638). The S4 segments, which have repeated motifs of one positively charged amino acid residue followed by two hydrophobic residues, are thought to serve as the primary voltage sensors (9, 39). Mutations of these charged amino acid residues have large effects on gating (38, 40, 41). The outward movement and rotation of these S4 segments has been observed directly by studies of state-dependent chemical modification and by fluorescent labeling of substituted cysteine residues (38, 4045). Most structure-function studies support a spiral or rotational motion of the S4 or S3 plus S4 α helices through the channel protein in order to move gating charges across the membrane electric field (9, 38, 39). In contrast, a strikingly different sweeping transmembrane paddle movement of these S1 through S4 α helices through the surrounding membrane lipid is suggested from the x-ray crystal structure of a bacterial KV channel (46). Precisely how these two distinct views of movement of the voltage sensors can be reconciled is the subject of active investigation and debate.

Addition of regulatory domains to the carboxyl terminals of 2-TM Kir channels, 6-TM calcium-activated potassium (KCa) channels, CNG channels, and hyperpolarization-activated and cyclic nucleotide–gated (HCN) channels (Fig. 1) yields gating by binding of small intracellular ligands such as calcium, magnesium, adenosine 5′-triphosphate (ATP), and cyclic nucleotides or by interactions with protein ligands (4750). Ligand binding to these domains is thought to exert a torque on the S6 segments that opens the pore by bending them (32, 51, 52). For KCa and HCN family members, ligand binding and membrane depolarization act in concert to open the pore (48, 53). The four-domain CaV channels have regulatory sites in their C-terminal intracellular segments that may exert a similar torque on the S6 segment in domain IV and may modulate the opening of the pore in response to membrane depolarization (54).

Identifying the Human VGL-Chanome

In order to identify all of the structurally related ion channel proteins encoded in the human genome, we used the common pore region as a template for a genome-wide search for related amino acid sequences. We built profiles based on hidden Markov models (HMMs) of each ion channel family (Fig. 1), using the sequences corresponding to the minimal pore structure (the pore loop and the flanking M1/S5 and M2/S6 transmembrane segments). We interrogated the nonredundant protein database, RefSeq Releases 2 and 6, of National Center for Biotechnology Information (NCBI), using each HMM profile [see Appendix and Tables S1 (;2004/253/re15/DC1) and S2 (;2004/253/re15/DC2) for detailed procedures]. This search revealed 143 genes that encode related ion channel proteins. The relations among the amino acid sequences of their minimal pore structures are illustrated in Fig. 1. We found 21 proteins related to four-domain NaV and CaV channels; two novel two-domain relatives of ion channel proteins [two-pore channels (TPCs)]; 90 proteins related to one-domain voltage-gated potassium channels [including 40 KV channels, 8 KCa channels, 10 cyclic nucleotide–modulated (CNG and HCN) channels, and 32 transient receptor potential (TRP) channels and relatives]; and 30 proteins related to the inwardly rectifying (Kir) and two-pore (K2P) potassium channels. We verified that these families are members of a common superfamily by determining their nearest neighbors in amino acid sequence space (see legend to Fig. 1 and Appendix). In each case, the closest relative is a family within the VGL-chanome, and the relation is highly significant. The molecular relations and physiological functions of these ion channel protein families are considered below, using the nomenclature adopted by the Nomenclature Committee of the International Union of Pharmacology (IUPHAR) (1).

Evolutionary Origins

How did the structural motifs of this large ion channel superfamily arise in evolution? Although the answer to this question is necessarily speculative, some insight can be gained by comparing ion channels in different species. Many bacteria have 2-TM potassium channels resembling Kir channels, and some bacteria have 6-TM voltage-gated potassium channels (55). Thus, it seems possible that the primordial members of this superfamily were 2-TM bacterial potassium channels. Addition of the S1 to S4 segments to this founding pore structure yielded voltage-gated potassium channels in bacteria. Bacteria also contain a novel 6-TM sodium channel with only a single homologous domain that functions as a tetramer in the same way a potassium channel does (56). This channel has striking similarities to vertebrate sodium and calcium channels, and a homolog may be their ancestor. The simplest organism expressing a four-domain calcium channel is yeast, which has a single calcium channel gene (57). To date, only multicellular organisms, including jellyfish, cnidarians, squid, and fruit fly (but not the roundworm C. elegans), have been found to express four-domain sodium channels (5861). These four-domain channels may have arisen by two cycles of gene duplication and fusion from an ancestral bacterial one-domain sodium channel.

Comparison of the VGL-chanomes of human, roundworm (C. elegans), and fruit fly (Drosophila) reveals some interesting relations [(Table 1), (Table S3,;2004/253/re15/DC3)]. All of the major families of human ion channels are represented in worms and flies except for NaV and HCN, which are missing in worms. The lack of NaV channels implies that CaV channels provide all of the inward current for conducted action potentials in C. elegans. K2P channels are remarkably expanded in the worm, with 44 members, compared with 15 in human and 11 in fruit fly, whereas all of the other channel families have the same or fewer members in these invertebrate species. The functional significance of this expanded K2P family in C. elegans is not understood at present.

Because of their ancient origins, the ion channel genes are spread throughout the genome. Only the late-evolving four-domain sodium channels have a large fraction(5 of the 10 family members) in a single cluster on human chromosome 2. The pattern of spread of the sodium channel genes among chromosomes has been correlated with the spread of homeobox genes during the vertebrate radiation and suggests that similar chromosomal dynamics affected both gene families (62). In contrast, most of the other ion channel genes were probably sorted to distinct chromosomes much earlier in evolution.

