Rat Skeletal Muscle Sodium Channel Research Articles

Probing Voltage-Dependent Structural Changes of the VSD in Mammalian Nav with LRET

Voltage-gated sodium channels (Nav) play an essential role in the generation and propagation of action potentials. Mammalian Nav alpha subunits are large single polypeptide chains organized in four different domains (DI-DIV). Each domain is composed of 6 transmembrane segments (S1-S6) in which S1-S4 constitute the voltage sensing domain (VSD) and S5 and S6 form the pore. Although Nav function has been studied comprehensively, the precise structural basis for the gating mechanisms has not been fully elucidated. Recently, the crystal structures of several prokaryotic sodium channels have been solved, but they are homo-tetrameric proteins in contrast to the mammalian Navs and these studies only provide a single snapshot of the channels in one of many possible conformational states. Therefore, techniques that provide dynamic structural information are needed. In this work we have used Lanthanide-based resonance energy transfer (LRET) to obtain structural information and conformational dynamics of the VSD from each domain of the rat skeletal muscle sodium channel (Nav1.4). We prepared Nav1.4 constructs with a genetically encoded lanthanide binding tag (LBT), which binds a lanthanide (Tb3+) ion with high affinity, inserted in several strategic places at the top of the S4 segment in each domain. Two new conotoxin analogs were synthesized to function as LRET acceptors: KIIIA-Bodipy and GIIIA-Bodipy. We calculated multiple distances from each one of the domains to the pore region where the labeled toxin binds in both resting and slow inactivated states in voltage-clamped Xenopus laevis oocytes expressing our Nav1.4 constructs, which remained functionally active. The results give a geometrical map of the S4 positions of each domain in the mammalian Nav channels and provide evidence for voltage-dependent structural changes. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

Congruent Pattern of Accessibility within the Pore of a Voltage-Gated Na+ Channel

Voltage-gated ion channels open a gate in response to membrane depolarization which allows ions to pass through the transmembrane pore of the channel. MTSET accessibility studies within the Shaker potassium channel revealed that K+ permeation is controlled by an intracellular gate, whereas Cd2+ and Ag+ accessibility measurements in the cyclic nucleotide-gated channel suggests that this process occurs at the selectivity filter. Modification data of cysteine residues introduced into the DIV S6 of a fast-inactivation removed voltage-gated sodium channel (VGSC) by MTSET indicate that sodium channel gating is also regulated by an intracellular gate. However, unlike the Shaker potassium channel, VGSCs are not composed of four identical subunits. Despite sequence similarity between each S6 helix, the multi-domain nature of VGSCs implies that there may be asymmetry within the pore that may have consequences in regard to channel gating. Thus, we sought to determine if analogous positions within the S6 helices of a VGSC act together to form an intracellular gate which occludes the pore while channels are closed. We scanned the MTSET accessibility of substituted cysteines in the pore lining helices of the first three domains (DI-DIII) in the rat skeletal muscle sodium channel. The modification data at these sites, selected on the basis of sequence alignment and MTSET accessibility data of DIV-S6, confirms that DII and DIII follow the same pattern of accessibility as that of DIV; however, the extent of block is quite varied across each domain. Along with DI accessibility data, these findings will be discussed in the context of pore gating in related voltage-gated ion channels.

