The VGL-Chanome: A Superfamily Specialized for Electrical Signaling and Ionic Homeostasis Frank H. Yu and William A. Catterall (5 October 2004) Sci. STKE 2004 (253), re15. [DOI: 10.1126/stke.2532004re15]

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

Frank H. Yu1 and William A. Catterall1* (Published 5 October 2004)

Complex multicellular organisms require rapid and voltage-gated protein superfamily (Fig. 1) (1). This accurate transmission of information among cells protein superfamily of 143 members is one of the largest groups and tissues and tight coordination of distant of signal transduction , ranking third after the G pro- functions. Electrical signals and resulting intracellu- tein–coupled receptors and the protein kinases in number. In lar calcium transients, in vertebrates, control con- analogy to the term “kinome” introduced for the protein kinase traction of muscle, secretion of hormones, sensation superfamily (2), we propose the term “chanome” for the of the environment, processing of information in the universe of ion channel proteins and the VGL-chanome for the brain, and output from the brain to peripheral superfamily of voltage-gated and related cation channels: the tissues. In nonexcitable cells, calcium transients sig- voltage-gated–like ion channels. Other large groups of ion

nal many key cellular events, including secretion, channels, such as the ligand-gated ion channels and chloride Downloaded from expression, and cell division. In epithelial cells, channels, might be referred to by similar shorthand names such huge ion fluxes are conducted across tissue bound- as LG-chanome and Cl-chanome. Here we review the molecular aries. All of these physiological processes are medi- and evolutionary relations among the members of the VGL- ated in part by members of the voltage-gated ion chanome and describe their functional roles in cell physiology. channel protein superfamily. This protein superfami- ly of 143 members is one of the largest groups of Structural and Functional Motifs

signal transduction proteins, ranking third after the The architectures of the ion channel families consist of four stke.sciencemag.org –coupled receptors and the protein kinases variations built on a common pore-forming structural theme. in number. Each member of this superfamily con- The founding members of this superfamily are the voltage- tains a similar pore structure, usually covalently at- gated sodium channels (Fig. 1, NaV) (3–6). Their principal α tached to regulatory domains that respond to subunits are composed of four homologous domains (I to IV) changes in membrane voltage, intracellular signaling that form the common structural motif for this family (7, 8) molecules, or both. Eight families are included in this (Fig. 2A). Each of these domains contains six regions that are protein superfamily—voltage-gated sodium, calcium, thought to form membrane-spanning α helices (termed seg-

and potassium channels; calcium-activated potassi- ments S1 to S6) and a membrane-reentrant loop between the S5 on July 7, 2009 um channels; cyclic nucleotide–modulated ion chan- and S6 segments (9). Structural analysis by high-resolution EM nels; transient receptor potential (TRP) channels; in- and image reconstruction shows that the four homologous do- wardly rectifying potassium channels; and two-pore mains surround a central pore and suggests laterally oriented potassium channels. This article identifies all of the entry ports in each domain for ion transit toward the central members of this protein superfamily in the human pore (10) (Fig. 2A). Voltage-gated calcium (CaV) channels have genome, reviews the molecular and evolutionary re- a similar structure (see below). Voltage-gated potassium lations among these ion channels, and describes channels, first identified as the product of the gene encoding their functional roles in cell physiology. the mutation in the fruit fly Drosophila, exemplify the second architectural theme in the ion channel superfamily. They Introduction are composed of tetramers of α subunits that each resemble one Complex multicellular organisms require rapid and accurate homologous domain of sodium and calcium channels (Fig. 1, transmission of information among cells and tissues and tight KV) (11–13). Several other families of ion channels also have coordination of distant functions. Electrical signals and the re- this tetrameric architecture (Fig. 1; see below). The inwardly sulting intracellular calcium transients, in vertebrates, control rectifying potassium channels constitute the simplest structural contraction of muscle, secretion of hormones, sensation of the motif in the ion channel protein superfamily. These channels are environment, processing of information in the brain, and output complexes of four subunits that each have only two transmem- from the brain to peripheral tissues. In nonexcitable cells, calci- brane segments, termed M1 and M2, which are analogous in um transients signal many key cellular events, including secre- structure and function to the S5 and S6 segments of voltage- tion, , and cell division. In epithelial cells, huge gated sodium, calcium, and potassium channels (Fig. 1, Kir) ion fluxes are conducted across tissue boundaries. All of these (14, 15). Two of these pore motifs are linked together to gener- physiological processes are mediated in part by members of the ate the fourth structural architecture, the two-pore potassium channels (Fig. 1, K2P) (16, 17). 1Department of Pharmacology, Mailstop 357280, University of Wash- The functional elements of the ion channel family of proteins can ington, Seattle, WA 98195–7280, USA. be divided into three complementary aspects: ion conductance, pore *Corresponding author. E-mail: [email protected]. gating, and regulation. The ion-conducting pore and selectivity filter

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R CNG HCN Cav

Kv10-12 Nav

K2p

TPC Downloaded from

TRP stke.sciencemag.org Kv1-9 on July 7, 2009 R 0.05 substitutions/site KCa

Kir 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 highlights seven groups of ion channel families and their mem- brane topologies. Four-domain channels (CaV and NaV) are shown as blue branches, potassium selective channels are shown as red branch- es, 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 chan- nels; 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 homolo- gous 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 set- tings. 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 −3 sequence of its pore. For example, for the KV channel profile, the nearest neighbor was CNGA1 (HMM E value of 2.6 × 10 ); for the cyclic −6 −3 nucleotide–modulated channel profile, KV11.2 (HMM E value of 1.5 × 10 ; for the TRP channel profile, CaV3.1 (HMM E value of 1.8 × 10 ); −6 and for the NaV/CaV profile, CatSper3 (HMM E value of 9.4 × 10 ). 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.

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Fig. 2. Structure of voltage-gated sodium channels and the mini- mal pore region. (A) Left, Schematic repre- sentation of the α subunit. Roman numerals indicate the homologous domains. The α-helical transmembrane segments S5 and S6 and the pore loop re- gion 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 inacti- vation are highlighted in yellow. The proba- ble N-linked glycosylation sites are indicat- ed 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 struc- ture of the NaV channel α subunit at 20 Å resolution, compiled from electron micro-

graph reconstructions. [Adapted from Sato Downloaded from et al.(10 )] (B) Open and closed conforma- tions of the potassium channel pore struc- tures. Two subunits of the bacterial K+ channels of KcsA (28), representing a “closed” conformation, and MthK (32), rep- resenting an “open” conformation, are shown in the figure. The selectivity filter is stke.sciencemag.org 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.