Voltage-Gated Sodium and Calcium Channels

NaV channels are specialized for electrical signaling and are responsible for the rapid, transient influx of sodium ions that underlies the rising phase of the action potential in nerve, muscle, and endocrine cells. Drugs that block NaV channels are used for local and spinal anesthesia and for treatment of pain, cardiac arrhythmias, and epilepsy (Table 1). There are 10 genes for NaV α subunits in the human genome, and 9 of them fall into a single family with greater than 70% identity of amino acid sequence in their transmembrane regions (63) (Fig. 3). These NaV channels are expressed in different excitable cell types and are localized in different subcellular compartments in neurons (6, 64). NaV channels conduct Na+ more than 10 times faster than K+ or Ca2+ (18). Their selectivity is determined by the side chains of four amino acid residues [Asp-Glu-Lys-Ala (DEKA)], one residue at the tip of each reentrant loops between the S5 and S6 segments of each domain, which are conserved in all 10 channels (65, 66). This arrangement contrasts with the selectivity filter of potassium channels in which the main-chain carbonyl groups interact with the conducted potassium ions (Fig. 2) (67).

Fig. 3.

Phylogenetic relations of the four-domain ion channels, NaV and CaV. The consensus tree was inferred by maximum parsimony analysis of the minimal pore regions from the fourth homologous domain of sodium and calcium channels, and summarizes 567 bootstrapped replicates from heuristic searches with random stepwise additions and TBR branch–swapping settings. Gapped sequences in the alignment were not analyzed. Tree was rooted to the bacterial sodium channel (NachBac), but this branch is not shown in the figure. Bootstrap values of 50 to 74% are depicted by an asterisk (*); values of 75 to 100% are depicted by **; and those that are <49% are not shown. The scale bar corresponds to the number of changes needed to explain the differences in the protein sequences. Vertical branch length is not significant.

In addition to voltage-gated channel activation, NaV channels undergo a second fast-gating process, voltage-dependent fast inactivation, which occludes the pore within 1 to 2 ms after opening. Inactivation is mediated by closure of a cytoplasmic inactivation gate formed by the hydrophobic motif isoleucine-phenylalanine-methionine-threonine (IFMT) within the intracellular linker between domains III and IV (41, 6870), which has a rigid loop-loop-helix structure (70) that is highly conserved in all ten NaV proteins. Inactivation exerts crucial control over sodium channel conductance, allowing the channel to open in rapid succession to generate trains of brief electrical signals.

CaV channels are the key signal transducers of electrical signaling, converting depolarization of the cell membrane to an influx of calcium ions that initiates contraction, secretion, neurotransmission, and other intracellular regulatory events. They have a pore-forming α1 subunit (7173), which resembles the α subunits of NaV channels in amino acid sequence and topological organization (Fig. 1) (74). CaV channels are more than 1000-fold selective for calcium over sodium or potassium (75, 76), and this selectivity is determined by a pore motif that has the amino acid residues EEEE precisely in place of the DEKA of NaV channels (65). Ten human genes encode pore-forming CaV α1 subunits that fall into three distinct subfamilies (CaV1 to CaV3). They have greater than 75% identity within a subfamily and as little as 25% amino acid sequence identity between subfamilies (77) (Fig. 3).

CaV1 channels conduct L-type calcium currents, which are important in excitation-contraction coupling, endocrine secretion, sensory transduction, and regulation of enzyme activity and gene expression (54, 78, 79). Drugs that block CaV1.2 channels selectively are important in the therapy of cardiovascular diseases, including hypertension, cardiac arrhythmia, and angina pectoris (Table 1). CaV2 channels conduct N-, P/Q-, and R-type calcium currents, which are primarily responsible for initiation of synaptic transmission at fast synapses in the nervous system (8082). Related to this function, they have a larger intracellular loop connecting domains II and III, which contains a synaptic protein interaction (synprint) site that binds SNARE proteins involved in exocytosis (82). The CaV2 channels are the targets for many paralytic peptide neurotoxins produced by spiders and cone snails, which specifically block fast synaptic transmission (83). CaV3 channels conduct T-type calcium currents, which are important for setting the frequency of action potential firing in repetitively active cells like the sino-atrial pacemaker cells in the heart and the pacemaker neurons of the thalamus (84). In addition to these well-characterized calcium channels, our genome scan revealed a single novel CaV-like protein that defines a fourth subfamily (85). This protein is most similar to CaV channels in the pore region, but probably is not highly calcium-selective because the signature motif for ion selectivity is Glu-Glu-Lys-Glu (EEKE) rather than EEEE.

Voltage-Gated and Calcium-Activated Potassium Channels.

The voltage-gated potassium channel family is the largest ion channel family in the human genome. KV channels are products of 40 genes in 12 subfamilies (86), and the related KCa channels are encoded by 8 genes in 4 subfamilies (1, 87, 88)) (Fig. 4). The KV1–9 subfamilies are the most closely related. The principal function of these channels is selective efflux of K+. In electrically excitable cells, KV channels set the resting membrane potential, repolarize cells during the falling phase of action potentials, terminate trains of action potentials, and hyperpolarize the cell between bursts of action potentials (86). In nonexcitable cells such as lymphocytes (89), KV channels control the cell membrane potential and thereby control the driving force for entry of calcium, which regulates many cell functions. In epithelial cells of the kidney, auditory cochlea, and other tissues, KV channels participate in transepithelial ion transport, as well as cell signaling (90, 91).

Fig. 4.

