Physico-chemical model of a voltage-gated sodium channel

A permeant ion is known to create in the channel pore a local electrical field, the intensity of which exceeds the intensity of an electrical field produced by the membrane potential. In our study, we consider a sodium channel model, in which the effects of a permeant ion, an inactivating particle, and pharmacological agents on mobile charged groups of the channel are semi-phenomenologically taken into account by using motion equations for a generalized structural variable. Stationary solutions for the equation correspond to “open,” “closed,” and “inactivated” channel states. Because of this, the channel free energy profile, as a function of the structural variable, has three local minima. The three energy values of these states depend both on the electrical field applied externally and on the near-membrane concentrations of permeant ions and acting pharmacological agents. Sodium channel activation and inactivation kinetics are considered resulting from relative changes of the free energy typical of the above three states of the channel. The results we obtained in the course of channel activation and inactivation modeling and their voltage dependence are qualitatively consistent with the commonly known experimental data. The proposed model allows one to qualitatively predict the dependence of the sodium channel kinetic characteristics on the concentrations of permeant ions and pharmacological agents.     

Hinge-bending motions in the pore domain of a bacterial voltage-gated sodium channel

Computational methods and experimental data are used to provide structural models for NaChBac, the homo-tetrameric voltage-gated sodium channel from the bacterium Bacillus halodurans, with a closed and partially open pore domain. Molecular dynamic (MD) simulations on membrane-bound homo-tetrameric NaChBac structures, each comprising six helical transmembrane segments (labeled S1 through S6), reveal that the shape of the lumen, which is defined by the bundle of four alpha-helical S6 segments, is modulated by hinge bending motions around the S6 glycine residues. Mutation of these glycine residues into proline and alanine affects, respectively, the structure and conformational flexibility of the S6 bundle. In the closed channel conformation, a cluster of stacked phenylalanine residues from the four S6 helices hinders diffusion of water molecules and Na(+) ions. Activation of the voltage sensor domains causes destabilization of the aforementioned cluster of phenylalanines, leading to a more open structure. The conformational change involving the phenylalanine cluster promotes a kink in S6, suggesting that channel gating likely results from the combined action of hinge-bending motions of the S6 bundle and concerted reorientation of the aromatic phenylalanine side-chains.     

A model of the putative pore region of the cardiac ryanodine receptor channel

Using the bacterial K+ channel KcsA as a template, we constructed models of the pore region of the cardiac ryanodine receptor channel (RyR2) monomer and tetramer. Physicochemical characteristics of the RyR2 model monomer were compared with the template, including homology, predicted secondary structure, surface area, hydrophobicity, and electrostatic potential. Values were comparable with those of KcsA. Monomers of the RyR2 model were minimized and assembled into a tetramer that was, in turn, minimized. The assembled tetramer adopts a structure equivalent to that of KcsA with a central pore. Characteristics of the RyR2 model tetramer were compared with the KcsA template, including average empirical energy, strain energy, solvation free energy, solvent accessibility, and hydrophobic, polar, acid, and base moments. Again, values for the model and template were comparable. The pores of KcsA and RyR2 have a common motif with a hydrophobic channel that becomes polar at both entrances. Quantitative comparisons indicate that the assembled structure provides a plausible model for the pore of RyR2. Movement of Ca2+, K+, and tetraethylammonium (TEA+) through the model RyR2 pore were simulated with explicit solvation. These simulations suggest that the model RyR2 pore is permeable to Ca2+ and K+ with rates of translocation greater for K+. In contrast, simulations indicate that tetraethylammonium blocks movement of metal cations.     

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.     

Analysis of the selectivity filter of the voltage-gated sodium channel NavRh

NaChBac is a bacterial voltage-gated sodium (Na_v) channel that shows sequence similarity to voltage-gated calcium channels. To understand the ion-permeation mechanism of Na_v channels, we combined molecular dynamics simulation, structural biology and electrophysiological approaches to investigate the recently determined structure of Na_vRh, a marine bacterial NaChBac ortholog. Two Na⁺ binding sites are identified in the selectivity filter (SF) in our simulations: The extracellular Na⁺ ion first approaches site 1 constituted by the side groups of Ser181 and Glu183, and then spontaneously arrives at the energetically more favorable site 2 formed by the carbonyl oxygens of Leu179 and Thr178. In contrast, Ca²⁺ ions are prone to being trapped by Glu183 at site 1, which then blocks the entrance of both Na⁺ and Ca²⁺ to the vestibule of the SF. In addition, Na⁺ permeates through the selective filter in an asymmetrical manner, a feature that resembles that of the mammalian Na_v orthologs. The study reported here provides insights into the mechanism of ion selectivity on Na⁺ over Ca²⁺ in mammalian Na_v channels.     