(18) of the six-transmembrane–segment (6-TM) channels (for exam- ions. Substitution of proline for this glycine, which should greatly on July 7, 2009 ple, the NaV, CaV, and KV families) are formed by their S5 and S6 favor the bent conformation, dramatically enhances activation and segments and the membrane-reentrant pore loop (P) between them slows pore closure of a bacterial sodium channel, which provides (19–27) (Fig. 2A). The analogous M1 and M2 segments and pore functional evidence for bending at this position as a key step in loop form the complete transmembrane structure of the 2-TM potas- opening the pore (34). sium channels (14, 15). X-ray crystallographic analysis of the three- Addition of the S1 to S4 segments to the pore structure in the dimensional structure of a 2-TM bacterial potassium channel NaV, CaV, and KV channels confers voltage-dependent pore open- (KcsA), analogous in overall topology to the inwardly rectifying ing. Although the mechanism of voltage-dependent gating is not potassium channels, reveals an “inverted teepee” arrangement of the known in detail, an overall view of the process has emerged from a M1 and M2 segments in a square array around a central pore (Fig. combination of biophysical, mutagenesis, and structural studies. 2B) (28). The narrow outer mouth of the pore is formed by the inter- Movement of charged amino acid residues associated with NaV and vening membrane-reentrant pore loop. This structure is cradled in a KV channel gating [gating currents (35)] are consistent with the out- cone formed by the tilted M1 and M2 transmembrane segments, with ward translocation of about 12 positive charges during channel acti- the M2 segments lining most of the inner pore, surrounded by the vation (36–38). The S4 segments, which have repeated motifs of M1 segments. A cavity in the center of the structure is water-filled one positively charged amino acid residue followed by two hy- and contains permeating potassium ions. The pore appears to be con- drophobic residues, are thought to serve as the primary voltage sen- stricted at the intracellular end by crossing of the M2 α helices. sors (9, 39). Mutations of these charged amino acid residues have Insight into gating of the pore has come from structural and large effects on gating (38, 40, 41). The outward movement and ro- functional experiments. Biophysical studies revealed gated access of tation of these S4 segments has been observed directly by studies of substituted amine blockers to the pore from the intracellular side of state-dependent chemical modification and by fluorescent labeling voltage-gated sodium and potassium channels (29–31). In the of substituted cysteine residues (38, 40–45). Most structure-function three-dimensional structure of a bacterial 2-TM calcium-activated studies support a spiral or rotational motion of the S4 or S3 plus S4 potassium channel (MthK) (Fig. 2B) analyzed in its calcium-bound, α helices through the channel protein in order to move gating presumably activated form, the M2 α helices are bent at a highly charges across the membrane electric field (9, 38, 39). In contrast, a conserved glycine residue (32, 33). This bend appears to open the strikingly different sweeping transmembrane paddle movement of intracellular mouth of the pore sufficiently to allow permeation of these S1 through S4 α helices through the surrounding membrane

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lipid is suggested from the x-ray crystal structure of a bacterial KV teria have 6-TM voltage-gated potassium channels (55). Thus, it channel (46). Precisely how these two distinct views of movement seems possible that the primordial members of this superfamily of the voltage sensors can be reconciled is the subject of active in- were 2-TM bacterial potassium channels. Addition of the S1 to vestigation and debate. S4 segments to this founding pore structure yielded voltage- Addition of regulatory domains to the carboxyl terminals of gated potassium channels in bacteria. Bacteria also contain a 2-TM Kir channels, 6-TM calcium-activated potassium (KCa) novel 6-TM sodium channel with only a single homologous do- channels, CNG channels, and hyperpolarization-activated and main that functions as a tetramer in the same way a potassium cyclic nucleotide–gated (HCN) channels (Fig. 1) yields gating channel does (56). This channel has striking similarities to ver- by binding of small intracellular ligands such as calcium, tebrate sodium and calcium channels, and a homolog may be magnesium, adenosine 5′-triphosphate (ATP), and cyclic nu- their ancestor. The simplest organism expressing a four-domain cleotides or by interactions with protein ligands (47–50). Lig- is yeast, which has a single calcium channel and binding to these domains is thought to exert a torque on the gene (57). To date, only multicellular organisms, including jel- S6 segments that opens the pore by bending them (32, 51, 52). lyfish, cnidarians, squid, and fruit fly (but not the roundworm For KCa and HCN family members, ligand binding and mem- C. elegans), have been found to express four-domain sodium brane depolarization act in concert to open the pore (48, 53). channels (58–61). These four-domain channels may have arisen The four-domain CaV channels have regulatory sites in their C- by two cycles of gene duplication and fusion from an ancestral terminal intracellular segments that may exert a similar torque bacterial one-domain sodium channel. on the S6 segment in domain IV and may modulate the opening Comparison of the VGL-chanomes of human, roundworm (C. of the pore in response to membrane depolarization (54). elegans), and fruit fly (Drosophila) reveals some interesting rela- tions [(Table 1), (Table S3, http://stke.sciencemag.org/

Identifying the Human VGL-Chanome cgi/content/full/sigtrans;2004/253/re15/DC3)]. All of the major Downloaded from In order to identify all of the structurally related ion channel families of human ion channels are represented in worms and flies proteins encoded in the , we used the common except for NaV and HCN, which are missing in worms. The lack of pore region as a template for a genome-wide search for related NaV channels implies that CaV channels provide all of the inward amino acid sequences. We built profiles based on hidden current for conducted action potentials in C. elegans. K2P channels Markov models (HMMs) of each ion channel family (Fig. 1), are remarkably expanded in the worm, with 44 members, compared using the sequences corresponding to the minimal pore struc- with 15 in human and 11 in fruit fly, whereas all of the other chan-

ture (the pore loop and the flanking M1/S5 and M2/S6 trans- nel families have the same or fewer members in these invertebrate stke.sciencemag.org membrane segments). We interrogated the nonredundant protein species. The functional significance of this expanded K2P family in database, RefSeq Releases 2 and 6, of National Center for C. elegans is not understood at present. Biotechnology Information (NCBI), using each HMM profile Because of their ancient origins, the ion channel genes are [see Appendix and Tables S1 (http://stke.sciencemag.org/ spread throughout the genome. Only the late-evolving four- cgi/content/full/sigtrans;2004/253/re15/DC1) and S2 domain sodium channels have a large fraction (5 of the 10 fami- (http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/253/r ly members) in a single cluster on human 2. The e15/DC2) for detailed procedures]. This search revealed 143 pattern of spread of the sodium channel genes among chromo-

genes that encode related ion channel proteins. The relations somes has been correlated with the spread of homeobox genes on July 7, 2009 among the amino acid sequences of their minimal pore struc- during the vertebrate radiation and suggests that similar chro- tures are illustrated in Fig. 1. We found 21 proteins related to mosomal dynamics affected both gene families (62). In con- four-domain NaV and CaV channels; two novel two-domain rel- trast, most of the other ion channel genes were probably sorted atives of ion channel proteins [two-pore channels (TPCs)]; 90 to distinct much earlier in evolution. proteins related to one-domain voltage-gated potassium channels [including 40 KV channels, 8 KCa channels, 10 cyclic Voltage-Gated Sodium and Calcium Channels nucleotide–modulated (CNG and HCN) channels, and 32 tran- NaV channels are specialized for electrical signaling and are re- sient receptor potential (TRP) channels and relatives]; and 30 sponsible for the rapid, transient influx of sodium ions that under- proteins related to the inwardly rectifying (Kir) and two-pore lies the rising phase of the action potential in nerve, muscle, and (K2P) potassium channels. We verified that these families are endocrine cells. Drugs that block NaV channels are used for local members of a common superfamily by determining their near- and spinal anesthesia and for treatment of pain, cardiac arrhyth- est neighbors in amino acid sequence space (see legend to Fig. mias, and epilepsy (Table 1). There are 10 genes for NaV α sub- 1 and Appendix). In each case, the closest relative is a family units in the human genome, and 9 of them fall into a single family within the VGL-chanome, and the relation is highly significant. with greater than 70% identity of amino acid sequence in their The molecular relations and physiological functions of these ion transmembrane regions (63) (Fig. 3). These NaV channels are ex- channel protein families are considered below, using the pressed in different excitable cell types and are localized in differ- nomenclature adopted by the Nomenclature Committee of the ent subcellular compartments in neurons (6, 64). NaV channels International Union of Pharmacology (IUPHAR) (1). conduct Na+ more than 10 times faster than K+ or Ca2+ (18). Their selectivity is determined by the side chains of four amino acid Evolutionary Origins residues [Asp-Glu-Lys-Ala (DEKA)], one residue at the tip of How did the structural motifs of this large ion channel super- each reentrant loop, between the S5 and S6 segments of each do- family arise in evolution? Although the answer to this question main, which are conserved in all 10 channels (65, 66). This ar- is necessarily speculative, some insight can be gained by com- rangement contrasts with the selectivity filter of potassium chan- paring ion channels in different species. Many bacteria have 2- nels in which the main-chain carbonyl groups interact with the TM potassium channels resembling Kir channels, and some bac- conducted potassium ions (Fig. 2) (67).