Tracking Voltage-Dependent Conformational Changes of the VSD in Nav with LRET

Voltage-gated sodium channels (Nav) are fundamental for the generation and the propagation of action potentials. Mammalian Nav alpha subunits are single macromolecules organized in four different domains (DI-DIV). Each is composed of 6 transmembrane segments (S1-S6) from which S1-S4 constitute the voltage sensing domain (VSD) and with S5 and S6 constituting the pore. While Nav function has been studied extensively, the exact structural mechanisms of gating are not fully understood. Recently, the crystal structure of the prokaryotic sodium channel, NavAb, has been solved, but NavAb is a homotetrameric protein in contract to the mammalian Navs. Thus many questions were not answered by the prokaryotic channel structures. To resolve the voltage dependent conformational changes of Nav, we tracked conformational changes of the VSD from each domain of the rat skeletal muscle sodium channel (Nav1.4) using Lanthanide-based Resonance Energy Transfer (LRET), a FRET technique that allows for precise measurement of intermolecular distances by taking advantage of the special properties of lanthanide as an energy donor. We prepared Nav1.4 constructs with a genetically encoded lanthanide binding tag (LBT), which holds a lanthanide (Tb3+) ion with high affinity, inserted at the top of the S4 segment in each domain. Also, we synthesized two toxins conjugated to dyes to function as acceptors: the pore-blocking small molecule tetrodotoxin conjugated with a HiLyte fluor488 (TTX-F), and the peptide β scorpion toxin Ts1, from the Brazilian scorpion Tityus serrulatus, conjugated with Alexa488 (Ts1-Alexa488). Having several donor positions (Tb3+ ions in LBT's) and two different acceptor positions (TTX-F and Ts1-Alexa488), we calculated multiple distances in voltage-clamped Xenopus laevis oocytes expressing our Nav1.4 constructs that remained functionally active. The results provide new insight to structure-function information in mammalian Nav channels. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

Searching for the Interaction Sites of the Beta1 Subunit with the Voltage-Sensing Domains of Sodium Channels Using LRET

Mammalian voltage-gated sodium channels (Nav) are composed of two subunits: a monomeric pore-forming subunit (α-subunit), that contains four domains (DI-DIV), each composed of a voltage-sensing domain (VSD, S1-S4) and a pore domain (S5-S6) and auxiliary subunits (β-subunits) that are transmembrane proteins with type I topology: containing an extracellular amino-terminus, a single transmembrane segment and an intracellular carboxyl terminus. A large body of literature has shown that interaction between α- and β-subunits results in Nav with altered gating kinetics suggesting that β-subunits may directly interact with VSD. However, aspects of the stoichiometry, arrangement and molecular interaction between α and β subunits remain unclear. In this study, we explored the location of β1 in relation to the rat skeletal muscle sodium channel α-subunit (Nav1.4) using lanthanide-based resonance energy transfer (LRET) via two strategies. 1) Four Nav1.4 constructs were designed to encode a Tb3+ binding-tag (Nav1.4-LBT) on top of the S4 of each domain (DI-LBT, DII-LBT, DIII-LBT and DIV-LBT) as energy donor. A hexa-histidine-tag was inserted in β1 (6His-β1), which binds a Cu2+ ion to act as acceptor. 2) β1 constructs were designed to encode at an extracellular site an LBT that binds Tb3+ as energy donor while the acceptor was Alexa488 conjugated to Ts1, a β scorpion toxin which binds to DII-VSD in Nav1.4. For both experiments, we used Xenopus laevis oocytes co-expressing Nav1.4 and β1 constructs injected with the cRNA at 1:1 molar ratio. In preliminary results, DIV-LBT + 6His-β1 oocytes displayed robust energy transfer between Tb3+ and Cu2+, indicating that at least one β1-subunit is located at ∼28 A of S4-DIV. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

A residue in the transmembrane segment 6 of domain I in insect and mammalian sodium channels regulate differential sensitivities to pyrethroid insecticides

Voltage-gated sodium channels are critical for electrical signaling in the nervous system. Pyrethroid insecticides exert their toxic action by modifying the gating of sodium channels. A valine to methionine mutation in the transmembrane segment 6 of domain I (IS6) of sodium channels from tobacco budworms (Heliothis virescens) has been shown to alter channel gating and reduce insect sodium channel sensitivity to pyrethroids. A valine to leucine substitution was subsequently reported in pyrethroid-resistant bedbug populations. Intriguingly, pyrethroid-resistant mammalian sodium channels possess an isoleucine at the corresponding position. To determine whether different substitutions at this position alter channel gating and confer pyrethroid resistance, we made valine to methionine, isoleucine or leucine substitutions at the corresponding position, V409, in a cockroach sodium channel and examined the gating properties and pyrethroid sensitivity of the three mutants in Xenopus oocytes. All three mutations reduced the channel sensitivity to three pyrethroids (permethrin, cismethrin and deltamethrin). V409M, but not V409I or V409L, caused 6–7mV depolarizing shifts in the voltage dependences of both activation and inactivation. V409M and V409L slowed channel activation kinetics and accelerated open-state deactivation kinetics, but V409I did not. Furthermore, the substitution of isoleucine with valine, but not with methionine nor leucine, at the corresponding position in a rat skeletal muscle sodium channel, rNav1.4, enhanced channel sensitivity to deltamethrin. Collectively, our study highlights an important role of residues at 409 in regulating not only sodium channel gating, but also the differential sensitivities of insect and mammalian sodium channels to pyrethroids.

