Conserved motifs in voltage-sensing and pore-forming modules of voltage-gated ion channel proteins

Voltage-gated ion channels (VGCs) mediate selective diffusion of ions across cell membranes to enable many vital cellular processes. Three-dimensional structure data are lacking for VGC proteins; hence, to better understand their function, there is a need to identify the conserved motifs using sequence analysis methods. In this study, we have used a profile-to-profile alignment method to identify several new conserved motifs specific to each transmembrane segment (TMS) of the voltage-sensing and the pore-forming modules of Ca2+, Na+, and K+ channel subfamilies. For Ca2+ and Na+, the functional theme of motif conservation is similar in all segments while they differ with those of the K+ channel proteins. Nevertheless, the conservation is strikingly similar in the S4 segment of the voltage-sensing module across all subfamilies. In each subfamily and for each TMS, we have identified conserved motifs/residues and correlated their functional significance and disease associations in human, using mutational data from the literature.     

Sp-tetraKCNG: A novel cyclic nucleotide gated K(+) channel

The sequence of a novel cGMP-regulated, tetrameric, K(+) selective channel (Sp-tetraKCNG) was discovered in the sea urchin Strongylocentrotus purpuratus. The Sp-tetraKCNG is a single polypeptide made of four KCNG domains similar to voltage-dependent Na(+) and Ca(2+) channels. Each KCNG domain has six transmembrane segments (S1-S6), the ion pore having the K(+) selectivity signature GYGD and a cyclic nucleotide-binding domain (CNBD). This novel channel is evolutionary located between K(+)-selective and voltage-dependent EAG channels and voltage-independent cationic CNG channels. Bilayer reconstitutions demonstrate such a cGMP-regulated K(+) selective channel in sea urchin spermatozoa.     

Convergent evolution of sodium ion selectivity in metazoan neuronal signaling

Ion selectivity of metazoan voltage-gated Na(+) channels is critical for neuronal signaling and has long been attributed to a ring of four conserved amino acids that constitute the ion selectivity filter (SF) at the channel pore. Yet, in addition to channels with a preference for Ca(2+) ions, the expression and characterization of Na(+) channel homologs from the sea anemone Nematostella vectensis, a member of the early-branching metazoan phylum Cnidaria, revealed a sodium-selective channel bearing a noncanonical SF. Mutagenesis and physiological assays suggest that pore elements additional to the SF determine the preference for Na(+) in this channel. Phylogenetic analysis assigns the Nematostella Na(+)-selective channel to a channel group unique to Cnidaria, which diverged >540 million years ago from Ca(2+)-conducting Na(+) channel homologs. The identification of Cnidarian Na(+)-selective ion channels distinct from the channels of bilaterian animals indicates that selectivity for Na(+) in neuronal signaling emerged independently in these two animal lineages.     

Site-specific mutations in a minimal voltage-dependent K+ channel alter ion selectivity and open-channel block

MinK is a small membrane protein of 130 amino acids with a single potential membrane-spanning alpha-helical domain. Its expression in Xenopus oocytes induces voltage-dependent, K(+)-selective channels. Using site-directed mutagenesis of a synthetic gene, we have identified residues in the hydrophobic region of minK that influence both ion selectivity and open-channel block. Single amino acid changes increase the channel's relative permeability for NH4+ and Cs+ without affecting its ability to exclude Na+ and Li+. Blockade by two common K+ channel pore blockers, tetraethylammonium and Cs+, was also modified. These results suggest that an ion selectivity region and binding sites for the pore blockers within the conduction pathway have been modified. We conclude that the gene encoding minK is a structural gene for a K+ channel protein.     

Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability

We have analyzed nucleic acid and amino acid sequence alignments of a variety of voltage-sensitive ion channels, using several methods for phylogenetic tree reconstruction. Ancient duplications within this family gave rise to three distantly related groups, one consisting of the Na+ and Ca++ channels, another the K+ channels, and a third including the cyclic nucleotide-binding channels. A series of gene duplications produced at least seven mammalian homologues of the Drosophila Shaker K+ channel; clones of only three of these genes are available from all three mammalian species examined (mouse, rat, and human), pointing to specific genes that have yet to be recovered in one or another of these species. The Shaw-related K+ channels and the Na+ channel family have also undergone considerable expansion in mammals, relative to flies. These expansions presumably reflect the needs of the high degree of physiological and neuronal complexity of mammals. Analysis of the separate domains of the four-domain channels (Ca++ and Na+) supports their having evolved by two sequential gene duplications and implies the historical existence of a functional two-domain channel.      

Construction of a cyclic nucleotide-gated KcsA K+ channel

The ability of an ion channel to open in response to a defined stimulus is central to its function. In ligand-gated channels, pore opening is conferred through transduction of a conformational change in a gating domain to the helices of the pore. Here, we present the construction of a designed cyclic nucleotide-gated (CNG) channel, named KcsA-CNG, by addition of a prokaryotic cyclic nucleotide-binding domain to a KcsA-derived K+ channel. This channel is functional in lipid bilayers at physiological pH and has the combined properties of both of its parent-derived components. It conducts K+ and is blocked by the K+ channel inhibitors Na+ and agitoxin-2. Channel open times are increased by about two orders of magnitude compared to wild-type KcsA. The average number of open channels increases by approximately 50% upon addition of cAMP. Although the absolute open probabilities are somewhat variable from one channel to the next, the property of cyclic nucleotide sensitivity is very reproducible. An apparent Kd value of approximately 90 nM was estimated. The successful construction of a cyclic nucleotide-gated KcsA K+ channel suggests that it should be possible to produce channels that will respond to novel ligands.     

Sodium permeability of a cloned small-conductance calcium-activated potassium channel

Small-conductance Ca2+-activated potassium channels (SK(Ca) channels) are heteromeric complexes of pore-forming main subunits and constitutively bound calmodulin. SK(Ca) channels in neuronal cells are activated by intracellular Ca2+ that increases during action potentials, and their ionic currents have been considered to underlie neuronal afterhyperpolarization. However, the ion selectivity of neuronal SK(Ca) channels has not been rigorously investigated. In this study, we determined the monovalent cation selectivity of a cloned rat SK(Ca) channel, rSK2, using heterologous expression and electrophysiological measurements. When extracellular K+ was replaced isotonically with Na+, ionic currents through rSK2 reversed at significantly more depolarized membrane potentials than the value expected for a Nernstian relationship for K+. We then determined the relative permeability of rSK2 for monovalent cations and compared them with those of the intermediate- and large-conductance Ca2+-activated K+ channels, IK(Ca) and BK(Ca) channels. The relative permeability of the rSK2 channel was determined as K+(1.0)>Rb+(0.80)>NH(4)+(0.19) approximately Cs+(0.19)>Li+(0.14)>Na+(0.12), indicating substantial permeability of small ions through the channel. Although a mutation near the selectivity filter mimicking other K+-selective channels influenced the size-selectivity for permeant ions, Na+ permeability of rSK2 channels was still retained. Since the reversal potential of endogenous SK(Ca) current is determined by Na+ permeability in a physiological ionic environment, the ion selectivity of native SK(Ca) channels should be reinvestigated and their in vivo roles may need to be restated.     

On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel.

