Ultraviolet-induced alterations of the sodium inactivation in myelinated nerve fibres.

Ultraviolet radiation irreversibly reduces the sodium permeability in nerve membranes and, in addition, induces a change of the potential dependence of the kinetic parameters of sodium inactivation in the node of Ranvier. This second ultraviolet effect shifts the kinetic parameters of sodium inactivation h infinity (V), alpha h (V), and beta h (V) to more negative potentials (no changes of the slopes of the curves). The amount of the displacement delta V along the potential axis is equal for the three parameters and depends on the ultraviolet dose. It is about delta V = --10 mV after an irradiation dose of 0.7 Ws/cm2 at 280 nm. Both ultraviolet-induced effects depend on membrane potential and on the wavelength of the applied radiation. But while the potential shift is enhanced at more negative holding potentials, the ultraviolet blocking is diminished and vice versa. Further, the ultraviolet-induced potential shift is greater at 260 nm than at 280 nm, whereas a maximum sensitivity of ultraviolet blocking is found at 280 nm. Therefore, the two radiation effects are the result of two separate photoreactions. For explanation of the radiation-induced potential shift it is assumed that ultraviolet radiation decreases the density of negative charges at the inner surface of the nodal membrane. From this hypothesis a value for the inner surface potential psii was derived. --19 mV less than or equal to psii less than or equal to --14 mV.     

Investigation of the relation between structure and function in myelinated nerve fibres with the aid of ultraviolet radiation

Ultraviolet radiation induces two photochemical alterations relevant to excitability in the nodal membranes: A selective blocking of the sodium permeability and a potential translation of the voltage dependent kinetic parameters of sodium inactivation and activation along the potential axis in the negative direction. The underlying processes are two different photoreactions, since 1) the action spectrum of the blocking effect shows a marked peak near 280 nm and rapidly decreasing sensitivity towards higher and lower wavelengths, while the action spectrum of the potential shift increases with lower wavelengths; 2) the blocking effect is enhanced by a more positive holding potential, while the potential shift is decreased; 3) the potential shift can be prevented intraaxonal application of l-cysteine or 2-mercaptoethanol, but the blocking effect is not affected.Paper presented at the Biomembrane Symposium of the Deutsche Gesellschaft für Biophysik, Freiburg, April 1975.     

Asymmetric electrostatic effects on the gating of rat brain sodium channels in planar lipid membranes.

The effects of ionic strength (10-1,000 mM) on the gating of batrachotoxin-activated rat brain sodium channels were studied in neutral and in negatively charged lipid bilayers. In neutral bilayers, increasing the ionic strength of the extracellular solution, shifted the voltage dependence of the open probability (gating curve) of the sodium channel to more positive membrane potentials. On the other hand, increasing the intracellular ionic strength shifted the gating curve to more negative membrane potentials. Ionic strength shifted the voltage dependence of both opening and closing rate constants of the channel in analogous ways to its effects on gating curves. The voltage sensitivities of the rate constants were not affected by ionic strength. The effects of ionic strength on the gating of sodium channels reconstituted in negatively charged bilayers were qualitatively the same as in neutral bilayers. However, important quantitative differences were noticed: in low ionic strength conditions (10-150 mM), the presence of negative charges on the membrane surface induced an extra voltage shift on the gating curve of sodium channels in relation to neutral bilayers. It is concluded that: (a) asymmetric negative surface charge densities in the extracellular (1e-/533A2) and intracellular (1e-/1,231A2) sides of the sodium channel could explain the voltage shifts caused by ionic strength on the gating curve of the channel in neutral bilayers. These surface charges create negative electric fields in both the extracellular and intracellular sides of the channel. Said electric fields interfere with gating charge movements that occur during the opening and closing of sodium channels; (b) the voltage shifts caused by ionic strength on the gating curve of sodium channels can be accounted by voltage shifts in both the opening and closing rate constants; (c) net negative surface charges on the channel's molecule do not affect the intrinsic gating properties of sodium channels but are essential in determining the relative position of the channel's gating curve; (d) provided the ionic strength is below 150 mM, the gating machinery of the sodium channel molecule is able to sense the electric field created by surface changes on the lipid membrane. I propose that during the opening and closing of sodium channels, the gating charges involved in this process are asymmetrically displaced in relation to the plane of the bilayer. Simple electrostatic calculations suggest that gating charge movements are influenced by membrane electrostatic potentials at distances of 48 and 28 A away from the plane of the membrane in the extracellular sides of the channel, respectively.     