Enhancement of Closed-State Inactivation by Neutralization of S4 Arginines in Domain IV of a Sodium Channel

In PNAS, Capes et al. (2012) showed that the voltage sensor of domain IV in the skeletal muscle voltage-dependent sodium channel is energetically coupled to the channel gate located at the outer side of the pore. For this, the authors elicited gating pore currents, also known as omega currents, through each of the channel's four voltage sensors, one at a time. They inventively showed that sodium channel blockers TTX and μ-CTX, which bind to the outer pore in the selectivity filter region of the channel, affect the gating pore currents through domain IV, suggesting a coupling between conformational transitions in the selectivity filter and the movement of S4 in the voltage sensor of domain IV. The authors did not refer to two important aspects, which are tightly related to their experimental approach. The first is that neutralization of arginines on S4 (by R to Q substitutions) used to elicit gating pore currents through each of the voltage sensor mutants, may abolish the voltage sensitivity of the voltage sensor (Bao et al., 1999), and position it in the activated or partial activated state (Gagnon and Bezanilla, 2009). The second aspect relates to the predominant role that domain IV voltage sensor plays in coupling activation with fast inactivation in sodium channels. The voltage sensor of domain IV is specialized in energizing a cytoplasmic inactivation element (IFM), connecting the third and fourth homologous domains, into the inner mouth of the pore via a mechanism known as “ball and chain” fast inactivation. Further, it was shown that a neutralizing mutation at the outermost S4 arginine in domain IV, which is associated with the paramyotonia congenital disease, greatly affects the coupling between activation and inactivation in the rat and human skeletal muscle sodium channels (Chahine et al., 1994). It is therefore likely that activation of the voltage sensor of domain IV by the neutralizing mutations shifts the channel into a closed-state inactivation (Armstrong, 2006), which is associated with gating transitions that favor TTX binding to the outer pore (Patton and Goldin, 1991). If this is indeed the case, it points out to coupling between closed-state gating transitions at the outer pore gates with virtually all of the four voltage sensors, and not only with that of domain IV. Accelerated transitions in the selectivity filter at the outer gate due to occlusion of the cytoplasmic side of the pore by N-type inactivation particles is well established in homotetrameric K+ channels, though the parallel process in eukaryotic sodium channels is still under debate. The technical limitation arises by channel entrance into a closed-state gating transitions due to neutralization of S4 arginines should be addressed to refine the experimental approach used by Capes et al. in order to make it more reliable. For example, it is possible to bring each of the mutant channels into a uniform gating state using a conditioning protocol that imposes channel entrance into a closed-state inactivation prior to recording of the gating pore currents, or use a mutant channel in which its fast inactivation is removed by mutating the IFM motif between domains III to IV.

Trans-Channel Interactions in Batrachotoxin-Modified Rat Skeletal Muscle Sodium Channels: Kinetic Analysis of Mutual Inhibition between μ-Conotoxin GIIIA Derivatives and Amine Blockers

R13X derivatives of μ-conotoxin GIIIA bind externally to single sodium channels and block current incompletely with mean “blocked” durations of several seconds. We studied interactions between two classes of blockers ( μ-conotoxins and amines) by steady state, kinetic analysis of block of BTX-modified Na channels in planar bilayers. The amines cause all-or-none block at a site internal to the selectivity filter. TPrA and DEA block single Na channels with very different kinetics. TPrA induces discrete, all-or-none, blocked events (mean blocked durations, ∼100 ms), whereas DEA produces a concentration-dependent reduction of the apparent single channel amplitude (“fast” block). These distinct modes of action allow simultaneous evaluation of block by TPrA and DEA, showing a classical, competitive interaction between them. The apparent affinity of TPrA decreases with increasing [DEA], based on a decrease in the association rate for TPrA. When an R13X μ-conotoxin derivative and one of the amines are applied simultaneously on opposite sides of the membrane, a mutually inhibitory interaction is observed. Dissociation constants, at +50 mV, for TPrA (∼4 mM) and DEA (∼30 mM) increase by ∼20%–50% when R13E (nominal net charge, +4) or R13Q (+5) is bound. Analysis of the slow blocking kinetics for the two toxin derivatives showed comparable decreases in affinity of the μ-conotoxins in the presence of an amine. Although this mutual inhibition seems to be qualitatively consistent with an electrostatic interaction across the selectivity filter, quantitative considerations raise questions about the mechanistic details of the interaction.