Voltage-sensitive sodium channels and calcium channels are homologous proteins with distinctly different selectivity for permeation of inorganic cations. This difference in function is specified by amino acid residues located within P-region segments that link presumed transmembrane elements S5 and S6 in each of four repetitive Domains I, II, III, and IV. By analyzing the selective permeability of Na+, K+, and Ca2+ in various mutants of the mu 1 rat muscle sodium channel, the results in this paper support the concept that a conserved motif of four residues contributed by each of the Domains I-IV, termed the DEKA locus in sodium channels and the EEEE locus in calcium channels, determines the ionic selectivity of these channels. Furthermore, the results indicate that the Lys residue in Domain III of the sodium channel is the critical determinant that specifies both the impermeability of Ca2+ and the selective permeability of Na+ over K+. We propose that the alkylammonium ion of the Lys(III) residue acts as an endogenous cation within the ion binding site/selectivity filter of the sodium channel to tune the kinetics and affinity of inorganic cation binding within the pore in a manner analogous to ion-ion interactions that occur in the process of multi-ion channel conduction.     

Plants do it differently. A new basis for potassium/sodium selectivity in the pore of an ion channel

Understanding of the molecular architecture necessary for selective K(+) permeation through the pore of ion channels is based primarily on analysis of the crystal structure of the bacterial K(+) channel KcsA, and structure:function studies of cloned animal K(+) channels. Little is known about the conduction properties of a large family of plant proteins with structural similarities to cloned animal cyclic nucleotide-gated channels (CNGCs). Animal CNGCs are nonselective cation channels that do not discriminate between Na(+) and K(+) permeation. These channels all have the same triplet of amino acids in the channel pore ion selectivity filter, and this sequence is different from that of the selectivity filter found in K(+)-selective channels. Plant CNGCs have unique pore selectivity filters; unlike those found in any other family of channels. At present, the significance of the unique pore selectivity filters of plant CNGCs, with regard to discrimination between Na(+) and K(+) permeation is unresolved. Here, we present an electrophysiological analysis of several members of this protein family; identifying the first cloned plant channel (AtCNGC1) that conducts Na(+). Another member of this ion channel family (AtCNGC2) is shown to have a selectivity filter that provides a heretofore unknown molecular basis for discrimination between K(+) and Na(+) permeation. Specific amino acids within the AtCNGC2 pore selectivity filter (Asn-416, Asp-417) are demonstrated to facilitate K(+) over Na(+) conductance. The selectivity filter of AtCNGC2 represents an alternative mechanism to the well-known GYG amino acid triplet of K(+) channels that has been identified as the critical basis for K(+) over Na(+) permeation through the pore of ion channels.     

Ion solvation and structural stability in a sodium channel investigated by molecular dynamics calculations

The stability and ion binding properties of the homo-tetrameric pore domain of a prokaryotic, voltage-gated sodium channel are studied by extensive all-atom molecular dynamics simulations, with the channel protein being embedded in a fully hydrated lipid bilayer. It is found that Na(+) ion presents in a mostly hydrated state inside the wide pore of the selectivity filter of the sodium channel, in sharp contrast to the nearly fully dehydrated state for K(+) ions in potassium channels. Our results also indicate that Na(+) ions make contact with only one or two out of the four polypeptide chains forming the selectivity filter, and surprisingly, the selectivity filter exhibits robust stability for various initial ion configurations even in the absence of ions. These findings are quite different from those in potassium channels. Furthermore, an electric field above 0.5V/nm is suggested to be able to induce Na(+) permeation through the selectivity filter.     

A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel

Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.     

Calcium-Activated K+ Channels and Calcium-Induced Calcium Release by Slow Vacuolar Ion Channels in Guard Cell Vacuoles Implicated in the Control of Stomatal Closure