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Channel Topology Human subfamilies Fly family Worm family Primary cellular functions Disease Current family and members members members in vertebrates mutations drugs

Na 24TM/4P 1:10 2 0 Initiate action potentials Periodic paralysis, Local anesthetics, V cardiac arrhythmia, antiarrhythmic drugs, epilepsy antiepileptic drugs

Ca 24TM/4P 4:11 4 5 Couple depolarization to Ca2+ Cardiac arrhythmia, Calcium channel V influx and intracellular signal migraine, episodic blockers used for transduction ataxia, stationary cardiovascular night blindness, disorders periodic paralysis

K 6TM/1P 12:40 9 16 Repolarize membrane potential; Cardiac arrhythmia, Antiarrhythmic drugs V transepithelial ion flux deafness

K 6TM/1P 4:8 3 6 Repolarize membrane potential Ca in responses to depolarization and Ca2+ influx

CNG 6TM/1P 2:6 4 6 Sensory signal transduction

HCN 6TM/1P 1:4 1 0 Regulate frequency of action potentials

TRP and 6TM/1P 7:32 12 21 Receptor-regulated cation influx, Mucolipidosis, Downloaded from relatives including Ca2+; sensory signal polycystic kidney transduction; fertilization by disease, deafness sperm

TCP 12TM/2P 2:2 0 0 Unknown

K 2TM/1P 7:15 3 3 Set and regulate resting Hyperinsulinemia, Sulfonylureas ir membrane potential; renal dysfunction transepithelial ion flux stke.sciencemag.org

K 4TM/2P 15:15 11 44 Set and regulate resting General anesthetics 2p membrane potential

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 sub- families and members are coded as total number of subfamilies: total number of protein family members. Human mutations in the on July 7, 2009 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.

In addition to voltage-gated channel activation, NaV chan- of NaV channels (65). Ten human genes encode pore-forming nels undergo a second fast-gating process, voltage-dependent CaV α1 subunits that fall into three distinct subfamilies (CaV1 fast inactivation, which occludes the pore within 1 to 2 ms after to CaV3). They have greater than 75% identity within a subfam- opening. Inactivation is mediated by closure of a cytoplasmic ily and as little as 25% amino acid sequence identity between inactivation gate formed by the hydrophobic motif isoleucine- subfamilies (77) (Fig. 3). phenylalanine-methionine-threonine (IFMT) within the intra- CaV1 channels conduct L-type calcium currents, which are cellular linker between domains III and IV (41, 68–70), which important in excitation-contraction coupling, endocrine secre- has a rigid loop-loop-helix structure (70) that is highly con- tion, sensory transduction, and regulation of enzyme activity served in all ten NaV proteins. Inactivation exerts crucial control and gene expression (54, 78, 79). Drugs that block CaV1.2 over sodium channel conductance, allowing the channel to open channels selectively are important in the therapy of cardiovascu- in rapid succession to generate trains of brief electrical signals. lar diseases, including hypertension, cardiac arrhythmia, and CaV channels are the key signal transducers of electrical angina pectoris (Table 1). CaV2 channels conduct N-, P/Q-, and signaling, converting depolarization of the cell membrane to an R-type calcium currents, which are primarily responsible for influx of calcium ions that initiates contraction, secretion, neu- initiation of synaptic transmission at fast synapses in the ner- rotransmission, and other intracellular regulatory events. They vous system (80–82). Related to this function, they have a larg- have a pore-forming α1 subunit (71–73), which resembles the α er intracellular loop connecting domains II and III, which con- subunits of NaV channels in amino acid sequence and topologi- tains a synaptic protein interaction (synprint) site that binds cal organization (Fig. 1) (74). CaV channels are more than SNARE proteins involved in exocytosis (82). The CaV2 chan- 1000-fold selective for calcium over sodium or potassium (75, nels are the targets for many paralytic peptide neurotoxins pro- 76), and this selectivity is determined by a pore motif that has duced by spiders and cone snails, which specifically block fast the amino acid residues EEEE precisely in place of the DEKA synaptic transmission (83). CaV3 channels conduct T-type calci-

www.stke.org/cgi/content/full/sigtrans;2004/253/re15 Page 5 R EVIEW um currents, which are important for setting the frequency of channels presents minimal energy barrier for K+ ions to occupy action potential firing in repetitively active cells like the sino- positions in the permeation pathway, leading to permeation rates atrial pacemaker cells in the heart and the pacemaker neurons for K+ ~1000 times those for Na+ (98). of the thalamus (84). In addition to these well-characterized cal- Like the NaV and CaV channels, the signal to open KV chan- cium channels, our genome scan revealed a single novel CaV- nels is membrane depolarization, which leads to an outward like protein that defines a fourth subfamily (85). This protein is movement of their S4 voltage-sensing segment (40, 43, 99). In most similar to CaV channels in the pore region, but probably is addition to voltage-dependent gating, the KCa1, 4, and 5 chan- not highly calcium-selective because the signature motif for ion nels (also termed large conductance, BK, or maxiK) respond to selectivity is Glu-Glu-Lys-Glu (EEKE) rather than EEEE. cytosolic ions that bind to an intrinsic regulatory domain in the intracellular C-terminal region. These channels are unique in Voltage-Gated and Calcium-Activated Potassium having a seventh transmembrane segment (S0) of unknown Channels. function at their N terminus (100). Direct binding of Ca2+ to an The voltage-gated potassium channel family is the largest ion RCK domain and a conserved group of negatively charged channel family in the human genome. KV channels are products amino acid residues designated the “calcium-bowl” in the of 40 genes in 12 subfamilies (86), and the related KCa channels C-terminal domain enhances channel opening by shifting the are encoded by 8 genes in 4 subfamilies (1, 87, 88)) (Fig. 4). voltage dependence of activation to more negative membrane The KV1–9 subfamilies are the most closely related. The princi- potentials (33, 101, 102). Other intracellular signals, including + + + pal function of these channels is selective efflux of K . In elec- Na and H , also mediate their effects on KCa channel function trically excitable cells, KV channels set the resting membrane through the C terminus (103, 104). Integration of the intracellu- potential, repolarize cells during the falling phase of action lar Ca2+ and Na+ signals with membrane potential changes is an

potentials, terminate trains of action potentials, and hyperpolar- important aspect of the KCa channel functions of regulating the Downloaded from ize the cell between bursts of action potentials (86). In nonex- contractility of smooth muscle (105), action potential firing in citable cells such as lymphocytes (89), KV channels control the neuronal cell bodies, and neurotransmitter release at synapses cell membrane potential and thereby control the driving force for entry of calcium, which regulates many cell functions. In Na 1.1 epithelial cells of the kidney, auditory cochlea, and other tis- v Nav1.2 sues, KV channels participate in transepithelial ion transport, as * Nav1.7 well as cell signaling (90, 91). Na 1.3 stke.sciencemag.org K channels are assemblies of four 6-TM subunits that v V α Na 1.6 surround a central ion-conducting pore. How do these four sub- v Na 1.4 units recognize each other and form specific tetrameric com- v * Na 1.5 plexes? For K 1–4 channels, a tetramerization domain (T1) lo- v V Na 1.8 cated within the N terminus is the primary determinant of spe- ** ** v Na 1.9 cific homo- and heteromultimeric α subunit associations that v Na generate functional K channels (92, 93). The x-ray crystal x V Ca 1.3 structure of the T1 domain shows that it forms a fourfold * v on July 7, 2009 Ca 1.4 square-symmetric structure in the absence of the remainder of v Ca 1.1 the protein (94); this suggests that each α subunit interacts with ** v Ca 1.2 a single β subunit. Heteromultimers of KV1–4 channels are * ** v formed within single subfamilies but not between subfamilies, Cav2.1 Ca 2.2 and this specificity lies in the T1 domains (95, 96). The KV5, 6, ** v 8, and 9 subfamilies do not form functional channels by them- Cav2.3 VGCNL1 selves but modify or inhibit the function of KV2 heteromers into which they are assembled, thereby functioning in vivo as modu- Cav3.1 ** Ca 3.2 lators or silencers of KV2 channels (1). Tetramers of KV7 and v Ca 3.3 KCa channels are formed through interactions of the C-terminal ** v RCK (regulators of conductance of K+) domains (96, 97), but the rules for formation of tetramers are less well established. 20 changes The three-dimensional structure of the bacterial KcsA potas- sium channel (Fig. 2B) has provided detailed insight into the Fig. 3. Phylogenetic relations of the four-domain ion channels, NaV and Ca . The consensus tree was inferred by maximum parsimony common mechanism of K channel ion permeation and selec- V V analysis of the minimal pore regions from the fourth homologous do- tivity. The selectivity filter is formed by the amino acids main of sodium and calcium channels, and summarizes 567 boot- Thr-Val-Gly-Tyr-Gly (TVGYG), which comprise the signature se- strapped replicates from heuristic searches with random stepwise quence of KV channels in the P loops of the four subunits (67). additions and TBR branch–swapping settings. Gapped sequences in Main-chain carbonyl oxygen atoms of these residues are regularly the alignment were not analyzed. Tree was rooted to the bacterial aligned in the 12 Å long, 2.5 Å wide lumen of the selectivity filter sodium channel (NachBac), but this branch is not shown in the + to coordinate multiple dehydrated K ions during passage. These figure. Bootstrap values of 50 to 74% are depicted by an asterisk (*); oxygen atoms are ideally positioned to substitute for the water values of 75 to 100% are depicted by **; and those that are <49% are molecules that normally surround each K+ ion in solution, but are not shown. The scale bar corresponds to the number of changes not optimally positioned for Na+, Ca2+, and Mg2+, which have needed to explain the differences in the protein sequences. smaller ionic radii (67). In this manner, the selectivity filter of K+ Vertical branch length is not significant.