ATP-induced stimulation of calcium binding to cardiac sarcolemma.

The influence of β-93 sulfhydryl groups on the oxidation of human hemoglobin by sodium nitrite was studied. It is shown that the blocking of these groups by iodoacetamine counteracts the inhibition of the hemoglobin oxidation reaction caused by inositol hexaphosphate. This effect is not present under anaerobic condition. However, in the absence of free oxygen (deoxyhemoglobin), blocking of the β-93 sulfhydryl groups accelerates markedly the rate of oxidation which is otherwise very slow. In the light of these observations, it is concluded that the hemoglobin β-93 free-SH groups play a protective role for the heme iron against oxidation. The rapid oxidation of modified hemoglobin by nitrite under anaerobic condition as well as the abolishment of the effect of IHP under aerobic condition by β-93-SH groups blockage argue against the assumption that R conformation is primarily responsible for the rapid oxidation of oxyhemoglobin by nitrite.     

Effects of Redox and Sulfhydryl Reagents on the Bioelectric Properties of the Giant Axon of the Squid

The effects of internally and externally applied sulfhydryl reagents on the bioelectric properties of the giant axon of the squid Loligo pealeii and Dosidicus gigas were studied. Cysteine-HCl (400 mM, pH 7.3) was used to remove axoplasm from the perfusion channel. Oxidizing agents (1 to 60 mM) tended to increase the duration of the action potential and had a slow, irreversible blocking effect when perfused internally; the membrane potential was little affected. Reducing agents applied internally caused a decrease in the spike duration without affecting its height or the membrane potential, although at high concentrations there was reversible deterioration of the action potential. Both external and internal perfusion of mercaptide-forming reagents caused deterioration in the action and membrane potentials with conduction block occurring in 5 to 45 min. 2-mercaptoethanol reversed the effects. Thiol alkylating reagents, iodoacetate and iodoacetamide, were without effect. N-ethylmaleimide did, however, block. Tests with chelating agents for nonheme iron in the membrane brought about no change in the electrical parameters. The implications of the present findings with regard to the macromolecular mechanism of excitation are discussed.     

Simple shifts in the voltage dependence of sodium channel gating caused by divalent cations

The effect of elevated divalent cation concentration on the kinetics of sodium ionic and gating currents was studied in voltage-clamped frog skeletal muscle fibers. Raising the Ca concentration from 2 to 40 mM resulted in nearly identical 30-mV shifts in the time courses of activation, inactivation, tail current decay, and ON and OFF gating currents, and in the steady state levels of inactivation, charge immobilization, and charge vs. voltage. Adding 38 mM Mg to the 2 mM Ca bathing a fiber produced a smaller shift of approximately 20 mV in gating current kinetics and the charge vs. voltage relationship. The results with both Ca and Mg are consistent with the hypothesis that elevated concentrations of these alkali earth cations alter Na channel gating by changing the membrane surface potential. The different shifts produced by Ca and Mg are consistent with the hypothesis that the two ions bind to fixed membrane surface charges with different affinities, in addition to possible screening.     