Stomatal closing requires the efflux of K+ from the large vacuolar organelle into the cytosol and across the plasma membrane of guard cells. More than 90% of the K+ released from guard cells during stomatal closure originates from the guard cell vacuole. However, the corresponding molecular mechanisms for the release of K+ from guard cell vacuoles have remained unknown. Rises in the cytoplasmic Ca2+ concentration have been shown to trigger ion efflux from guard cells, resulting in stomatal closure. Here, we report a novel type of largely voltage-independent K+-selective ion channel in the vacuolar membrane of guard cells that is activated by physiological increases in the cytoplasmic Ca2+ concentration. These vacuolar K+ (VK) channels had a single channel conductance of 70 pS with 100 mM KCI on both sides of the membrane and were highly selective for K+ over NH4+ and Rb+. Na+, Li+, and Cs+ were not measurably permeant. The Ca2+, voltage, and pH dependences, high selectivity for K+, and high density of VK channels in the vacuolar membrane of guard cells suggest a central role for these K+ channels in the initiation and control of K+ release from the vacuole to the cytoplasm required for stomatal closure. The activation of K+-selective VK channels can shift the vacuolar membrane to more positive potentials on the cytoplasmic side, sufficient to activate previously described slow vacuolar cation channels (SV-type). Analysis of the ionic selectivity of SV channels demonstrated a Ca2+ over K+ selectivity (permeability ratio for Ca2+ to K+ of ~3:1) of these channels in broad bean guard cells and red beet vacuoles, suggesting that SV channels play an important role in Ca2+-induced Ca2+ release from the vacuole during stomatal closure. A model is presented suggesting that the interaction of VK and SV channel activities is crucial in regulating vacuolar K+ and Ca2+ release during stomatal closure. Furthermore, the possibility that the ubiquitous SV channels may represent a general mechanism for Ca2+-induced Ca2+ release from higher plant vacuoles is discussed.     

Functional architecture of the inner pore of a voltage-gated Ca2+ channel

The inner pore of voltage-gated Ca2+ channels (VGCCs) is functionally important, but little is known about the architecture of this region. In K+ channels, this part of the pore is formed by the S6/M2 transmembrane segments from four symmetrically arranged subunits. The Ca2+ channel pore, however, is formed by four asymmetric domains of the same (alpha1) subunit. Here we investigated the architecture of the inner pore of P/Q-type Ca2+ channels using the substituted-cysteine accessibility method. Many positions in the S6 segments of all four repeats of the alpha1 subunit (Ca(v)2.1) were modified by internal methanethiosulfonate ethyltrimethylammonium (MTSET). However, the pattern of modification does not fit any known sequence alignment with K+ channels. In IIS6, five consecutive positions showed clear modification, suggesting a likely aqueous crevice and a loose packing between S6 and S5 segments, a notion further supported by the observation that some S5 positions were also accessible to internal MTSET. These results indicate that the inner pore of VGCCs is indeed formed by the S6 segments but is different from that of K+ channels. Interestingly some residues in IIIS6 and IVS6 whose mutations in L-type Ca2+ channels affect the binding of dihydropyridines and phenylalkylamines and are thought to face the pore appeared not to react with internal MTSET. Probing with qBBr, a rigid thiol-reactive agent with a dimension of 12 angstroms x 10 angstroms x 6 angstroms suggests that the inner pore can open to >10 angstroms. This work provides an impetus for future studies on ion permeation, gating, and drug binding of VGCCs.     

CRAC channels: activation,permeation, and the search for a molecular identity

The Ca2+ release-activated Ca2+ (CRAC) channel is a highly Ca2+-selective store-operated channel that is expressed in T lymphocytes, mast cells, and other hematopoietic cells. In T cells, CRAC channels are essential for generating the prolonged intracellular Ca2+ ([Ca2+](i)) elevation required for the expression of T-cell activation genes. Here we review recent work addressing CRAC channel regulation, pore properties, and the search for CRAC channel genes. Of the current models for CRAC current (I(CRAC)) activation, several new studies argue against a conformational coupling mechanism in which IP(3) receptors communicate store depletion to CRAC channels through direct physical interaction. The study of CRAC channels has been complicated by the fact that they lose activity in the absence of extracellular Ca2+. Attempts to maintain current size by removing intracellular Mg2+ have been found to unmask Mg2+-inhibited cation (MIC/MagNuM/TRPM7) channels, which have been mistaken in several studies for the CRAC channel. Recent studies under conditions that prevent MIC activation reveal that CRAC channels use high-affinity binding of Ca2+ in the pore to achieve high Ca2+ selectivity but have a surprisingly low conductance for both Ca2+ (approximately 10fS) and Na+ (approximately 0.2pS). Pore properties provide a unique fingerprint that provides a stringent test for potential CRAC channel genes and suggest models for the ion selectivity mechanism.     