Phylogenetic relations of the potassium channels and the cyclic nucleotide–modulated ion channels (CNG, HCN). The consensus tree was inferred by maximum parsimony analysis of the minimal pore regions from KV, KCa, Kir, CNG, and HCN, and of the first pore region from K2P ion channels, and summarizes 226 bootstrapped replicates from heuristic searches with random stepwise additions, and TBR branch swapping settings. Gapped sequences in the alignment were not analyzed. Tree was rooted to the bacterial sodium channel (NachBac), but this branch is not shown in the figure. Bootstrap values of 50 to 74% are depicted by *; values of 75 to 100% are depicted by **; and not shown are those that are <49%. The scale bar corresponds to the number of changes needed to explain the differences in the protein sequences. Vertical branch length is not precise.

KV channels are assemblies of four 6-TM α subunits that surround a central ion-conducting pore. How do these four subunits recognize each other and form specific tetrameric complexes? For KV1–4 channels, a tetramerization domain (T1) located within the N terminus is the primary determinant of specific homo- and heteromultimeric α subunit associations that generate functional KV channels (92, 93). The x-ray crystal structure of the T1 domain shows that it forms a fourfold square-symmetric structure in the absence of the remainder of the protein (94); this suggests that each α subunit interacts with a single β subunit. Heteromultimers of KV1–4 channels are formed within single subfamilies but not between subfamilies, and this specificity lies in the T1 domains (95, 96). The KV5, 6, 8, and 9 subfamilies do not form functional channels by themselves but modify or inhibit the function of KV2 heteromers into which they are assembled, thereby functioning in vivo as modulators or silencers of KV2 channels (1). Tetramers of KV7 and KCa channels are formed through interactions of the C-terminal RCK (regulators of conductance of K+) domains (96, 97), but the rules for formation of tetramers are less well established.

The three-dimensional structure of the bacterial KcsA potassium channel (Fig. 2B) has provided detailed insight into the common mechanism of KV channel ion permeation and selectivity. The selectivity filter is formed by the amino acids Thr-Val-Gly-Tyr-Gly (TVGYG), which comprise the signature sequence of KV channels in the P loops of the four subunits (67). Main-chain carbonyl oxygen atoms of these residues are regularly aligned in the 12 Å long, 2.5 Å wide lumen of the selectivity filter to coordinate multiple dehydrated K+ ions during passage. These oxygen atoms are ideally positioned to substitute for the water molecules that normally surround each K+ ion in solution, but are not optimally positioned for Na+, Ca2+, and Mg2+, which have smaller ionic radii (67). In this manner, the selectivity filter of K+ channels presents minimal energy barrier for K+ ions to occupy positions in the permeation pathway, leading to permeation rates for K+ ~1000 times those for Na+ (98).

Like the NaV and CaV channels, the signal to open KV channels is membrane depolarization, which leads to an outward movement of their S4 voltage-sensing segment (40, 43, 99). In addition to voltage-dependent gating, the KCa1, 4, and 5 channels (also termed large conductance, BK, or maxiK) respond to cytosolic ions that bind to an intrinsic regulatory domain in the intracellular C-terminal region. These channels are unique in having a seventh transmembrane segment (S0) of unknown function at their N terminus (100). Direct binding of Ca2+ to an RCK domain and a conserved group of negatively charged amino acid residues designated the "calcium-bowl" in the C-terminal domain enhances channel opening by shifting the voltage dependence of activation to more negative membrane potentials (33, 101, 102). Other intracellular signals, including Na+ and H+, also mediate their effects on KCa channel function through the C terminus (103, 104). Integration of the intracellular Ca2+ and Na+ signals with membrane potential changes is an important aspect of the KCa channel functions of regulating the contractility of smooth muscle (105), action potential firing in neuronal cell bodies, and neurotransmitter release at synapses (106). For the KCa2 and KCa3 channels that form small conductance (SK) and intermediate conductance (IK) calcium-activated potassium channels, calmodulin associated with a specific regulatory site in the C-terminal domain is the calcium sensor (107). Binding of Ca2+ to calmodulin drives channel activation, apparently by cross-linking the C-terminal domains of two adjacent subunits and exerting a torque on their S6 segments to open them (51). Voltage-dependent gating is not observed, despite the presence of a typical S4 transmembrane segment. These channels respond to increases in intracellular calcium by opening and repolarizing the cell membrane potential to end action potential trains in excitable cells and to increase the driving force for calcium influx in nonexcitable cells (106).

KV channels inactivate during prolonged depolarizations, but much more slowly than do NaV channels. Two types of inactivation have been defined for the Drosophila Shaker channel, which is a homolog of the KV1 subfamily in vertebrates. In N-type inactivation, the N-terminal of the α subunit occludes the intracellular mouth of the pore after it opens, whereas in C-type inactivation the pore itself is thought to close (108, 109). In contrast, the N terminus of the auxiliary KVβ subunit of vertebrate KV channels (see below) mediates their N-type inactivation in most cases (110, 111). The mechanism of C-type inactivation of vertebrate KV channels has not yet been well defined (112).

The KV10–12 subfamilies are related to Drosophila ether-a-go-go (eag) channels (113, 114). These channels are primarily voltage-gated and have high selectivity for K+. Like other KV channels, they contribute to repolarization of electrically excitable cells to terminate action potentials and are involved in signal transduction in nonexcitable cells (115). In spite of these functions in common with other KV channels, the pore sequences of KV10–12 channels are more closely related in amino acid sequence to the cyclic nucleotide–modulated family (see below) than to KV1–9 channels (Fig. 4). Moreover, their C-terminal domains contain a cyclic nucleotide–binding motif whose function has not been fully defined (116118). KV10–12 channels also form heteromultimers within, but not between, subfamilies (119). An N-terminal PAS (Per-Arnt-Sim) domain may have a role in membrane targeting or protein interactions (120).