Effects of membrane surface charge and calcium on the gating of rat brain sodium channels in planar bilayers [published erratum appears in J Gen Physiol 1989 Apr;93(4):following 760]

The voltage-dependent gating of single, batrachotoxin-activated Na channels from rat brain was studied in planar lipid bilayers composed of negatively charged or neutral phospholipids. The relationship between the probability of finding the Na channel in the open state and the membrane potential (Po vs. Vm) was determined in symmetrical NaCl, both in the absence of free Ca2+ and after the addition of Ca2+ to the extracellular side of the channel, the intracellular side, or both. In the absence of Ca2+, neither the midpoint (V0.5) of the Po vs. Vm relation, nor the steepness of the gating curve, was affected by the charge on the bilayer lipid. The addition of 7.5 mM Ca2+ to the external side caused a depolarizing shift in V0.5. This depolarizing shift was approximately 17 mV in neutral bilayers and approximately 25 mV in negatively charged bilayers. The addition of the same concentration of Ca2+ to only the intracellular side caused hyperpolarizing shifts in V0.5 of approximately 7 mV (neutral bilayers) and approximately 14 mV (negatively charged bilayers). The symmetrical addition of Ca2+ caused a small depolarizing shift in Po vs. Vm. We conclude that: (a) the Na channel protein possesses negatively charged groups on both its inner and outer surfaces. Charges on both surfaces affect channel gating but those on the outer surface exert a stronger influence. (b) Negative surface charges on the membrane phospholipid are close enough to the channel's gating machinery to substantially affect its operation. Charges on the inner and outer surfaces of the membrane lipid affect gating symmetrically. (c) Effects on steady-state Na channel activation are consistent with a simple superposition of contributions to the local electrostatic potential from charges on the channel protein and the membrane lipid.     

Barium modulates the gating of batrachotoxin-treated Na+ channels in high ionic strength solutions.

Batrachotoxin-activated rat brain Na+ channels were reconstituted in neutral planar phospholipid bilayers in high ionic strength solutions (3 M NaCl). Under these conditions, diffuse surface charges present on the channel protein are screened. Nevertheless, the addition of extracellular and/or intracellular Ba2+ caused the following alterations in the gating of Na+ channels: (a) external (or internal) Ba2+ caused a depolarizing (or hyperpolarizing) voltage shift in the gating curve (open probability versus membrane potential curve) of the channels; (b) In the concentration range of 10-120 mM, extracellular Ba2+ caused a larger voltage shift in the gating curve of Na+ channels than intracellular Ba2+; (c) voltage shifts of the gating curve of Na+ channels as a function of external or internal Ba2+ were fitted with a simple binding isotherm with the following parameters: for internal Ba2+, delta V0.5,max (maximum voltage shift) = -11.5 mV, KD = 64.7 mM; for external Ba2+, delta V0.5,max = 13.5 mV, KD = 25.8 mM; (d) the change in the open probability of the channel caused by extracellular or intracellular Ba2+ is a consequence of alterations in both the opening and closing rate constants. Extracellular and intracellular divalent cations can modify the gating kinetics of Na+ channels by a specific modulatory effect that is independent of diffuse surface potentials. External or internal divalent cations probably bind to specific charges on the Na+ channel glycoprotein that modulate channel gating.     

Potential-dependent interaction of chemical reagents with the gating mechanism of the nerve fiber sodium channel

The action of two water-soluble carbodiimides and of Woodward's reagent K on the properties of the gating mechanism of sodium channels of the Ranvier node membrane was investigated. Treatment with carbodiimide solution (pH 4.8–5.2) at a potential of –80 to –100 mV was shown to delay activation and inactivation of the channels considerably and to reduce the sensitivity of both gating functions to changes in membrane potential. The effective activation charge, determined relative to the limiting logarithmic slope of the activation curve (z_ef) was reduced by 1.7 times. Treatment of the membrane under the same conditions, but at zero holding potential, induced much smaller changes in properties of the gating mechanism; under these conditions z_ef remained unchanged. Woodward's reagent at high negative potential induced the same changes in the gating system as carbodiimide at 0 mV. The action of Woodward's reagent also depended on potential, but by a lesser degree than when carbodiimides were used. The results suggest that two types of carboxyl groups exist on the outer surface of the membrane: "mobile," which perform the role of gating particles and which are moved from the surface when the channel changes into the open or inactivated state, and "immobile," which face the external solution whatever the state of the channel.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 16, No. 5, pp. 577–590, September–October, 1984.     