Structure and function of voltage-gated sodium and calcium channels.

The primary structures of the Na+ channel alpha-subunits from several species have now been deduced from cDNA sequences, and complete primary structures of all of the subunits of skeletal muscle L-type Ca2+ channels have been defined. Current research on voltage-gated ion channels is focusing on defining the structural components responsible for specific aspects of channel function. Recent experiments have identified regions of these channels that are important for voltage-dependent activation and inactivation, ion conductance, regulation by protein phosphorylation, and modulation by drugs and neurotoxins using a combination of antibody mapping and site-directed mutagenesis approaches. The results form the outlines of a structural map of ion channel function.     

New structure and function in plant K+ channels: KCO1, an outward rectifier with a steep Ca2+ dependency.

Potassium (K+) channels mediating important physiological functions are characterized by a common pore-forming (P) domain. We report the cloning and functional analysis of the first higher plant outward rectifying K+ channel (KCO1) from Arabidopsis thaliana. KCO1 belongs to a new class of ''two-pore'' K+ channels recently described in human and yeast. KCO1 has four putative transmembrane segments and tandem calcium-binding EF-hand motifs. Heterologous expression of KCO1 in baculovirus-infected insect (Spodoptera frugiperda) cells resulted in outwardly rectifying, K+-selective currents elicited by depolarizing voltage pulses in whole-cell measurements. Activation of KCO1 was strongly dependent on the presence of nanomolar concentrations of cytosolic free Ca2+ [Ca2+]cyt. No K+ currents were detected when [Ca2+]cyt was adjusted to <150 nM. However, KCO1 strongly activated at increasing [Ca2+]cyt, with a saturating activity observed at approximately 300 nM [Ca2+]cyt. KCO1 single channel analysis on excised membrane patches, resulting in a single channel conductance of 64 pS, confirmed outward rectification as well as Ca2+-dependent activation. These data suggest a direct link between calcium-mediated signaling processes and K+ ion transport in higher plants. The identification of KCO1 as the first plant K+ outward channel opens a new field of structure-function studies in plant ion channels.     

How do calcium channels transport calcium ions?

Calcium channel activity is crucial for many fundamental physiological processes ranging from the heart beat to synaptic transmission. The channel-forming protein, of about 2000 amino acids, comprises four domains internally homologous to each other. Voltage-dependent Ca2+ channels are the most selective ion channels known. Under physiological conditions, they prefer Ca2+ over Na+ by a ratio of about 1000:1. To explain at the same time the exquisite ion selectivity and the large Ca2+ ion turnover rate of Ca2+ channels (approximately 3 x 10(6) ions/s), two kind models have been proposed. In one, the conduction pathway possesses two high-affinity binding sites. When two Ca2+ ions are bound to each site, the mutual repulsion between them speeds the exit rate for the ions, causing greater ion permeation through the pore. The second model hypothesizes the existence of a single site having a charged structure able to attract multiple, interacting ions, simultaneously. Recent studies that combine mutagenesis and electrophysiology show that the high-affinity binding site is formed by a ring of glutamate residues located in the pore forming region of the Ca2+ channel. As proposed in the second class of models, the results suggest that four glutamate residues, one glutamate donated by each repeat, combine to form a single high-affinity site. In this review the different conduction models for Ca2+ channels are discussed and confronted with structural data.     

Ion channels in mammalian proximal renal tubules.