Cyclic Nucleotide–Modulated Channels

Cyclic nucleotide–gated channels were first purified and cloned from the retina (121). Six distinct genes in two subfamilies encode subunits for CNG channels, four CNGA subunits and two CNGB subunits (122) (Fig. 4). In heterologous cell expression systems, the A subunits form functional channels by themselves, whereas the B subunits cannot. Functional CNG channels in the retina are heteromers assembled in 3A:1B ratio (123, 124). Under physiological conditions, CNG channels in photoreceptors and olfactory sensory neurons produce depolarizing currents carried principally by Na+ and Ca2+ (122). Different subunits compose CNG channels in rod (A1 and B1a subunits) and cone (A3 and B3 subunits) photoreceptors and in olfactory sensory neurons (A2, A4 and B1b subunits) (125, 126). The amino acid sequences of CNG and K+ channels diverge exactly at the K+ channel TVGYG selectivity motif; in CNG channels hydrophilic residues replace the Tyr residue. Although these channels contain three or four positively charged amino acid residues in their S4 segment, they are not responsive to membrane potential. Instead, they are gated by direct binding of adenosine 3′,5′-monophosphate (cAMP) and guanosine 3′,5′-monophosphate (cGMP) to the cyclic nucleotide–binding domain in the C terminus (47, 121), which is thought to promote conformational changes to open and close the pore, perhaps by exerting a torque on the S6 segment.

The HCN channel family is encoded by four genes in the human genome (127, 128) (Fig. 4) and is closely related to the CNG family. HCN channels discriminate Na+ from K+ poorly (129, 130), despite having a partial selectivity sequence (GYG) similar to that of the KV channels (TVGYG). Evidently, the TV residues and other sequence differences between KV and HCN channels in amino acid residues that flank the selectivity filter motif modify the ion selectivity of HCN channels. Under physiological conditions, Na+ carries most of the depolarizing current through HCN channels, and divalent cations neither permeate nor block these channels (127). Unique among vertebrate voltage-gated ion channels, the reverse voltage–dependence of the HCN channels leads to activation upon hyperpolarization. The S4 segments of HCN channels contain up to 10 positively charged residues, whereas most voltage-gated channels contain 5 to 7 positively charged amino acids. The molecular mechanism of this reverse voltage–dependence of gating is not yet clear, but outward movement of the highly charged S4 segment is important as evidenced by mutagenesis experiments and fluorescence labeling (131, 132). Similar to CNG channels, a cyclic nucleotide–binding domain is present in the C-terminal region of HCN channels (118). Binding of cAMP acts synergistically with membrane hyperpolarization to regulate channel gating, similar to the synergistic gating of KCa1 channels by membrane depolarization and intracellular calcium binding. Deletion of the cyclic nucleotide–binding domain produces a depolarizing shift in the voltage-dependence of activation, suggesting that the unoccupied cyclic nucleotide–binding domain has an autoinhibitory function (133). HCN channels can form heteromultimers in vitro, but it is not clear whether this also occurs in situ (128) These channels are crucial to control of rhythmic electrical discharge in repetitively firing pacemaker cells in the sino-atrial node of the heart and the thalamus in the brain (129, 130). Their ion conductance increases as the membrane is repolarized following an action potential, and this increase provides a regenerative boost that activates sodium channels and begins a new action potential. Direct binding of cAMP and cGMP regulates their activation, which links second messenger signaling and pacemaking in rhythmically firing cells.

Molecular Relations Between KV Channels, KCa Channels, and Cyclic Nucleotide–Modulated Channels

A careful look at the phylogenetic tree in Fig. 4 shows that the KV1 to KV9 channels form one closely related cluster, positioned adjacent to clusters of K2P channels that may have evolved later and Kir channels that may have evolved earlier. Within the KV1–9 cluster, the KV1–4 channels form one related group, the KV5, 6, 8, and 9 channels a second related group, and the KV7 channels (formerly KCNQ or KVLQT) form a third more distant group of KV channels.

Surprisingly, the CNG, HCN, and KV10–12 channels form the fourth large cluster when the molecular relations are determined by comparison of pore-forming sequences (Fig. 4). This is a robust result that is obtained by all methods of analysis of molecular relations that we have used. Evidently, these channels arose from a common evolutionary pathway despite their diversity of ion selectivity (K+, Ca2+, or nonselective cation channels) and primary gating mechanism (voltage, cyclic nucleotides, or both).

An additional surprise is the distant relation of the sequences of the KCa channels and the KV channels in the S5, P loop, and S6 segments (Fig. 4). Evidently, these two groups of channels with similar transmembrane topologies separated from each other early in evolution and their pore domains have become distinct in amino acid sequence.

Transient Receptor Potential Channels

Although it is the second largest among those related to voltage-gated ion channels, the TRP channel family was underappreciated until recently. Beginning with a founding member identified in the Drosophila phototransduction system (134, 135), 32 genes that encode one-domain TRP channels and structural relatives have been defined and classified into at least six distinct subfamilies (Figs. 1 and 5) (136). TRP channels are 6-TM proteins that resemble KV channels in overall architecture, but show limited conservation of the S4 positive charges and P loop sequences that are the hallmarks of the KV, NaV, and CaV families. Although some show preferential permeability to Ca2+, TRP channels in general are nonselective cation channels whose opening leads to influx of extracellular cations that depolarizes the membrane potential and increases intracellular Ca2+ (137). This heterogeneous group of ion channel proteins may generate additional diversity through formation of heteromeric channel complexes, as has been demonstrated for TRPC (137, 138). In addition, some family members have ankyrin repeats in their N-terminal domains that may interact with intracellular proteins (137).

Fig. 5.