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(106). For the KCa2 and KCa3 channels that form small conduc- (cAMP) and guanosine 3′,5′-monophosphate (cGMP) to the tance (SK) and intermediate conductance (IK) calcium- cyclic nucleotide–binding domain in the C terminus (47, 121), activated potassium channels, calmodulin associated with a which is thought to promote conformational changes to open and specific regulatory site in the C-terminal domain is the calcium close the pore, perhaps by exerting a torque on the S6 segment. sensor (107). Binding of Ca2+ to calmodulin drives channel The HCN channel family is encoded by four genes in the hu- activation, apparently by cross-linking the C-terminal domains man genome (127, 128) (Fig. 4) and is closely related to the of two adjacent subunits and exerting a torque on their S6 seg- CNG family. HCN channels discriminate Na+ from K+ poorly ments to open them (51). Voltage-dependent gating is not (129, 130), despite having a partial selectivity sequence (GYG) observed, despite the presence of a typical S4 transmembrane similar to that of the KV channels (TVGYG). Evidently, the TV segment. These channels respond to increases in intracellular residues and other sequence differences between KV and HCN calcium by opening and repolarizing the cell membrane poten- channels in amino acid residues that flank the selectivity filter tial to end action potential trains in excitable cells and to in- motif modify the ion selectivity of HCN channels. Under physi- crease the driving force for calcium influx in nonexcitable cells ological conditions, Na+ carries most of the depolarizing cur- (106). rent through HCN channels, and divalent cations neither perme- KV channels inactivate during prolonged depolarizations, but ate nor block these channels (127). Unique among vertebrate much more slowly than do NaV channels. Two types of inactivation voltage-gated ion channels, the reverse voltage–dependence of have been defined for the Drosophila Shaker channel, which is a the HCN channels leads to activation upon hyperpolarization. homolog of the KV1 subfamily in vertebrates. In N-type inactiva- The S4 segments of HCN channels contain up to 10 positively tion, the N-terminal of the α subunit occludes the intracellular charged residues, whereas most voltage-gated channels contain mouth of the pore after it opens, whereas in C-type inactivation the 5 to 7 positively charged amino acids. The molecular mecha-

pore itself is thought to close (108, 109). In contrast, the N terminus nism of this reverse voltage–dependence of gating is not yet Downloaded from of the auxiliary KVβ subunit of vertebrate KV channels (see below) clear, but outward movement of the highly charged S4 segment mediates their N-type inactivation in most cases (110, 111). The is important as evidenced by mutagenesis experiments and fluo- mechanism of C-type inactivation of vertebrate KV channels has not rescence labeling (131, 132). Similar to CNG channels, a cyclic yet been well defined (112). nucleotide–binding domain is present in the C-terminal region The KV10–12 subfamilies are related to Drosophila ether-a- of HCN channels (118). Binding of cAMP acts synergistically go-go (eag) channels (113, 114). These channels are primarily with membrane hyperpolarization to regulate channel gating, + voltage-gated and have high selectivity for K . Like other KV similar to the synergistic gating of KCa1 channels by membrane stke.sciencemag.org channels, they contribute to repolarization of electrically ex- depolarization and intracellular calcium binding. Deletion of citable cells to terminate action potentials and are involved in the cyclic nucleotide–binding domain produces a depolarizing signal transduction in nonexcitable cells (115). In spite of these shift in the voltage-dependence of activation, suggesting that functions in common with other KV channels, the pore se- the unoccupied cyclic nucleotide–binding domain has an au- quences of KV10–12 channels are more closely related in amino toinhibitory function (133). HCN channels can form heteromul- acid sequence to the cyclic nucleotide–modulated family (see timers in vitro, but it is not clear whether this also occurs in situ below) than to KV1–9 channels (Fig. 4). Moreover, their C-ter- (128) These channels are crucial to control of rhythmic electri-

minal domains contain a cyclic nucleotide–binding motif whose cal discharge in repetitively firing pacemaker cells in the sino- on July 7, 2009 function has not been fully defined (116–118). KV10–12 chan- atrial node of the heart and the thalamus in the brain (129, 130). nels also form heteromultimers within, but not between, sub- Their ion conductance increases as the membrane is repolarized families (119). An N-terminal PAS (Per-Arnt-Sim) domain may following an action potential, and this increase provides a re- have a role in membrane targeting or protein interactions (120). generative boost that activates sodium channels and begins a new action potential. Direct binding of cAMP and cGMP regu- Cyclic Nucleotide–Modulated Channels lates their activation, which links second messenger signaling Cyclic nucleotide–gated channels were first purified and cloned and pacemaking in rhythmically firing cells. from the retina (121). Six distinct genes in two subfamilies en- code subunits for CNG channels, four CNGA subunits and two Molecular Relations Between KV Channels, KCa CNGB subunits (122) (Fig. 4). In heterologous cell expression Channels, and Cyclic Nucleotide–Modulated Channels systems, the A subunits form functional channels by themselves, A careful look at the phylogenetic tree in Fig. 4 shows that the whereas the B subunits cannot. Functional CNG channels in the KV1 to KV9 channels form one closely related cluster, posi- retina are heteromers assembled in 3A:1B ratio (123, 124). Under tioned adjacent to clusters of K2P channels that may have physiological conditions, CNG channels in photoreceptors and evolved later and Kir channels that may have evolved earlier. olfactory sensory neurons produce depolarizing currents carried Within the KV1–9 cluster, the KV1–4 channels form one related + 2+ principally by Na and Ca (122). Different subunits compose group, the KV5, 6, 8, and 9 channels a second related group, CNG channels in rod (A1 and B1a subunits) and cone (A3 and and the KV7 channels (formerly KCNQ or KVLQT) form a B3 subunits) photoreceptors and in olfactory sensory neurons third more distant group of KV channels. (A2, A4 and B1b subunits) (125, 126). The amino acid sequences Surprisingly, the CNG, HCN, and KV10–12 channels form of CNG and K+ channels diverge exactly at the K+ channel the fourth large cluster when the molecular relations are deter- TVGYG selectivity motif; in CNG channels hydrophilic residues mined by comparison of pore-forming sequences (Fig. 4). This replace the Tyr residue. Although these channels contain three or is a robust result that is obtained by all methods of analysis of four positively charged amino acid residues in their S4 segment, molecular relations that we have used. Evidently, these channels they are not responsive to membrane potential. Instead, they are arose from a common evolutionary pathway despite their diver- gated by direct binding of adenosine 3′,5′-monophosphate sity of ion selectivity (K+, Ca2+, or nonselective cation