Mechanisms of cation permeation in cardiac sodium channel: description by dynamic pore model.

The selective permeability to monovalent metal cations, as well as the relationship between cation permeation and gating kinetics, was investigated for native tetrodotoxin-insensitive Na-channels in guinea pig ventricular myocytes using the whole-cell patch clamp technique. By the measurement of inward unidirectional currents and biionic reversal potentials, we demonstrate that the cardiac Na-channel is substantially permeable to all of the group Ia and IIIa cations tested, with the selectivity sequence Na(+) >/= Li(+) > Tl(+) > K(+) > Rb(+) > Cs(+). Current kinetics was little affected by the permeant cation species and concentrations tested (相似文献   

The selectivity filter of the voltage-gated sodium channel is involved in channel activation

Amino acids located in the outer vestibule of the voltage-gated Na+ channel determine the permeation properties of the channel. Recently, residues lining the outer pore have also been implicated in channel gating. The domain (D) IV P-loop residue alanine 1529 forms a part of the putative selectivity filter of the adult rat skeletal muscle (mu1) Na+ channel. Here we report that replacement of alanine 1529 by aspartic acid enhances entry to an ultra-slow inactivated state. Ultra-slow inactivation is characterized by recovery time constants on the order of approximately 100 s from prolonged depolarizations and by the fact that entry to this state can be reduced by binding to the pore of a mutant mu-conotoxin GIIIA, suggesting that ultra-slow inactivation may reflect a structural rearrangement of the outer vestibule. The voltage dependence of ultra-slow inactivation in DIV-A1529D is U-shaped, with a local maximum near -60 mV, whereas activation is maximal only above -20 mV. Furthermore, a train of brief depolarizations produces more ultra-slow inactivation than a single maintained depolarization of the same duration. These data suggest that ultra-slow inactivation emanates from "partially activated" closed states and that the P-loop in DIV may undergo a conformational change during channel activation, which is accentuated by DIV-A1529D.     

Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish

Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82 % similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue.     

Analysis of a putative voltage-gated prokaryotic potassium channel.

Most of the completely sequenced prokaryotic genomes contain genes of potassium channel homologues, but there is still not much known about the role of these proteins in prokaryotes. Here we describe the large-scale overproduction and purification of a prokaryotic voltage-gated potassium channel homologue, Kch, from Escherichia coli. After successful overproduction of the protein, a specific increase in the potassium permeability of the cells was found. Kch could be purified in large amounts using classical purification methods to prevent aggregation of the protein. The physiological state of the protein was revealed to be a homotetramer and the protein was shown to be localized to the cytoplasmic membrane of the cells. In the course of the localization studies, we found a specific increase in the density of the cytoplasmic membrane on Kch production. This was linked to the observed increase in the protein to lipid ratio in the membranes. Another observed change in the membrane composition was an increase in the cardiolipin to phosphatidylglycerol ratio, which may indicate a specific cardiolipin requirement of Kch. On the basis of some of our results, we discuss a function for Kch in the maintenance of the membrane potential in E. coli.     

A series-parallel model of the voltage-gated sodium channel

A series-parallel model of the kinetics of the voltage-gated sodium channel is described. It goes some way towards reconciling the time-courses of the gating and macroscopic sodium currents in the squid giant axon with the molecular structure of the channel.     

Use-dependent block of the voltage-gated Na+ channel by tetrodotoxin and saxitoxin: Effect of pore mutations that change ionic selectivity

Voltage-gated Na⁺ channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca²⁺ permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca²⁺ or Na⁺ ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca²⁺ permeability, suggesting that ion–toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.     

Modeling of ion permeation in calcium and sodium channel selectivity filters

Structure-function studies have shown that it is possible to convert a sodium channel to a calcium-selective channel by a single amino acid substitution in the selectivity filter locus. Ion permeation through the "model selectivity filter" was modeled with a reduced set of functional groups representative of the constituent amino acid side chains. Force-field minimizations were conducted to obtain the energy profile of the cations as they get desolvated and bind to the "model selectivity filter." The calculations suggest that the ion selectivity in the calcium channel is due to preferential binding, whereas in the sodium channel it is due to exclusion. Energetics of displacement of a bound cation from the calcium "model selectivity filter" by another cation suggest that "multi-ion mechanism" reduces the activation barrier for ion permeation. Thus, the simple model captures qualitatively most of the conduction characteristics of sodium and calcium channels. However, the computed barriers for permeation are fairly large, suggesting that ion interaction with additional residues along the transport path may be essential to effect desolvation.     