Phylogenetic relations of the transient receptor potential (TRP) channels and their related family members. The consensus tree was inferred by maximum parsimony analysis of the minimal pore regions from TRPC, TRPM, TRPV, ANKTM1, TRPP, and TRPML, and of the pore region from second homologous domain of TPC ion channels, and it summarizes 578 bootstrapped replicates from heuristic searches with random stepwise additions, and TBR branch–swapping settings. Gapped sequences in the alignment were not analyzed. Tree was rooted to the bacterial sodium channel (NachBac), but this branch is not shown in the figure. Bootstrap values of 50 to 74% are depicted by *; values of 75 to 100% are depicted by **; and not shown are those that are <49%. The scale bar corresponds to the number of changes needed to explain the differences in the protein sequences. Vertical branch length is not significant.

Physiological triggers that activate TRP channels are diverse and incompletely understood. Sustained Ca2+ entry through TRPC channels is stimulated by hormones that activate phospholipase C isozymes and thereby generate diacylglycerol and other lipid messengers; these channels are an important component of receptor-operated and possibly also store-operated cation channels that play a fundamental role in cellular Ca2+ signaling and homeostasis (139141).

Both TRPV and TRPM channels are involved in sensory signaling. Members of the TRPV subfamily can be activated by the hot chili pepper ingredient, capsaicin; high osmolarity; low pH; and low intracellular Ca2+ (142). In addition, members of TRPV family respond to thermal stimuli. TRPV1 and TRPV2 are activated by differing levels of noxious heat, whereas TRPV3 and TRPV4 are gated by innocuous warmth <42°C (143146). Mice lacking TRPV1 are deficient in both thermal sensation and response to thermal hyperalgesia (147). Sensation of cold temperatures involves the TRPM family. TRPM1 channels were first identified as an up-regulated mRNA in melanoma cells, but their physiological function is unknown. The menthol-sensitive TRPM8 channels are gated by cool temperatures (<25°C), whereas the distantly related ANKTM1 is activated by even colder temperatures (148150). The discovery that TRPV and TRPM channels are responsible for transduction of thermal sensation and thermal pain is a major breakthrough in understanding temperature sensing at the molecular level.

It is likely that other TRPM subfamily members have different roles in sensory physiology involving distinct molecular mechanisms (136). TRPM2 is activated by ADP-ribose, an intracellular second messenger. TRPM4 and TRPM5 are activated by G protein–coupled receptor-mediated release of Ca2+ from intracellular stores. TRPM6 and TRPM7 are unique among ion channels in that they are controlled through autophosphorylation by a phospholipase C–interacting kinase that is fused to their intracellular C termini (151). These channels appear to play a crucial role in Mg2+ transport in cells (152).

The TRP channels have S4 segments with repeated motifs of one positively charged amino acid residue followed by two hydrophobic residues, but there are fewer positive charges than in most voltage-gated ion channels. Are TRP channels also voltage-sensitive? A recent report argues persuasively that they are. Voets et al. (153) report that TRPV1 and TRPM8 channels are activated by depolarization to potentials above 100 mV, far beyond the physiological range of membrane potentials. However, the voltage dependence of activation is shifted negatively by cooling or menthol treatment for TRPM8 or by heating or capsaicin treatment for TRPV1, allowing activation at negative membrane potentials. This synergistic gating by voltage plus either temperature or ligand binding resembles the synergistic gating of KCa1 channels by voltage and Ca2+ and HCN channels by voltage and cyclic nucleotides. These results suggest similar allosteric gating mechanisms for these distantly related members of the ion channel superfamily.

The TRPP and TRPML subfamilies of TRP channels (136) are incompletely characterized physiologically, because most of them have not been successfully expressed in heterologous cells. TRPP1, the PKD2 protein (154), whose defect is associated with inherited polycystic kidney disease, is thought to be a Ca2+-permeable channel activated by bending of the apical cilium of certain kidney epithelia, presumably to sense fluid flow through renal tubules (155). Mutations in TRPML1 (mucolipin-1) cause mucolipidosis type IV, a neurodegenerative, lysosomal storage disorder with defects in membrane sorting or endocytosis (156). Mutation of the TRPML3 channel, expressed in the cochleal hair cells of the ear, causes deafness and vestibular dysfunction in the varitint-waddler mouse from which this gene was cloned (157). The putative one-domain Ca2+ channels of sperm, CatSper1–4 channels (158160), are also distantly related to the TRP family. Although the presence of positively charged residues in S4 suggests that depolarization may activate these channels, voltage-gated CatSper currents have yet to be recorded directly. However, ablation of CatSper1 in mouse causes defects in cAMP-mediated Ca2+ influx and sperm motility (158). Most recently, a two-pore channel (TPC) protein with two repeats of a 6-TM domain, resembling a fusion of two TRP-like channels, has been cloned from rat kidney (161), and there are two orthologous human genes, TPCN1 and TPCN2, that are expressed in human liver, lung, and kidney. These two-domain channels may add to the remarkable diversity of form and function of the TRP family of channels.

Inwardly Rectifying and Two-Pore Potassium Channels

The inwardly rectifying Kir channels derive their name from their capacity to conduct K+ inward more readily than outward. However, at physiological membrane potentials, these channels mediate K+ efflux, which repolarizes and hyperpolarizes the membrane potential. Kir channels have the simplest transmembrane topology in the voltage-gated–like ion channel family, namely, two transmembrane segments (M1 and M2) that are equivalent to the S5 and S6 of KV channels with an intervening reentrant P loop (Fig. 2B). All 15 members of the 7 Kir subfamilies [(Figs. 1 and 4), (Table S2,;2004/253/re15/DC2)] contain the conserved (T/S)IG(Y/F)G motif of KV channels in the P loop, consistent with their highly selective permeation of K+ (1). Because of the absence of the S4 voltage-sensor, the Kir channels are not gated by changes in transmembrane voltage per se. However, changes in membrane potential are still important to their function, as they regulate physical occlusion of the pore of the Kir channels by intracellular Mg2+ and polyamines, which is the mechanistic basis for the block of the pore at positive membrane potentials that causes inward rectification (162, 163).