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Fig. 4. Phylogenetic relations of the Kv1.1 (KCNA1) potassium channels and the cyclic Kv1.2 (KCNA2) Kv1.8 (KCNA8) nucleotide–modulated ion channels Kv1.3 (KCNA3) (CNG, HCN). The consensus tree Kv1.4 (KCNA4) KCNA5 was inferred by maximum parsimo- Kv1.5 ( ) ** Kv1.6 (KCNA6) ny analysis of the minimal pore re- Kv1.7 (KCNA7) K 3.1 (KCNC1) gions from KV, KCa, Kir, CNG, and v Kv3.2 (KCNC2) HCN, and of the first pore region ** Kv3.3 (KCNC3) from K ion channels, and summa- Kv3.4 (KCNC4) 2P KCND2 rizes 226 bootstrapped replicates Kv4.2 ( ) ** Kv4.3 (KCND3) from heuristic searches with ran- Kv4.1 (KCND1) dom stepwise additions, and TBR Kv2.1 (KCNB1) K 2.2 (KCNB2) branch swapping settings. Gapped ** v ** Kv9.1 (KCNS1) sequences in the alignment were * * Kv9.2 (KCNS2) not analyzed. Tree was rooted to Kv9.3 (KCNS3) Kv8.1 (KCNV1) the bacterial sodium channel Kv8.2 (KCNV2) (NachBac), but this branch is not Kv6.3 (KCNG3) KCNG1 shown in the figure. Bootstrap val- * Kv6.1 ( ) ** Kv6.2 (KCNG2) ues of 50 to 74% are depicted by *; Kv6.4 (KCNG4) values of 75 to 100% are depicted Kv5.1 (KCNF1) K 7.2 (KCNQ2) by **; and not shown are those that v Kv7.3 (KCNQ3) are <49%. The scale bar corre- Kv7.4 (KCNQ4) ** Downloaded from sponds to the number of changes Kv7.5 (KCNQ5) K 7.1 (KCNQ1) v KCNK1 needed to explain the differences in K2P1.1 ( ) * KCNK7 the protein sequences. Vertical K2P7.1 ( ) KCNK6 K2P6.1 ( ) branch length is not precise. KCNK12 K2P12.1 ( ) ** KCNK13 K2P13.1 ( ) KCNK2 K2P2.1 ( ) ** KCNK10 * ** K2P10.1 ( ) KCNK4 K2P4.1 ( ) KCNK16 stke.sciencemag.org K2P16.1 ( ) KCNK17 K2P17.1 ( ) KCNK5 * K2P5.1 ( ) KCNK3 K2P3.1 ( ) * KCNK9 ** K2P9.1 ( ) KCNK15 K2P15.1 ( ) K 18.1 (TRIK) 2P ** Kir4.1 (KCNJ10) Kir4.2 (KCNJ15) Kir7.1 (KCNJ13) Kir1.1 (KCNJ1) Kir2.2 (KCNJ12) on July 7, 2009 Kir2.3 (KCNJ4) Kir2.4 (KCNJ14) * Kir2.1 (KCNJ2) Kir5.1 (KCNJ16) ** Kir6.1 (KCNJ8) ** Kir6.2 (KCNJ11) Kir3.2 (KCNJ6) * Kir3.4 (KCNJ5) * Kir3.3 (KCNJ9) Kir3.1 (KCNJ3) KCNH1 Kv10.1 ( ) ** KCNH5 Kv10.2 ( ) KCNH6 Kv11.2 ( ) ** ** KCNH7 Kv11.3 ( ) ** KCNH2 Kv11.1 ( ) KCNH8 * Kv12.1 ( ) KCNH4 ** Kv12.3 ( ) KCNH3 Kv12.2 ( ) CNGA2 * CNGA3 * CNGA1 * CNGA4 CNGB1 ** CNGB3 HCN3 ** HCN4 HCN1 KCNN2* HCN2 KCa2.2 ( ) * KCNN3 ** KCa2.3 ( ) KCNN1 ** KCa2.1 ( ) KCNN4 KCa3.1 ( ) KCa1.1 (KCNMA1) ** KCa5.1 (KCNMC1) ** Kca4.1 (KCNT1) KCa4.2 (KCNT2) 20 changes

www.stke.org/cgi/content/full/sigtrans;2004/253/re15 Page 8 R EVIEW channels) and primary gating mechanism (voltage, cyclic channels in that they are controlled through autophosphoryla- nucleotides, or both). tion by a phospholipase C–interacting kinase that is fused to An additional surprise is the distant relation of the sequences their intracellular C termini (151). These channels appear to 2+ of the KCa channels and the KV channels in the S5, P loop, and play a crucial role in Mg transport in cells (152). S6 segments (Fig. 4). Evidently, these two groups of channels The TRP channels have S4 segments with repeated motifs of with similar transmembrane topologies separated from each one positively charged amino acid residue followed by two hy- other early in evolution and their pore domains have become drophobic residues, but there are fewer positive charges than in distinct in amino acid sequence. most voltage-gated ion channels. Are TRP channels also voltage-sensitive? A recent report argues persuasively that they are. Transient Receptor Potential Channels Voets et al. (153) report that TRPV1 and TRPM8 channels are acti- Although it is the second largest among those related to voltage- vated by depolarization to potentials above 100 mV, far beyond the gated ion channels, the TRP channel family was underappreciated physiological range of membrane potentials. However, the voltage until recently. Beginning with a founding member identified in the dependence of activation is shifted negatively by cooling or menthol Drosophila phototransduction system (134, 135), 32 genes that treatment for TRPM8 or by heating or capsaicin treatment for encode one-domain TRP channels and structural relatives have TRPV1, allowing activation at negative membrane potentials. This been defined and classified into at least six distinct subfamilies synergistic gating by voltage plus either temperature or ligand bind- (Figs. 1 and 5) (136). TRP channels are 6-TM proteins that resem- ing resembles the synergistic gating of KCa1 channels by voltage 2+ ble KV channels in overall architecture, but show limited conser- and Ca and HCN channels by voltage and cyclic nucleotides. vation of the S4 positive charges and P loop sequences that are the These results suggest similar allosteric gating mechanisms for these hallmarks of the KV, NaV, and CaV families. Although some show distantly related members of the ion channel superfamily. 2+