Epithelial sodium channel pore region. structure and role in gating

Epithelial sodium channels (ENaC) have a crucial role in the regulation of extracellular fluid volume and blood pressure. To study the structure of the pore region of ENaC, the susceptibility of introduced cysteine residues to sulfhydryl-reactive methanethiosulfonate derivatives ((2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) and [(2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET)) and to Cd(2+) was determined. Selected mutants within the amino-terminal portion (alphaVal(569)-alphaTrp(582)) of the pore region responded to MTSEA, MTSET, or Cd(2+) with stimulation or inhibition of whole cell Na(+) current. The reactive residues were not contiguous but were separated by 2-3 residues where substituted cysteine residues did not respond to the reagents and line one face of an alpha-helix. The activation of alphaS580Cbetagamma mENaC by MTSET was associated with a large increase in channel open probability. Within the carboxyl-terminal portion (alphaSer(583)-alphaSer(592)) of the pore region, only one mutation (alphaS583C) conferred a rapid, nearly complete block by MTSEA, MTSET, and Cd(2+), whereas several other mutant channels were partially blocked by MTSEA or Cd(2+) but not by MTSET. Our data suggest that the outer pore of ENaC is formed by an alpha-helix, followed by an extended region that forms a selectivity filter. Furthermore, our data suggest that the pore region participates in ENaC gating.     

Coupled motions between pore and voltage-sensor domains: a model for Shaker B, a voltage-gated potassium channel

A high-resolution crystal structure of KvAP, an archeabacterial voltage-gated potassium (Kv) channel, complexed with a monoclonal Fab fragment has been recently determined. Based on this structure, a mechanism for the activation (opening) of Kv channels has been put forward. This mechanism has since been criticized, suggesting that the resolved structure is not representative of the family of voltage-gated potassium channels. Here, we propose a model of the transmembrane domain of Shaker B, a well-characterized Kv channel, built by homology modeling and docking calculations. In this model, the positively charged S4 helices are oriented perpendicular to the membrane and localized in the groove between segments S5 and S6 of adjacent subunits. The structure and the dynamics of the full atomistic model embedded in a hydrated lipid bilayer were investigated by means of two large-scale molecular dynamics simulations under transmembrane-voltage conditions known to induce, respectively, the resting state (closed) and the activation (opening) of voltage-gated channels. Upon activation, the model undergoes conformational changes that lead to an increase of the hydration of the charged S4 helices, correlated with an upward translation and a tilting of the latter, concurrently with movements of the S5 helices and the activation gate. Although small, these conformational changes ultimately result in an alteration of the ion-conduction pathway. Our findings support the transporter model devised by Bezanilla and collaborators, and further underline the crucial role played by internal hydration in the activation of the channel.     

The pore of the voltage-gated proton channel

In classical tetrameric voltage-gated ion channels four voltage-sensing domains (VSDs), one from each subunit, control one ion permeation pathway formed by four pore domains. The human Hv1 proton channel has a different architecture, containing?a VSD, but lacking a pore domain. Since its location is not known, we searched for the Hv permeation pathway. We find that mutation of the S4 segment's third arginine R211 (R3) compromises proton selectivity, enabling conduction of a metal cation and even of the large organic cation guanidinium, reminiscent of Shaker's omega pore. In the open state, R3 appears to interact with an aspartate (D112) that is situated in the middle of S1 and is unique to Hv channels. The double mutation of both residues further compromises cation selectivity. We propose that membrane depolarization reversibly positions R3 next to D112 in?the transmembrane VSD to form the ion selectivity filter in the channel's open conformation.     

Peregrination of the selectivity filter delineates the pore of the human voltage-gated proton channel hHV1

Extraordinary selectivity is crucial to all proton-conducting molecules, including the human voltage-gated proton channel (hH_V1), because the proton concentration is >10⁶ times lower than that of other cations. Here we use “selectivity filter scanning” to elucidate the molecular requirements for proton-specific conduction in hH_V1. Asp¹¹², in the middle of the S1 transmembrane helix, is an essential part of the selectivity filter in wild-type (WT) channels. After neutralizing Asp¹¹² by mutating it to Ala (D112A), we introduced Asp at each position along S1 from 108 to 118, searching for “second site suppressor” activity. Surprisingly, most mutants lacked even the anion conduction exhibited by D112A. Proton-specific conduction was restored only with Asp or Glu at position 116. The D112V/V116D channel strikingly resembled WT in selectivity, kinetics, and ΔpH-dependent gating. The S4 segment of this mutant has similar accessibility to WT in open channels, because R211H/D112V/V116D was inhibited by internally applied Zn²⁺. Asp at position 109 allowed anion permeation in combination with D112A but did not rescue function in the nonconducting D112V mutant, indicating that selectivity is established externally to the constriction at F150. The three positions that permitted conduction all line the pore in our homology model, clearly delineating the conduction pathway. Evidently, a carboxyl group must face the pore directly to enable conduction. Molecular dynamics simulations indicate reorganization of hydrogen bond networks in the external vestibule in D112V/V116D. At both positions where it produces proton selectivity, Asp frequently engages in salt linkage with one or more Arg residues from S4. Surprisingly, mean hydration profiles were similar in proton-selective, anion-permeable, and nonconducting constructs. That the selectivity filter functions in a new location helps to define local environmental features required to produce proton-selective conduction.     