In addition to voltage-dependent blocking by Mg2+ and polyamines, the ion conductance activity of Kir channels is regulated by a remarkable diversity of intracellular messengers. Different subfamilies are regulated by direct binding of different sets of ligands: phosphatidylinositol bisphosphate (PIP2), H+, and ATP, for the Kir1 subfamily; H+ and PIP2 for Kir2; G protein βγ subunits and PIP2 for Kir3; and ATP and ADP for Kir6 (164166). Outward K+ conductance through Kir channels maintains the resting membrane potential near the K+ reversal potential to control a wide spectrum of cellular processes, including transepithelial transport of K+ in the kidney and other epithelial tissues (90), secretion of insulin in the beta cells of the pancreas (167, 168), regulation of maximum diastolic potential and beating rate in the heart (164), and determination and modulation of the resting membrane potential in neurons (169). Kir subunits can form heteromeric channels, but auxiliary subunit association has been demonstrated only for the Kir6 subunits, which assemble with regulatory sulfonylurea receptor subunits (SUR) of the ATP-binding cassette (ABC) family (see below) to form the functional KATP channel complex (165, 167). Direct binding of cellular ATP or sulfonylurea drugs to the SUR subunits inhibits the activity of the Kir6 subunits of the KATP channel complex, depolarizes the cell, and promotes the exocytosis of vesicular insulin by the pancreatic beta cells. This signaling pathway allows coupling of metabolic state to electrical excitability (165, 167, 170).

Members of the K2P channel family are composed of four transmembrane segments with two P loops, a topology similar to a tandem fusion of two Kir channels, and the pore selectivity sequence of the K2P channels is T(I/V)GYG or T(I/V)GFG similar to the K+ channels (Fig. 1) (171173). These channels are sometimes called "leak channels" or "open rectifiers" that conduct "background" K+ currents because they are constantly active at the resting membrane potential of cells and serve to hold the cell membrane potential near the equilibrium potential for K+. Low sequence homology among K2P channels suggests that the 15 genes in the human genome each constitute a distinct subfamily (1) (Fig. 4). K2P channels conduct background K+ currents in both excitable and nonexcitable cells (171173). There is no evidence of heteromer formation or association with auxiliary subunits. K2P channels are resistant to most K+ channel blockers, but their activity is modulated by various agents including mechanical stress, changes in pH, changes in temperature, and fatty acids (171173). Background K+ currents are important physiologically to stabilize the membrane potential in excitable cells. Moreover, K2P channels are molecular targets for inhalation anesthetics and enhanced activation of K2P currents by these agents leads to hyperpolarization of neurons and thus reduction of excitability (174).

Disease Mutations in the Ion Channel Superfamily

The many different genetic diseases caused by their mutations highlight the broad-ranging physiological roles of ion channels (Table 1). NaV channels are the molecular targets for many mutations that cause diseases of hyperexcitability, including periodic paralysis of skeletal muscle, cardiac arrhythmia, and epilepsy (175). Most often these mutations are dominant because they cause gain of function by blocking the normal inactivation process or by causing precocious activation of the channels. Similarly, mutations of KV channels cause cardiac arrhythmias due to impaired repolarization (176), sometimes accompanied by deafness or other specific deficits. Mutations of CaV channels cause cardiac arrhythmia, periodic paralysis of skeletal muscle, familial migraine, night blindness, and cerebellar ataxia (175). Mutations in cyclic nucleotide–gated channels in the retina cause inherited retinitis pigmentosa (177). Mutations of Kir channels cause inherited forms of hyperinsulinemia and renal dysfunction (178). The discovery that mutations of ion channels cause inherited diseases points to additional opportunities for these proteins as potential drug targets for therapy of diseases of hypo- or hyperexcitability and altered ion transport.

Auxiliary Subunits of the Ion Channel Protein Family

The principal pore-forming subunits of the voltage-gated ion channels and their structural relatives are primarily responsible for their characteristic gating, selective ion conductance, and regulation by second-messenger signaling and pharmacologic agents. However, many of these principal subunits are associated with auxiliary subunits that modify their expression, functional properties, and subcellular localization (179). In the exceptional case of KATP channels, the auxiliary subunit is a primary regulator of channel activity. In this section, we briefly review the structure, function, and genetic relations of the known auxiliary subunits.

Sodium Channels.

Purification and characterization of sodium channels provided the first evidence for ion channel auxiliary subunits (3, 5). Sodium channels have a single family of auxiliary subunits, NaVβ1 to NaVβ4, which interact with the different α subunits and alter their physiological properties and subcellular localization. These proteins have a single transmembrane segment, a large N-terminal extracellular domain that is homologous in structure to a variable chain (V-type) immunoglobulin-like fold, and a short C-terminal intracellular segment (Fig. 6) (180183). The NaVβ subunits interact with α subunits through their extracellular immunoglobulin-like domains, modulate α subunit function, and enhance their cell surface expression (184). Like other proteins with an extracellular immunoglobulin-like fold, they also serve as cell adhesion molecules by interacting with extracellular matrix proteins, cell adhesion molecules, and cytoskeletal linker proteins (185189). A mutation in a conserved cysteine in the immunoglobulin-like fold of the NaVβ1 subunit causes familial epilepsy (190). The NaVβ subunits are a recent evolutionary addition to the family of ion channel–associated proteins, as they have only been identified in vertebrates.