preferential permeability to Ca , TRP channels in general are Downloaded from nonselective cation channels whose opening leads to influx of ex- TRPP1 tracellular cations that depolarizes the membrane potential and in- *** TRPP3 creases intracellular Ca2+ (137). This heterogeneous group of ion TRPP2 channel proteins may generate additional diversity through forma- TRPC3 ** TRPC7 tion of heteromeric channel complexes, as has been demonstrated TRPC6 for TRPC (137, 138). In addition, some family members have ** TRPC4 ** TRPC5 ankyrin repeats in their N-terminal domains that may interact with TRPC1 stke.sciencemag.org intracellular proteins (137). mTrp2 Physiological triggers that activate TRP channels are diverse ** TRPM1 2+ TRPM3 and incompletely understood. Sustained Ca entry through ** TRPM6 TRPC channels is stimulated by hormones that activate phos- ** ** TRPM7 TRPM2 pholipase C isozymes and thereby generate diacylglycerol and * TRPM8 other lipid messengers; these channels are an important compo- ** TRPM4 nent of receptor-operated and possibly also store-operated * TRPM5 2+ ANKTM1 cation channels that play a fundamental role in cellular Ca TRPV1 on July 7, 2009 signaling and homeostasis (139–141). TRPV2 ** TRPV3 Both TRPV and TRPM channels are involved in sensory TRPV4 signaling. Members of the TRPV subfamily can be activated by ** TRPV5 the hot chili pepper ingredient, capsaicin; high osmolarity; low TRPV6 2+ TRPML1 pH; and low intracellular Ca (142). In addition, members of ** TRPML3 TRPV family respond to thermal stimuli. TRPV1 and TRPV2 TRPML2 TPC1 are activated by differing levels of noxious heat, whereas TR- TPC2 PV3 and TRPV4 are gated by innocuous warmth <42°C CatSper2 (143–146). Mice lacking TRPV1 are deficient in both thermal CatSper3 CatSper1 sensation and response to thermal hyperalgesia (147). Sensation * CatSper4 20 changes of cold temperatures involves the TRPM family. TRPM1 chan- nels were first identified as an up-regulated mRNA in Fig. 5. Phylogenetic relations of the transient receptor potential melanoma cells, but their physiological function is unknown. (TRP) channels and their related family members. The consensus The menthol-sensitive TRPM8 channels are gated by cool tem- tree was inferred by maximum parsimony analysis of the minimal pore regions from TRPC, TRPM, TRPV, ANKTM1, TRPP, and peratures (<25°C), whereas the distantly related ANKTM1 is TRPML, and of the pore region from second homologous domain activated by even colder temperatures (148–150). The discovery of TPC ion channels, and it summarizes 578 bootstrapped repli- that TRPV and TRPM channels are responsible for transduction cates from heuristic searches with random stepwise additions, of thermal sensation and thermal pain is a major breakthrough and TBR branch–swapping settings. Gapped sequences in the in understanding temperature sensing at the molecular level. alignment were not analyzed. Tree was rooted to the bacterial It is likely that other TRPM subfamily members have differ- sodium channel (NachBac), but this branch is not shown in the ent roles in sensory physiology involving distinct molecular figure. Bootstrap values of 50 to 74% are depicted by *; values of mechanisms (136). TRPM2 is activated by ADP-ribose, an in- 75 to 100% are depicted by **; and not shown are those that are tracellular second messenger. TRPM4 and TRPM5 are activated <49%. The scale bar corresponds to the number of changes by G protein–coupled receptor-mediated release of Ca2+ from needed to explain the differences in the protein sequences. intracellular stores. TRPM6 and TRPM7 are unique among ion Vertical branch length is not significant.

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The TRPP and TRPML subfamilies of TRP channels (136) are nation and modulation of the resting membrane potential in incompletely characterized physiologically, because most of them neurons (169). Kir subunits can form heteromeric channels, but have not been successfully expressed in heterologous cells. TRPP1, auxiliary subunit association has been demonstrated only for the PKD2 protein (154), whose defect is associated with inherited the Kir6 subunits, which assemble with regulatory sulfonylurea polycystic kidney disease, is thought to be a Ca2+-permeable chan- receptor subunits (SUR) of the ATP-binding cassette (ABC) nel activated by bending of the apical cilium of certain kidney ep- family (see below) to form the functional KAT P channel complex ithelia, presumably to sense fluid flow through renal tubules (155). (165, 167). Direct binding of cellular ATP or sulfonylurea drugs Mutations in TRPML1 (mucolipin-1) cause mucolipidosis type IV, to the SUR subunits inhibits the activity of the Kir6 subunits of a neurodegenerative, lysosomal storage disorder with defects in the KAT P channel complex, depolarizes the cell, and promotes membrane sorting or endocytosis (156). Mutation of the TRPML3 the exocytosis of vesicular insulin by the pancreatic beta cells. channel, expressed in the cochleal hair cells of the ear, causes deaf- This signaling pathway allows coupling of metabolic state to ness and vestibular dysfunction in the varitint-waddler mouse from electrical excitability (165, 167, 170). 2+ which this gene was cloned (157). The putative one-domain Ca Members of the K2P channel family are composed of four channels of sperm, CatSper1–4 channels (158–160), are also dis- transmembrane segments with two P loops, a topology similar tantly related to the TRP family. Although the presence of positively to a tandem fusion of two Kir channels, and the pore selectivity charged residues in S4 suggests that depolarization may activate sequence of the K2P channels is T(I/V)GYG or T(I/V)GFG sim- these channels, voltage-gated CatSper currents have yet to be ilar to the K+ channels (Fig. 1) (171–173). These channels are recorded directly. However, ablation of CatSper1 in mouse causes sometimes called “leak channels” or “open rectifiers” that con- defects in cAMP-mediated Ca2+ influx and sperm duct “background” K+ currents because they are constantly ac- motility (158). Most recently, a two-pore channel (TPC) protein tive at the resting membrane potential of cells and serve to hold

with two repeats of a 6-TM domain, resembling a fusion of two the cell membrane potential near the equilibrium potential for Downloaded from + TRP-like channels, has been cloned from rat kidney (161), and K . Low among K2P channels suggests that there are two orthologous human genes, TPCN1 and TPCN2, that the 15 genes in the human genome each constitute a distinct + are expressed in human liver, lung, and kidney. These two-domain subfamily (1) (Fig. 4). K2P channels conduct background K channels may add to the remarkable diversity of form and function currents in both excitable and nonexcitable cells (171–173). of the TRP family of channels. There is no evidence of heteromer formation or association with + auxiliary subunits. K2P channels are resistant to most K chan-

Inwardly Rectifying and Two-Pore Potassium Channels nel blockers, but their activity is modulated by various agents stke.sciencemag.org The inwardly rectifying Kir channels derive their name from including mechanical stress, changes in pH, changes in temper- their capacity to conduct K+ inward more readily than outward. ature, and fatty acids (171–173). Background K+ currents are However, at physiological membrane potentials, these channels important physiologically to stabilize the membrane potential in + mediate K efflux, which repolarizes and hyperpolarizes the excitable cells. Moreover, K2P channels are molecular targets membrane potential. Kir channels have the simplest transmem- for inhalation anesthetics and enhanced activation of K2P cur- brane topology in the voltage-gated–like ion channel family, rents by these agents leads to hyperpolarization of neurons and namely, two transmembrane segments (M1 and M2) that are thus reduction of excitability (174). equivalent to the S5 and S6 of KV channels with an intervening on July 7, 2009 reentrant P loop (Fig. 2B). All 15 members of the 7 Kir subfami- Disease Mutations in the Ion Channel Superfamily lies [(Figs. 1 and 4), (Table S2, http://stke.sciencemag.org/ The many different genetic diseases caused by their mutations high- cgi/content/full/sigtrans;2004/253/re15/DC2)] contain the con- light the broad-ranging physiological roles of ion channels (Table served (T/S)IG(Y/F)G motif of KV channels in the P loop, con- 1). NaV channels are the molecular targets for many mutations that sistent with their highly selective permeation of K+ (1). Because cause diseases of hyperexcitability, including periodic paralysis of of the absence of the S4 voltage-sensor, the Kir channels are not skeletal muscle, cardiac arrhythmia, and epilepsy (175). Most often gated by changes in transmembrane voltage per se. However, these mutations are dominant because they cause gain of function changes in membrane potential are still important to their func- by blocking the normal inactivation process or by causing preco- tion, as they regulate physical occlusion of the pore of the Kir cious activation of the channels. Similarly, mutations of KV chan- channels by intracellular Mg2+ and polyamines, which is the nels cause cardiac arrhythmias due to impaired repolarization (176), mechanistic basis for the block of the pore at positive mem- sometimes accompanied by deafness or other specific deficits. Mu- brane potentials that causes inward rectification (162, 163). tations of CaV channels cause cardiac arrhythmia, periodic paralysis In addition to voltage-dependent blocking by Mg2+ and of skeletal muscle, familial migraine, night blindness, and cerebellar polyamines, the ion conductance activity of Kir channels is regulat- ataxia (175). Mutations in cyclic nucleotide–gated channels in the ed by a remarkable diversity of intracellular messengers. Different retina cause inherited retinitis pigmentosa (177). Mutations of Kir subfamilies are regulated by direct binding of different sets of lig- channels cause inherited forms of hyperinsulinemia and renal dys- + ands: phosphatidylinositol bisphosphate (PIP2), H , and ATP, for the function (178). The discovery that mutations of ion channels cause + Kir1 subfamily; H and PIP2 for Kir2; G protein βγ subunits and inherited diseases points to additional opportunities for these pro- + PIP2 for Kir3; and ATP and ADP for Kir6 (164–166). Outward K teins as potential drug targets for therapy of diseases of hypo- or hy- conductance through Kir channels maintains the resting membrane perexcitability and altered ion transport. potential near the K+ reversal potential to control a wide spectrum of cellular processes, including transepithelial transport of K+ in the Auxiliary Subunits of the Ion Channel Protein Family kidney and other epithelial tissues (90), secretion of insulin in the The principal pore-forming subunits of the voltage-gated ion beta cells of the pancreas (167, 168), regulation of maximum dias- channels and their structural relatives are primarily responsible tolic potential and beating rate in the heart (164), and determi- for their characteristic gating, selective ion conductance, and