In the plasma membranes of mammalian proximal renal tubules single ion channels were investigated mainly in isolated tubules perfused on one side, in isolated nonperfused (collapsed) tubules and in primary cell cultures. With these techniques, the following results were obtained: in the luminal membrane of isolated one-sided perfused tubules of rabbit and mouse S3 segments, K(+)-selective channels with single-channel conductance (g) of 33 pS and 63 pS, respectively, were recorded. In primary cultures of rabbit S1 segments, a small-conductance (42 pS) as well as a large-conductance (200 pS) K+ channel were observed. The latter was Ca2(+)- and voltage-sensitive. In cultured cells a Ca2(+)-activated, nonselective cation channel with g = 25 pS was also recorded. On the other hand, an amiloride-sensitive channel with g = 12 pS, which was highly selective for Na+ over K+, was observed in the isolated perfused S3 segment. In the basolateral membrane of isolated perfused S3 segments, two types of K+ channels with g = 46 pS and 36 pS, respectively, were observed. The latter channel was not dependent on cytosolic Ca2+ in cell-excised patches. A K+ channel with g = 54 pS was recorded in isolated nonperfused S1 segments. This channel showed inward rectification and was more active at depolarizing potentials. In isolated perfused S3 segments, in addition to the K+ channels also a nonselective cation channel with g = 28 pS was observed. This channel was highly dependent on cytosolic Ca2+ in cell-free patches. It can be concluded that the K+ channels both in the luminal and contraluminal cell membrane are involved in the generation of the cell potential. Na+ channels in the luminal membrane may participate in Na+ reabsorption, whereas the function of a basolateral cation channel remains unclear. Recently, single anion-selective channels were recorded in membranes of endocytotic vesicles, isolated from rat proximal tubules. Vesicles were enlarged by the dehydration/rehydration method and investigated with the patch clamp technique. The Cl- channel had a conductance of 73 pS, the current-voltage curve was linear and the channel inactivated at high negative clamp potentials. It is suggested that this channel is responsible for charge neutrality during active H+ uptake into the endosomes.     

Comparative changes of levels of nitrendipine Ca2+ channels, of tetrodotoxin-sensitive Na+ channels and of ouabain-sensitive (Na+ + K+)-ATPase following denervation of rat and chick skeletal muscle

Three major ion transport systems, the nitrendipine-sensitive Ca2+ channels, the tetrodotoxin-sensitive Na+ channel and the ouabain-sensitive (Na+ + K+)-ATPase, have been studied in skeletal muscle from rat and chick after chronic denervation. It is shown that the situation found for the Ca2+ channel differs dramatically from that found for the Na+ channel and the (Na+ + K+)-ATPase and that regulation of the nitrendipine-sensitive Ca2+ channel in denervated muscle also differs widely from that of the tetrodotoxin-sensitive Na+ channel and the ouabain-sensitive (Na+ + K+)-ATPase which show a quite similar evolution.     

A putative two pore channel AtTPC1 mediates Ca(2+) flux in Arabidopsis leaf cells

The gene encoding voltage-gated channel with high affinity for Ca(2+) permeation has not been cloned from plants. In the present study, we isolated a full-length cDNA encoding a putative Ca(2+ )channel (AtTPC1) from Arabidopsis. AtTPC1 has two conserved homologous domains, both of which contain six transmembrane segments (S1-S6) and a pore loop (P) between S5 and S6 in each domain, and has the highest homology with the two pore channel TPC1 recently cloned from rat. The overall structure is similar to the half of the general structure of alpha-subunits of voltage-activated Ca(2+) channels from animals. AtTPC1 rescued the Ca(2+) uptake activity of a yeast mutant cch1. Sucrose-induced luminescence, which reflects a cytosolic free Ca(2+) increase in aequorin-expressing Arabidopsis leaves, was enhanced by overexpression of AtTPC1 and suppressed by antisense expression of it. Sucrose-H(+) symporters AtSUC1 and 2, which depolarize membrane potential of cells receiving sucrose, also depressed a Ca(2+) increase by their antisense expression. These results suggest that AtTPC1 mediates a voltage-activated Ca(2+ )influx in Arabidopsis leaf cells.