The μO-conotoxin MrVIA inhibits voltage-gated sodium channels by associating with domain-3

Several families of peptide toxins from cone snails affect voltage-gated sodium (Na V) channels: μ-conotoxins block the pore, δ-conotoxins inhibit channel inactivation, and μO-conotoxins inhibit Na V channels by an unknown mechanism. The only currently known μO-conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (Na V1.4) and brain (Na V1.2) sodium channels in mammalian cells. A systematic domain-swapping strategy identified the C-terminal pore loop of domain-3 as the major determinant for Na V1.4 being more potently blocked than Na V1.2 channels. μO-conotoxins therefore show an interaction pattern with Na V channels that is clearly different from the related μ- and δ-conotoxins, indicative of a distinct molecular mechanism of channel inhibition.

β1-subunit modulates the Nav1.4 sodium channel by changing the surface charge

Voltage-gated sodium channels, comprised of a pore-forming alpha-subunit and additional regulatory (beta) subunits, play a critical role in regulation of neuronal excitability. Mechanisms of regulation of beta-subunits remain elusive. We have tested the functional effects of beta1 sodium channel subunit on surface charges as a mechanism for channel modulation. HEK-293 cell lines permanently transfected with the sole rat skeletal muscle sodium channel alpha-subunit (Nav1.4), or co-expressing the sodium channel alpha-subunit and beta1-subunit were studied with the whole-cell mode of the patch-clamp technique. At physiological extracellular Ca2+ concentration (2 mM), expression of beta1-subunit did not produce any significant effect on the voltage-dependent properties of sodium currents. However, a shift of half-activation potentials of sodium channel by changing the extracellular Ca2+ was potentiated when beta1 was co-expressed with alpha-subunit. In contrast, the expression of beta1-subunit did not affect the Ca2+ binding to the open or to the closed sodium channel pore, difference of the effect provoked by extracellular Ca2+ could therefore be attributed to an increased in negative surface charge determined by the presence of beta1-subunit. These data are in agreement with the hypothesis of a modulation of the sodium current by the expression of the highly sialylated beta1-subunit, which would alter the channel gating by increasing the density of surface negative charges in the vicinity of the sodium channel voltage sensing machinery.

Modeling of benzocaine analog interactions with the D4S6 segment of Na V4.1 voltage-gated sodium channels

Local anesthetics (LAs) are compounds that inhibit the propagation of action potentials in excitable tissues by blocking voltage-gated Na + channels. Mutagenesis studies have demonstrated that several amino acid residues are important sites of LA interaction with the channel, but these studies provide little information regarding the molecular forces that govern drug-binding interactions, including the binding orientation of drugs. We used computational methods to construct a simple model of benzocaine analog binding with the D4S6 segment of rat skeletal muscle (Na V4.1) sodium channels. The model revealed that four hydrophobic residues form a binding cavity for neutral LAs, and docking studies indicated that increasing hydrophobicity among the benzocaine analogs allowed a better fit within the binding cavity. The similarities between our simple model and published experimental data suggested that modeling of LA interactions with sodium channels, along with experimental approaches, could further enhance our understanding of LA interactions with sodium channels.