Fig. 6.

Auxiliary subunits of the VGL-chanome. The transmembrane folding patterns of the auxiliary subunits of the VGL-chanome are illustrated, with cylinders representing predicted α helices. Note that the intracellular and extracellular sides of the proteins are oriented in opposite directions for the top and bottom membranes, as in a cell. N-linked carbohydrate chains are indicated by Ψ. The intracellular auxiliary subunits are illustrated by their representative three-dimensional structure data deposited in PDB and drawn using Cn3D: 1VYU for CaVβ, 1S6C for KchIp, and 1QRQ for KVβ.

Table 1.

Structure and function of the VGL-chanome. This table highlights the human VGL-ion channels’ transmembrane topology, physiological roles, human diseases caused by mutations of channel genes, current drug therapies that target respective ion channel families. Transmembrane topology is coded as: total number of transmembrane segments (TM)/number of P loops (P). Human subfamilies and members are coded as total number of subfamilies: total number of protein family members. Human mutations in the specified channel family that cause inherited forms of the indicated diseases. For most of these diseases, the inherited form is a small percentage of the entire disease population. Current drugs in wide therapeutic use that target the indicated ion channels are listed, not only those that target specific genetic diseases.

Calcium Channels.

CaV1 and CaV2 channels have four distinct auxiliary subunits, CaVα2, CaVβ, CaVγ, and CaVδ (Fig. 6) (191), which each form a small protein family. The CaVα2 and CaVδ subunits are encoded by the same gene (192), whose translation product is proteolytically cleaved and disulfide linked to yield the mature extracellular α2 subunit glycoprotein of 140 kD and transmembrane disulfide-linked δ subunit glycoprotein of 27 kD (193). Four CaVα2δ genes are known (194). The four CaVβ subunits are all intracellular proteins with a common pattern of α-helical and unstructured segments (194, 195). They have important regulatory effects on cell surface expression, and they also modulate the gating of calcium channels, which causes enhanced activation upon depolarization and altered rate and voltage dependence of inactivation (194). Recent structural modeling and x-ray crystallography studies have revealed that, like the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, these subunits contain conserved, interacting SH3 and guanylate kinase domains and, therefore, may interact with other intracellular proteins (196200). Eight CaVγ subunit genes encode glycoproteins with four transmembrane segments (194, 201). Although the CaVγ1 subunit is associated specifically with skeletal muscle CaV1.1 channels, other CaVγ subunits interact with other calcium channels, glutamate receptors, and possibly other membrane-signaling proteins (194). Thus, the γ subunits discovered as components of calcium channels apparently have a more widespread role in assembly and cell surface expression of other membrane signaling proteins.

Voltage-Gated Potassium Channels.

KV1 channels are often associated with an intracellular KVβ subunit (KVβ1–3) (Fig. 6) (110, 202, 203), which interacts with the N-terminal T1 domain and forms a symmetric tetramer on the intracellular surface of the channels (204). The three KVβ subunits are superficially similar to the CaVβ subunits in their cytoplasmic location, but are not related in amino acid sequence or structure. The N terminus of KVβ subunits of vertebrates serves as an inactivation gate for KV1 α subunits (203) and is thought to enter the pore and block it during sustained channel opening (111). This is a unique example of a direct physical role for an auxiliary subunit in channel gating, rather than a role modulating the gating process of its associated pore-forming α subunit.

KV4 channels interact with the K channel–interacting proteins KChIp1–4, which are members of the neuronal calcium sensor family of calmodulin-like calcium regulatory proteins and have four EF hand motifs (Fig. 6) (205). The KChIps enhance expression of KV4 channels and modify their functional properties by binding to a site in the intracellular T1 domain, similar to the interaction of KVβ subunits with KV1 channels.

The KV7, KV10, and KV11 channels associate with a different type of auxiliary subunit—the minK-like subunits. These five closely related proteins have a single transmembrane segment and small extracellular and intracellular domains (206, 207). Although these subunits are topologically similar to NaVβ and CaVδ subunits, they do not have substantial amino acid sequence similarity. The minK-like subunits are important regulators of KV7 channel function (208, 209), and mutations in one of these auxiliary subunits causes a form of familial long QT syndrome, which predisposes to dangerous cardiac arrhythmias (210). In addition, recent work indicates that these subunits also associate with KV3 and KV4 channels and are responsible for a form of inherited periodic paralysis (210, 211). In light of this work, it is possible that all KV channels associate with minK-related subunits. If this hypothesis is true, the KV channels would then resemble the NaV and CaV channels in having an associated subunit with a single transmembrane segment and short intracellular and extracellular domains. It will be intriguing to learn if there is a common function for these similar auxiliary ion channel subunits.

Calcium-Activated Potassium Channels.

The KCa1, 4, and 5 family of channels are associated with one of four auxiliary KCaβ subunits, which have two transmembrane segments and both N and C termini in the cytosol (Fig. 6) (212, 213). The KCaβ subunits contribute to the binding site for the peptide scorpion toxin charybdotoxin and related channel-blocking agents (214), but their effects on channel function and roles in cell physiology are still emerging in current research (213).

Inwardly Rectifying Potassium Channels.

Unique among the inwardly rectifying potassium channel subunits, the Kir6 subunits that form KATP channels are associated with SURs (215), which are crucial regulators of channel activity. They are also the molecular targets for the sulfonylurea class of KATP channel blockers, which are used to enhance insulin secretion in therapy of diabetes. The three SUR proteins are members of the ABC transporter family of membrane proteins. They have an amino terminal domain with five probable transmembrane segments, which is followed by two domains with six transmembrane segments and two ATP-binding motifs, a pattern similar to other members of the ABC transporter family (216). Sequential binding and hydrolysis of ATP at the two nucleotide-binding cassettes regulate Kir6 channel function in response to changes in the concentration and ratio of ATP and ADP and, thereby, regulate channel activity in response to the metabolic state of the cell. This form of channel regulation is crucial in control of insulin release from the beta cells of the pancreas. Mutations in SUR are responsible for some forms of familial hyperinsulinemia (217).