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regulation by second-messenger signaling and pharmacologic The three KVβ subunits are superficially similar to the CaVβ subunits agents. However, many of these principal subunits are in their cytoplasmic location, but are not related in amino acid se- associated with auxiliary subunits that modify their expression, quence or structure. The N terminus of KVβ subunits of vertebrates functional properties, and subcellular localization (179). In the serves as an inactivation gate for KV1 α subunits (203) and is exceptional case of KAT P channels, the auxiliary subunit is a thought to enter the pore and block it during sustained channel primary regulator of channel activity. In this section, we briefly opening (111). This is a unique example of a direct physical role for review the structure, function, and genetic relations of the an auxiliary subunit in channel gating, rather than a role modulating known auxiliary subunits. the gating process of its associated pore-forming α subunit. Sodium Channels. Purification and characterization of sodi- KV4 channels interact with the K channel–interacting pro- um channels provided the first evidence for ion channel auxil- teins KChIp1–4, which are members of the neuronal calcium iary subunits (3, 5). Sodium channels have a single family of sensor family of calmodulin-like calcium regulatory proteins auxiliary subunits, NaVβ1 to NaVβ4, which interact with the and have four EF hand motifs (Fig. 6) (205). The KChIps en- different α subunits and alter their physiological properties and hance expression of KV4 channels and modify their functional subcellular localization. These proteins have a single transmem- properties by binding to a site in the intracellular T1 domain, brane segment, a large N-terminal extracellular domain that is similar to the interaction of KVβ subunits with KV1 channels. homologous in structure to a variable chain (V-type) The KV7, KV10, and KV11 channels associate with a different immunoglobulin-like fold, and a short C-terminal intracellular type of auxiliary subunit—the minK-like subunits. These five close- segment (Fig. 6) (180–183). The NaVβ subunits interact with α ly related proteins have a single transmembrane segment and small subunits through their extracellular immunoglobulin-like extracellular and intracellular domains (206, 207). Although these domains, modulate α subunit function, and enhance their cell subunits are topologically similar to NaVβ and CaVδ subunits, they

surface expression (184). Like other proteins with an extracellu- do not have substantial amino acid sequence similarity. The minK- Downloaded from lar immunoglobulin-like fold, they also serve as cell adhesion like subunits are important regulators of KV7 channel function (208, molecules by interacting with extracellular matrix proteins, cell 209), and mutations in one of these auxiliary subunits causes a form adhesion molecules, and cytoskeletal linker proteins (185–189). of familial long QT syndrome, which predisposes to dangerous car- A mutation in a conserved cysteine in the immunoglobulin-like diac arrhythmias (210). In addition, recent work indicates that these fold of the NaVβ1 subunit causes familial epilepsy (190). The subunits also associate with KV3 and KV4 channels and are respon- NaVβ subunits are a recent evolutionary addition to the family sible for a form of inherited periodic paralysis (210, 211). In light of

of ion channel–associated proteins, as they have only been iden- this work, it is possible that all KV channels associate with minK- stke.sciencemag.org tified in vertebrates. related subunits. If this hypothesis is true, the KV channels would Calcium Channels. CaV1 and CaV2 channels have four dis- then resemble the NaV and CaV channels in having an associated tinct auxiliary subunits, CaVα2, CaVβ, CaVγ, and CaVδ (Fig. 6) subunit with a single transmembrane segment and short intracellular (191), which each form a small protein family. The CaVα2 and and extracellular domains. It will be intriguing to learn if there is a CaVδ subunits are encoded by the same gene (192), whose common function for these similar auxiliary ion channel subunits. translation product is proteolytically cleaved and disulfide Calcium-Activated Potassium Channels. The KCa1, 4, and 5 linked to yield the mature extracellular α2 subunit glycoprotein family of channels are associated with one of four auxiliary of 140 kD and transmembrane disulfide-linked δ subunit glyco- KCaβ subunits, which have two transmembrane segments and on July 7, 2009 protein of 27 kD (193). Four CaVα2δ genes are known (194). both N and C termini in the cytosol (Fig. 6) (212, 213). The The four CaVβ subunits are all intracellular proteins with a KCaβ subunits contribute to the binding site for the peptide common pattern of α-helical and unstructured segments (194, scorpion toxin charybdotoxin and related channel-blocking 195). They have important regulatory effects on cell surface ex- agents (214), but their effects on channel function and roles in pression, and they also modulate the gating of calcium chan- cell physiology are still emerging in current research (213). nels, which causes enhanced activation upon depolarization and Inwardly Rectifying Potassium Channels. Unique among the altered rate and voltage dependence of inactivation (194). inwardly rectifying potassium channel subunits, the Kir6 sub- Recent structural modeling and x-ray crystallography studies units that form KAT P channels are associated with SURs (215), have revealed that, like the membrane-associated guanylate ki- which are crucial regulators of channel activity. They are also nase (MAGUK) family of scaffolding proteins, these subunits the molecular targets for the sulfonylurea class of KAT P channel contain conserved, interacting SH3 and guanylate kinase do- blockers, which are used to enhance insulin secretion in therapy mains and, therefore, may interact with other intracellular pro- of diabetes. The three SUR proteins are members of the ABC teins (196-200). Eight CaVγ subunit genes encode glycoproteins transporter family of membrane proteins. They have an amino with four transmembrane segments (194, 201). Although the terminal domain with five probable transmembrane segments, CaVγ1 subunit is associated specifically with skeletal muscle which is followed by two domains with six transmembrane seg- CaV1.1 channels, other CaVγ subunits interact with other calci- ments and two ATP-binding motifs, a pattern similar to other um channels, glutamate receptors, and possibly other members of the ABC transporter family (216). Sequential bind- membrane-signaling proteins (194). Thus, the γ subunits discov- ing and hydrolysis of ATP at the two nucleotide-binding cas- ered as components of calcium channels apparently have a more settes regulate Kir6 channel function in response to changes in widespread role in assembly and cell surface expression of oth- the concentration and ratio of ATP and ADP and, thereby, regu- er membrane signaling proteins. late channel activity in response to the metabolic state of the Voltage-Gated Potassium Channels. KV1 channels are often asso- cell. This form of channel regulation is crucial in control of in- ciated with an intracellular KVβ subunit (KVβ1–3) (Fig. 6) (110, 202, sulin release from the beta cells of the pancreas. Mutations in 203), which interacts with the N-terminal T1 domain and forms a SUR are responsible for some forms of familial hyperinsuline- symmetric tetramer on the intracellular surface of the channels (204). mia (217).

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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 orient- ed in opposite directions for the top and bottom membranes, as in a cell. N-linked carbohydrate chains are indicated by Ψ. The intra- cellular 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.