Isolation, molecular cloning and functional characterization of a novel β-toxin from the Venezuelan scorpion, Tityus zulianus

Sting in children by Tityus zulianus scorpions (western Venezuela) often produces cardiorespiratory arrest and death by pulmonary oedema. To assess its toxicity, lethality in mice of T. zulianus soluble venom was determined. Toxin composition was studied by fractionating the crude venom through reversed-phase HPLC. The most abundant peptide, Tz1, was purified further and its N-terminal sequence, amino acid composition and molecular mass (by electron-spray ionization mass spectrometry) determined. In the presence of Tz1, activation of recombinant rat skeletal muscle sodium channels (Na V1.4) was shifted about 35 mV in the hyperpolarizing direction in a prepulse-dependent manner. This typical β-toxin effect had an apparent EC 50 of 3.5 μM. A cDNA sequence encoding Tz1 was isolated from T. zulianus venom gland RNA using a combination of 5′- and 3′-RACE PCR. Analysis of the encoded sequence indicated that Tz1 is the processed product of a precursor containing: (i) a 20-residue long leader peptide; (ii) the amino acid sequence of the mature toxin (64 residues); and (iii) an extra Gly-Lys tail at the C-terminus, probably removed post-translationally. A comparison of Tz1 with Tityus serrulatus β-toxin Ts1 revealed that some of the non-conservative replacements in Tz1 lie in regions potentially involved in receptor recognition.

Voltage-dependent inhibition of rat skeletal muscle sodium channels by aminoglycoside antibiotics.

Aminoglycoside (AG) antibiotics interact with numerous biological molecules, including some voltage-gated ion channels. The present study demonstrates that 4,5-disubstituted (neomycin class) and 4,6-disubstituted (kanamycin class) AGs inhibit whole-cell currents through cloned rat skeletal muscle sodium channels (mu1, Na(V)4.1). Increases in the amplitude of the step command reduced inhibition by extracellular AGs but increased inhibition by intracellularly applied AGs, indicating that the block was voltage dependent. Furthermore, intracellular neamine or sisomycin hastened the rate of macroscopic current decay at positive voltages. Extracellular solution containing sodium ions slowed the rate of current decay in the presence of intracellular sisomycin and decreased the apparent affinity of sisomycin from the intracellular side twofold. Current inhibition by extracellularly or intracellularly applied AGs was well fitted by the Woodhull model of pore block. The model indicated that most extracellularly applied AGs interact at a site that is an electrical distance of approximately 10-15% from the outside, whereas intracellularly applied neamine or sisomycin bind to sites that are approximately 49% and approximately 24%, respectively, into the electric field from the inside. Our data suggested that AG antibiotics induce a low-affinity, voltage-dependent block of mu1 channels.

Conotoxins as sensors of local pH and electrostatic potential in the outer vestibule of the sodium channel.

We examined the block of voltage-dependent rat skeletal muscle sodium channels by derivatives of μ-conotoxin GIIIA (μCTX) having either histidine, glutamate, or alanine residues substituted for arginine-13. Toxin binding and dissociation were observed as current fluctuations from single, batrachotoxin-treated sodium channels in planar lipid bilayers. R13X derivatives of μCTX only partially block the single-channel current, enabling us to directly monitor properties of both μCTX-bound and -unbound states under different conditions. The fractional residual current through the bound channel changes with pH according to a single-site titration curve for toxin derivatives R13E and R13H, reflecting the effect of changing the charge on residue 13, in the bound state. Experiments with R13A provided a control reflecting the effects of titration of all residues on toxin and channel other than toxin residue 13. The apparent pKs for the titration of residual conductance are shifted 2–3 pH units positive from the nominal pK values for histidine and glutamate, respectively, and from the values for these specific residues, determined in the toxin molecule in free solution by NMR measurements. Toxin affinity also changes dramatically as a function of pH, almost entirely due to changes in the association rate constant, kon. Interpreted electrostatically, our results suggest that, even in the presence of the bound cationic toxin, the channel vestibule strongly favors cation entry with an equivalent local electrostatic potential more negative than −100 mV at the level of the “outer charged ring” formed by channel residues E403, E758, D1241, and D1532. Association rates are apparently limited at a transition state where the pK of toxin residue 13 is closer to the solution value than in the bound state. The action of these unique peptides can thus be used to sense the local environment in the ligand-–receptor complex during individual molecular transitions and defined conformational states.