Ion selectivity predictions from a two-site permeation model for the cyclic nucleotide-gated channel of retinal rod cells.

We developed a two-site, Eyring rate theory model of ionic permeation for cyclic nucleotide-gated channels (CNGCs). The parameters of the model were optimized by simultaneously fitting current-voltage (IV) data sets from excised photoreceptor patches in electrolyte solutions containing one or more of the following ions: Na+, Ca2+, Mg2+, and K+. The model accounted well for 1) the shape of the IV relations; 2) the binding affinity for Na+; 3) reversal potential values with single-sided additions of Ca2+ or Mg2+ and biionic KCl; and 4) the K1 and voltage dependence for divalent block from the cytoplasmic side of the channel. The differences between the predicted K1's for extracellular block by Ca2+ and Mg2+ and the values obtained from heterologous expression of only the alpha-subunit of the channel suggest that the beta-subunit or a cell-specific factor affects the interaction of divalent cations at the external but not the internal face of the channel. The model predicts concentration-dependent permeability ratios with single-sided addition of Ca2+ and Mg2+ and anomalous mole fraction effects under a limited set of conditions for both monovalent and divalent cations. Ca2+ and Mg2+ are predicted to carry 21% and 10%, respectively, of the total current in the retinal rod cell at -60 mV.     

Asymmetric organization of the pore region of the epithelial sodium channel

Epithelial sodium channels (ENaCs) are composed of three homologous subunits that have regions preceding the second transmembrane domain (also referred as pre-M2) that form part of the channel pore. To identify residues within this region of the beta-subunit that line the pore, we systematically mutated residues Gln(523)-Ile(536) to cysteine. Wild type and mutant mouse ENaCs were expressed in Xenopus oocytes, and a two-electrode voltage clamp was used to examine the properties of mutant channels. Cysteine substitutions of 9 of 13 residues significantly altered Li(+) to Na(+) current ratios, whereas only cysteine replacement of beta Gly(529) resulted in K(+)-permeable channels. Besides beta G525C, large increases in the inhibitory constant of amiloride were observed with mutations at beta Gly(529) and beta Ser(531) within the previously identified 3-residue tract that restricts K(+) permeation. Cysteine substitution preceding (beta Phe(524) and beta Gly(525)), within (beta Gly(530)) or following (beta Leu(533)) this 3-residue tract, resulted in enhanced current inhibition by external MTSEA. External MTSET partially blocked channels with cysteine substitutions at beta Gln(523), beta Phe(524), and beta Trp(527). MTSET did not inhibit alpha beta G525C gamma, although previous studies showed that channels with cysteine substitutions at the corresponding sites within the alpha- and gamma-subunits were blocked by MTSET. Our results, placed in context with previous observations, suggest that pore regions from the three ENaC subunits have an asymmetric organization.     

Temperature dependence of proton permeation through a voltage-gated proton channel

Voltage-gated proton channels are found in many different types of cells, where they facilitate proton movement through the membrane. The mechanism of proton permeation through the channel is an issue of long-term interest, but it remains an open question. To address this issue, we examined the temperature dependence of proton permeation. Under whole cell recordings, rapid temperature changes within a few milliseconds were imposed. This method allowed for the measurement of current amplitudes immediately before and after a temperature jump, from which the ratios of these currents (I_ratio) were determined. The use of I_ratio for evaluating the temperature dependence minimized the contributions of factors other than permeation. Temperature jumps of various degrees (ΔT, −15 to 15°C) were applied over a wide temperature range (4–49°C), and the Q₁₀s for the proton currents were evaluated from the I_ratios. Q₁₀ exhibited a high temperature dependence, varying from 2.2 at 10°C to 1.3 at 40°C. This implies that processes with different temperature dependencies underlie the observed Q₁₀. A novel resistivity pulse method revealed that the access resistance with its low temperature dependence predominated in high temperature ranges. The measured temperature dependence of Q₁₀ was decomposed into Q₁₀ of the channel and of the access resistances. Finally, the Q₁₀ for proton permeation through the voltage-gated proton channel itself was calculated and found to vary from 2.8 at 5°C to 2.2 at 45°C, as expected for an activation enthalpy of 64 kJ/mol. The thermodynamic features for proton permeation through proton-selective channels were discussed for the underlying mechanism.