Movement and crevices around a sodium channel S3 segment

Voltage sensing is due mainly to the movement of positively charged S4 segments through the membrane electric field during changes of membrane potential. The roles of other transmembrane segments are under study. The S3 segment of domain 4 (D4/S3) in the sodium channel Na(v)1.4 carries two negatively charged residues and has been implicated in voltage-dependent gating. We substituted cysteines into nine putative "high impact" sites along the complete length of D4/S3 and evaluated their accessibilities to extracellular sulfhydryl reagents. Only the four outermost substituted cysteines (L1433C, L1431C, G1430C, and S1427C) are accessible to extracellular sulfhydryl reagents. We measured the voltage-dependent modification rates of the two cysteines situated at the extreme ends of this accessible region, L1433C and S1427C. Independent of the charge on the sulfhydryl reagents, depolarization increases the reactivity of both of these residues. Thus, the direction of the voltage dependence is opposite to that expected for a negatively charged voltage sensor, namely an inward translational movement in response to depolarization. Intrinsic electrostatic potentials were probed by charged sulfhydryl reagents and were either negative or positive, respectively, near L1433C and S1427C. The magnitude of the electrostatic potential near S1427C decreases with depolarization, suggesting that the extracellular crevice next to it widens during depolarization. S1427C experiences 44% of the electric field, as probed by charged cysteine reagents. To further explore movements around D4/S3, we labeled cysteines with the photoactivatable cross-linking reagent benzophenone-4-carboxamidocysteine methanethiosulfonate and examined the effects of UV irradiation on channel gating. After labeling with this reagent, all accessible cysteine mutants show altered gating upon brief UV irradiation. In each case, the apparent insertion efficiency of the photoactivated benzophenone increases with depolarization, indicating voltage-dependent movement near the extracellular end of D4/S3.     

Redox Regulation of Large Conductance Ca2+-activated K+ Channels in Smooth Muscle Cells

The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca²⁺-activated K⁺ (maxi-K) channels were examined in excised patches from tracheal myocytes. Channel activity was modified by sulfhydryl redox agents applied to the cytosolic surface, but not the extracellular surface, of membrane patches. Sulfhydryl reducing agents dithiothreitol, β-mercaptoethanol, and GSH augmented, whereas sulfhydryl oxidizing agents diamide, thimerosal, and 2,2′-dithiodipyridine inhibited, channel activity in a concentration-dependent manner. Channel stimulation by reduction and inhibition by oxidation persisted following washout of the compounds, but the effects of reduction were reversed by subsequent oxidation, and vice versa. The thiol-specific reagents N-ethylmaleimide and (2-aminoethyl)methanethiosulfonate inhibited channel activity and prevented the effect of subsequent sulfhydryl oxidation. Measurements of macroscopic currents in inside-out patches indicate that reduction only shifted the voltage/nP_o relationship without an effect on the maximum conductance of the patch, suggesting that the increase in nP_o following reduction did not result from recruitment of more functional channels but rather from changes of channel gating. We conclude that redox modulation of cysteine thiol groups, which probably involves thiol/disulfide exchange, alters maxi-K channel gating, and that this modulation likely affects channel activity under physiological conditions.     

比较不同类型白内障形成过程中非蛋白质巯基与蛋白质巯基的动态变化及关系

本文报道了在亚硒酸钠、平阳霉素及半乳糖诱发大鼠产生白内障过程中晶状体中非蛋白质巯基及蛋白质巯基的动态变化,并探讨了其变化机理及相互关系。在亚硒酸钠诱发白内障过程中,给药24h后晶状体中非蛋白质巯基减少到正常的二分之一,以后又逐渐回升,但始终未达到正常水平,至第7天,非蛋白质巯基又再度减少。在平阳霉素及半乳糖诱发白内障过程中,晶状体中非蛋白质巯基分别在给药后的第7天及第3天开始大量减少,以后继续减少,至第15天时,其含量分别为正常的十分之一及五分之一。在体外,亚硒酸钠有促进还原型谷胱甘肽自氧化的作用,半乳糖对此作用无影响,而平阳霉素可阻止其进行,但能加强亚硒酸钠的促进作用。在三种白内障晶状体中,蛋白质巯基开始减少的时间均较非蛋白质巯基为晚,这表明只有非蛋白质巯基减少到一定程度后蛋白质巯基才会被大量氧化,同时也说明非蛋白质巯基具有保护蛋白质巯基免受氧化的作用。只有这种保护作用减弱后,才会使蛋白质巯基遭受氧化而导致白内障。     