A comparison of the μ-conotoxins by [ 3H]saxitoxin binding assays in neuronal and skeletal muscle sodium channel

Sodium channels from rat brain, rat skeletal muscle, chick brain, and eel electroplax were compared by using the μ-conotoxins GIIIA, PIIIA, and StIII and [ 3H]saxitoxin. Rat skeletal muscle and eel electroplax sodium channels are equally sensitive to GIIIA, PIIIA, and StIII, displacing >90% of the [ 3H]saxitoxin binding sites in rat skeletal muscle and eel electroplax membranes and exhibiting inhibitory concentrations at half-maximal percentage specific binding (IC50) of 0.97 nM for GIIIA, 1.3 nM for PIIIA in rat skeletal muscle, and concentrations of 3.5 nM for GIIIA and 2.8 nM for PIIIA in eel electroplax. PIIIA and GIIIA at all concentrations inhibit only up to 10% of [ 3H]saxitoxin binding sites in chick brain membranes. μ-Conotoxin StIII at all concentrations inhibits only up to 10% of [ 3H]saxitoxin binding sites in rat brain membranes and displays two-site binding inhibition of the [ 3H]saxitoxin binding sites in rat skeletal muscle. PIIIA displaces >60% of the [ 3H]saxitoxin binding sites (IC50 of 44 nM) while GIIIA blocks out only 30% of the binding sites in rat brain sodium channels (IC50 of 69 nM). Thus, sodium channel subtypes can be classified into two categories: μ-conotoxin sensitive (i.e., subtypes predominantly found in rat skeletal muscle and eel electroplax) and insensitive (i.e., subtypes predominantly found in rat and chick brain).

Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation.

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.

Role of outer ring carboxylates of the rat skeletal muscle sodium channel pore in proton block.

Voltage-gated Na+ current is reduced by acid solution. Protons reduce peak Na+ conductance by lowering single channel conductance and shift the voltage range of gating by neutralizing surface charges. Structure-function studies identify six carboxyls and a lysine in the channel's outer vestibule. We examined the roles of the superficial ring of carboxyls in acid block of Na(v)1.4 (the rat skeletal muscle Na+ channel isoform) by measuring the effects of their neutralization or their substitution by lysine on sensitivity to acid solutions, using the two-micropipette voltage clamp in Xenopus oocytes. Alteration of the outer ring of carboxylates had little effect on the voltage for half-activation of Na+ current, as if they are distant from the channels' voltage sensors. The mutations did not abolish proton block; rather, they all shifted the pK(a) (-log of the dissociation constant) in the acid direction. Effects of neutralization on pK(a) were not identical for different mutations, with E758Q > D1241A > D1532N > E403Q. E758K showed double the effect of E758Q, and the other lysine mutations all produced larger effects than the neutralizing mutations. Calculation of the electrostatic potential produced by these carboxylates using a pore model showed that the pK(a) values of carboxylates of Glu-403, Glu-758, and Asp-1532 are shifted to values similar to the experimentally measured pK(a). Calculations also predict the experimentally observed changes in pK(a) that result from mutational neutralization or introduction of a positive charge. We propose that proton block results from partial protonation of these outer ring carboxylates and that all of the carboxylates contribute to a composite Na+ site.

Critical molecular determinants of voltage-gated sodium channel sensitivity to mu-conotoxins GIIIA/B.

GIIIA/B mu-conotoxins block the rat skeletal muscle sodium channel (rNa(v)1.4) with high affinity by binding to specific residues in the pore. However, human Na(v)1.4 (hNa(v)1.4) channels, which are resistant to block by GIIIA/B, have these same pore residues. We used chimera constructs, site-directed mutagenesis, and electrophysiological techniques to investigate which residues determine GIIIA/B selectivity. Exchange of serine 729 in the D2/S5-S6 linker of rat Na(v)1.4 with leucine (S729L), the corresponding residue in hNa(v)1.4, reduces the sensitivity of rNa(v)1.4 by approximately 20-fold and largely accounts for the differential sensitivity of rNa(v)1.4 and hNa(v)1.4 to both GIIIA and GIIIB. To determine whether D2/S5-S6 linker residues might contribute to the resistance of neuronal channels to GIIIA/B, we exchanged residues in this linker that differed between rNa(v)1.4 and neuronal channels. Substitution of aspargine 732 with lysine (N732K), the corresponding residue in rNa(v)1.1a and rNa(v)1.7, reduced the GIIIB sensitivity of rNa(v)1.4 by approximately 20-fold. The N732K substitution, however, only reduced GIIIA sensitivity of rNa(v)1.4 by approximately 4-fold, demonstrating that GIIIA and GIIIB have distinct interactions with the D2/S5-S6 linker. Our data indicate that naturally occurring variants in the extra-pore region of the D2/S5-S6 linker contribute to the isoform-specific sensitivity of sodium channels to GIIIA/B. Because S729 and N732 are not part of the high-affinity binding site for mu-conotoxins, these extra-pore residues probably influence the accessibility of the toxin to the binding site within the pore and/or the stability of the toxin-channel complex. Our results should aid the development of toxins that block specific neuronal sodium channel isoforms.