Polycystins. Polycystins (PKDs) 1 and 2 were both discov- been purified from native tissues, it is possible that many addi- ered as the targets of mutations that cause autosomal dominant tional ion channels have auxiliary subunits associated with polycystic kidney disease (154, 218). PKD2 is an ion channel them. As the auxiliary subunits that are known have diverse ef- protein similar in topology and amino acid sequence to TRP fects on ion channel function, expression, and localization, it channels (see TRPP channels above), whereas PKD1 is an aux- will be important to fully define the set of auxiliary subunits in iliary subunit that modifies expression and function of PKD2 order to fully understand the functional and regulatory charac- (219). Like SUR, it has a complex transmembrane structure teristics of ion channels in situ. (Fig. 6). Elucidating the functional relations between PKD1 and PKD2 and defining their roles in normal kidney function is an Conclusion active area of investigation. The voltage-gated ion channel superfamily is one of the largest More Auxiliary Subunits? Almost every ion channel that has families of signaling proteins, following the G protein–coupled been isolated from its native mammalian tissue has been found receptors and the protein kinases in the number of family mem- to have auxiliary subunits. Because most of the superfamily bers. The family is likely to have evolved from a 2-TM ancestor members have been identified by cDNA cloning and have never like the bacterial KcsA channel. The additions of intracellular

www.stke.org/cgi/content/full/sigtrans;2004/253/re15 Page 12 R EVIEW regulatory domains for ligand binding and a 4-TM domain for original screen of RefSeq Release 2 yielded 144 ion channel voltage-dependent gating have produced extraordinarily versa- proteins. To test for completeness of this set, we rescreened tile signaling molecules with the capacity to respond to voltage RefSeq Release 6 (12 July 2004), which has 21,429 confirmed signals and intracellular effectors and to integrate information protein records and 6553 predicted protein records. We found coming from these two distinct types of inputs. The resulting no new ion channel proteins in the most recent RefSeq release, signaling mechanisms control most aspects of cell physiology and one ion channel protein was deleted in the RefSeq database and underlie complex integrative processes like learning and owing to an error (TRPC8), yielding the final set of 143 ion memory in the brain and coordinated movements in muscles. channel proteins reported here. The latest estimates from gene- The evolutionary appearance and refinement of these signaling prediction programs suggest that there are fewer than 24,500 mechanisms is one of the landmark events that allowed the de- protein-coding genes, including ~3000 pseudogenes (220). velopment of complex multicellular organisms. Pseudogenes are not represented in the RefSeq database, so ~21,500 protein-encoding genes are expected to be in the Ref- Appendix Seq database when full coverage of the genome is achieved. The A complete file of the 143 human ion channel genes is provided extra RefSeq records reflect existence of naturally occurring in Table S1 (http://stke.sciencemag.org/cgi/content/full/ splice variants of gene transcripts. Although the exact coverage sigtrans;2004/253/re15/DC1) in the form of an Excel spreadsheet. of the human genes in the RefSeq database is not known The file contains the ion channel protein names as approved by the precisely, comparison of these numbers indicates that it is near- IUPHAR Nomenclature Committee (1), the corresponding gene ly complete. In any case, the increase of 2156 records from Ref- names as approved by HUGO, the complete amino acid sequence of Seq Release 2 to RefSeq Release 6, about 10% of the predicted a representative isolate, and the pore-forming segments of the hu- number of protein-encoding human genes, yielded no new ion channels. Therefore, we are confident that the small number of man genes that were used for molecular comparisons. A file of their Downloaded from relatives in C. elegans and Drosophila is shown in Table S3 human proteins that are not yet represented in the RefSeq (http://stke.sciencemag.org/cgi/content/full/sigtrans;2004/ database will include few, if any, new members of the ion chan- 253/re15/DC3). In Table S2 (http://stke.sciencemag.org/cgi/ nel protein family. content/full/sigtrans;2004/253/re15/DC2), the alignment of the pore-forming segments of the 143 ion channels used in the phyloge- References and Notes netic analyses is provided. In the alignment, mouse Trp2 protein se- 1. International Union of Pharmacology (IUPHAR), IUPHAR Compendium of Voltage-Gated Ion Channels, W. A. Catterall, G. A. Gutman, K. G. quence was included because the human ortholog is a pseudogene. Chandy, Eds. (IUPHAR Media, Leeds, UK, 2002); available at stke.sciencemag.org Comparison of these aligned sequences by minimum evolution and http://www.iuphar-db.org/iuphar-ic/. maximum parsimony methods yielded the phylogenetic trees pre- 2. G. Manning, D. B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam, The protein kinase complement of the human genome. Science 298, sented in the main text. 1912–1934 (2002). Our approach to genomic analysis of the voltage-gated ion 3. D. A. Beneski, W. A. Catterall, Covalent labeling of protein components channel superfamily proceeded in three steps. First, we con- of the sodium channel with a photoactivable derivative of scorpion toxin. Proc. Natl. Acad. Sci. U.S.A. 77, 639–643 (1980). structed profile HMMs for the conserved pore sequences of 4. W. S. Agnew, A. C. Moore, S. R. Levinson, M. A. Raftery, Identification of each of the families of ion channels identified in the IUPHAR a large molecular weight peptide associated with a tetrodotoxin binding proteins from the electroplax of Electrophorus electricus. Biochem. Bio- compendium (1). ClustalX alignments were made of human on July 7, 2009 protein sequences of 20 sodium and calcium channels (domain phys. Res. Commun. 92, 860–866 (1980). 5. W. A. Catterall, The molecular basis of neuronal excitability. Science IV), 75 potassium channels (first pore motif of K2P), 8 cyclic 223, 653–661 (1984). nucleotide–gated channels (CNG, HCN), and 29 TRP and relat- 6. W. A. Catterall, From ionic currents to molecular mechanisms: The ed channels. These minimum pore alignments were fed into the structure and function of voltage-gated sodium channels. Neuron 26, 13–25 (2000). HMMER 2.3 suite of programs (Washington University, St. 7. M. Noda, S. Shimizu, T. Tanabe, T. Takai, T. Kayano, T. Ikeda, H. Taka- Louis; http://hmmer.wustl.edu/) to generate and calibrate pro- hashi, H. Nakayama, Y. Kanaoka, N. Minamino, K. Kangawa, H. Matsuo, file HMMs by using default parameters and to screen the Ref- M. Raftery, T. Hirose, S. Inayama, H. Hayashida, T. Miyata, S. Numa, Primary structure of Electrophorus electricus sodium channel deduced Seq databases to identify every related family member. Second, from cDNA sequence. Nature 312, 121–127 (1984). to test whether each family is a member of the VGL-chanome, 8. M. Noda, T. Ikeda, T. Suzuki, H. Takeshima, T. Takahashi, M. Kuno, S. we determined its nearest neighbor in amino acid sequence Numa, Expression of functional sodium channels from cloned cDNA. Nature 322, 826–828 (1986). space as defined by the HMM screening results. In every case, 9. H. R. Guy, P. Seetharamulu, Molecular model of the action potential we found that the nearest neighbor is another family within the sodium channel. Proc. Natl. Acad. Sci. U.S.A. 508, 508–512 (1986). VGL-chanome, confirming that all families are members of a 10. C. Sato, Y. Ueno, K. Asai, K. Takahashi, M. Sato, A. Engel, Y. Fujiyoshi, The voltage-sensitive sodium channel is a bell-shaped molecule with single superfamily of ion channels. Finally, we fed these related several cavities. Nature 409, 1047–1051 (2001). sequences into the PAUP program and developed a phylogenetic 11. O. Pongs, N. Kecskemethy, R. Muller, I. Krah-Jentgens, A. Baumann, H. map of all 143 members of the VGL-chanome using maximum H. Kiltz, I. Canal, S. Llamazares, A. Ferrus, Shaker encodes a family of parsimony and neighbor-joining algorithms. These methods re- putative potassium channel proteins in the nervous system of Drosophila. EMBO J. 7, 1087–1096 (1988). sulted in the molecular relations illustrated in Fig. 1. 12. D. M. Papazian, T. L. Schwarz, B. L. Tempel, Y. N. Jan, L. Y. 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