Polycystins (PKDs) 1 and 2 were both discovered as the targets of mutations that cause autosomal dominant polycystic kidney disease (154, 218). PKD2 is an ion channel protein similar in topology and amino acid sequence to TRP channels (see TRPP channels above), whereas PKD1 is an auxiliary subunit that modifies expression and function of PKD2 (219). Like SUR, it has a complex transmembrane structure (Fig. 6). Elucidating the functional relations between PKD1 and PKD2 and defining their roles in normal kidney function is an active area of investigation.

More Auxiliary Subunits?

Almost every ion channel that has been isolated from its native mammalian tissue has been found to have auxiliary subunits. Because most of the superfamily members have been identified by cDNA cloning and have never been purified from native tissues, it is possible that many additional ion channels have auxiliary subunits associated with them. As the auxiliary subunits that are known have diverse effects on ion channel function, expression, and localization, it will be important to fully define the set of auxiliary subunits in order to fully understand the functional and regulatory characteristics of ion channels in situ.


The voltage-gated ion channel superfamily is one of the largest families of signaling proteins, following the G protein–coupled receptors and the protein kinases in the number of family members. The family is likely to have evolved from a 2-TM ancestor like the bacterial KcsA channel. The additions of intracellular regulatory domains for ligand binding and a 4-TM domain for voltage-dependent gating have produced extraordinarily versatile signaling molecules with the capacity to respond to voltage signals and intracellular effectors and to integrate information coming from these two distinct types of inputs. The resulting signaling mechanisms control most aspects of cell physiology and underlie complex integrative processes like learning and memory in the brain and coordinated movements in muscles. The evolutionary appearance and refinement of these signaling mechanisms is one of the landmark events that allowed the development of complex multicellular organisms.


A complete file of the 143 human ion channel genes is provided in Table S1 (;2004/253/re15/DC1) in the form of an Excel spreadsheet. The file contains the ion channel protein names as approved by the IUPHAR Nomenclature Committee (1), the corresponding gene names as approved by HUGO, the complete amino acid sequence of a representative isolate, and the pore-forming segments of the human genes that were used for molecular comparisons. A file of their relatives in C. elegans and Drosophila is shown in Table S3 (;2004/253/re15/DC3). In Table S2 (;2004/253/re15/DC2), the alignment of the pore-forming segments of the 143 ion channels used in the phylogenetic analyses is provided. In the alignment, mouse Trp2 protein sequence was included because the human ortholog is a pseudogene. Comparison of these aligned sequences by minimum evolution and maximum parsimony methods yielded the phylogenetic trees presented in the main text.

Our approach to genomic analysis of the voltage-gated ion channel superfamily proceeded in three steps. First, we constructed profile HMMs for the conserved pore sequences of each of the families of ion channels identified in the IUPHAR compendium (1). ClustalX alignments were made of human protein sequences of 20 sodium and calcium channels (domain IV), 75 potassium channels (first pore motif of K2P), 8 cyclic nucleotide–gated channels (CNG, HCN), and 29 TRP and related channels. These minimum pore alignments were fed into the HMMER 2.3 suite of programs (Washington University, St. Louis; to generate and calibrate profile HMMs by using default parameters and to screen the RefSeq databases to identify every related family member. Second, to test whether each family is a member of the VGL-chanome, we determined its nearest neighbor in amino acid sequence space as defined by the HMM screening results. In every case, we found that the nearest neighbor is another family within the VGL-chanome, confirming that all families are members of a single superfamily of ion channels. Finally, we fed these related sequences into the PAUP program and developed a phylogenetic map of all 143 members of the VGL-chanome using maximum parsimony and neighbor-joining algorithms. These methods resulted in the molecular relations illustrated in Fig. 1.

We tested the completeness of our set of 143 ion channel genes by interrogating multiple releases of data in the RefSeq database. The RefSeq protein database provides a complete, nonredundant set of open reading frames derived from the complete human genome DNA sequence. RefSeq Release 2(4 November 2003) had 19,872 confirmed protein records (with corresponding ESTs) and 5954 predicted protein records. Our original screen of RefSeq Release 2 yielded 144 ion channel proteins. To test for completeness of this set, we rescreened RefSeq Release 6(12 July 2004), which has 21,429 confirmed protein records and 6553 predicted protein records. We found no new ion channel proteins in the most recent RefSeq release, and one ion channel protein was deleted in the RefSeq database owing to an error (TRPC8), yielding the final set of 143 ion channel proteins reported here. The latest estimates from gene-prediction programs suggest that there are fewer than 24,500 protein-coding genes, including ~3000 pseudogenes (220). Pseudogenes are not represented in the RefSeq database, so ~21,500 protein-encoding genes are expected to be in the RefSeq database when full coverage of the genome is achieved. The extra RefSeq records reflect existence of naturally occurring splice variants of gene transcripts. Although the exact coverage of the human genes in the RefSeq database is not known precisely, comparison of these numbers indicates that it is nearly complete. In any case, the increase of 2156 records from RefSeq Release 2 to RefSeq Release 6, about 10% of the predicted number of protein-encoding human genes, yielded no new ion channels. Therefore, we are confident that the small number of human proteins that are not yet represented in the RefSeq database will include few, if any, new members of the ion channel protein family.


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