Outer and Central Charged Residues in DIVS4 of Skeletal Muscle Sodium Channels Have Differing Roles in Deactivation

We tested the effects of charge-neutralizing mutations of the eight arginine residues in DIVS4 of the rat skeletal muscle sodium channel (rNa V1.4) on deactivation gating from the open and inactivated states. We hypothesized that neutralization of outer or central charges would accelerate the I-to-C transition as measured by recovery delay because these represent a portion of the immobilizable charge. R1Q abbreviated recovery delay as a consequence of reduced charge content. R4Q increased delay, whereas R5Q abbreviated delay, and charge-substitutions at these residues indicated that each effect was allosteric. We also hypothesized that neutralization of any residue in DIVS4 would slow the O-to-C transition with reduction in positive charge. Reduction in charge at R1, and to a lesser extent at R5, slowed open-state deactivation, while charge neutralizations at R2, R3, R4, R6, and R7 accelerated open-state deactivation. Our findings suggest that arginine residues in DIVS4 in rNa V1.4 have differing roles in channel closure from open and inactivated states. Furthermore, they suggest that deactivation in DIVS4 is regulated by charge interaction between the electrical field with the outermost residue, and by local allosteric interactions imparted by central charges.

Ketamine Blockade of Voltage-gated Sodium Channels

The general anesthetic ketamine is known to be an N-methyl-D-aspartate receptor blocker. Although ketamine also blocks voltage-gated sodium channels in a local anesthetic-like fashion, little information exists on the molecular pharmacology of this interaction. We measured the effects of ketamine on sodium channels. Wild-type and mutant (F1579A) recombinant rat skeletal muscle sodium channels were expressed in Xenopus oocytes. The F1579A amino acid substitution site is part of the intrapore local anesthetic receptor. The effect of ketamine was measured in oocytes expressing wild-type or mutant sodium channels using two-electrode voltage clamp. Ketamine blocked sodium channels in a local anesthetic-like fashion, exhibiting tonic blockade (concentration for half-maximal inhibition [IC50] = 0.8 mm), phasic blockade (IC50 = 2.3 mm), and leftward shift of the steady-state inactivation; the parameters of these actions were strongly modified by alteration of the intrapore local anesthetic binding site (IC50 = 2.1 mm and IC50 = 10.3 mm for tonic and phasic blockade, respectively). Compared with lidocaine, ketamine showed greater tonic inhibition but less phasic blockade. Ketamine interacts with sodium channels in a local anesthetic-like fashion, including sharing a binding site with commonly used clinical local anesthetics.

A carboxy-terminal α-helical segment in the rat skeletal muscle voltage-dependent Na + channel is responsible for its interaction with the amino-terminus

Cytoplasmic segments of the adult rat skeletal muscle sodium channel α-subunit (rSkM1) comprise a major portion (∼40%) of the total protein and are involved in channel functions both general, such as inactivation, and isoform-specific, for example, protein kinase A modulation. Far ultraviolet circular dichroism measurements of synthetic peptides and overexpressed fusion proteins containing individual channel cytoplasmic segments suggest that cytoplasmic domains of rSkM1 contain ordered secondary structures even in the absence of adjoining transmembrane segments. Intrinsic fluorescence experiments with a nested set of carboxy-terminal deletion proteins confirm a specific interaction between the channel’s amino- and carboxy-termini and identify residues 1716–1737 in the carboxy-terminus as the region that binds to the amino-terminus. Circular dichroism measurements suggest that this same region is organized as an α-helix and that electrostatic forces may contribute to this association. The interaction of the amino- and carboxy-termini is not accompanied by secondary structure changes detectable by circular dichroism spectroscopy, but a decrease in intrinsic fluorescence indicates that this association is accompanied by a change in the environment of Trp 1617.