Sodium ionic and gating currents in mammalian cells

Ionic and gating currents from voltage-gated sodium channels were recorded in mouse neuroblastoma cells using the path-clamp technique. Displacement currents were measured from whole-cell recordings. The gating charge displaced during step depolarizations increased with the applied membrane potential and reached saturating levels above 20 mV Prolonged large depolarizations produced partial immobilization of the gating charge, and only about one third of the displaced charge was quickly reversed upon return to negative holding potentials. The activation and inactivation properties of macroscopic sodium currents were characterized by voltage-clamp analysis of large outside-out patches and the single-channel conductance was estimated from nonstationary noise analysis. The general properties of the sodium channels in mouse neuroblastoma cells are very similar to those previously reported for various preparations of invertebrate and vertebrate nerve cells. Offprint requests to: O. Moran     

Functional groups and quaternary structure of chicken liver xanthine dehydrogenase

Xanthine dehydrogenase from chicken liver is a dimeric enzyme, each hemimolecule containing one FAD and two Fe/S groups. Determination of sulfhydryl groups with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuribenzoic acid (PMB) showed a variable number of sulfhydryl groups depending onpH, ionic strength, and nature of the reaction medium and buffer. The number of disulfide bonds was determined with DTNB and reducing conditions. Amino groups were determined with 2,4,6,-trinitrobencensulfonic acid (TNBS). At constant temperature andpH the reaction of DTNB and TNBS with native xanthine dehydrogenase showed an exponential dependence on time. From the obtained parameters the number of available sulfhydryl and amino groups at infinite concentration of enzyme and the rate constant of the equation were determined. The absorption spectrum of the enzyme changed with time when a chaotropic agent (1 M sodium nitrate) was added to the medium. This difference was detected by measuring the absorbance in the range 450–550 nm. The absorption spectrum (between 350 and 600 nm) also changed when a denaturating agent (sodium dodecyl sulfate) was added. This modification increased with time and depended on the medium.     

Voltage-dependent membrane capacitance in rat pituitary nerve terminals due to gating currents

We investigated the voltage dependence of membrane capacitance of pituitary nerve terminals in the whole-terminal patch-clamp configuration using a lock-in amplifier. Under conditions where secretion was abolished and voltage-gated channels were blocked or completely inactivated, changes in membrane potential still produced capacitance changes. In terminals with significant sodium currents, the membrane capacitance showed a bell-shaped dependence on membrane potential with a peak at approximately -40 mV as expected for sodium channel gating currents. The voltage-dependent part of the capacitance showed a strong correlation with the amplitude of voltage-gated Na+ currents and was markedly reduced by dibucaine, which blocks sodium channel current and gating charge movement. The frequency dependence of the voltage-dependent capacitance was consistent with sodium channel kinetics. This is the first demonstration of sodium channel gating currents in single pituitary nerve terminals. The gating currents lead to a voltage- and frequency-dependent capacitance, which can be well resolved by measurements with a lock-in amplifier. The properties of the gating currents are in excellent agreement with the properties of ionic Na+ currents of pituitary nerve terminals.     

Time dependence of the effect ofp-chloromercuribenzoate on erythrocyte water permeability: A pulsed nuclear magnetic resonance study

Summary Pulsed nuclear magnetic resonance spectroscopy is employed to determine the time dependence of the change in erythrocyte water permeability following exposure top-chloromercuribenzoate (PCMB) orp-chloromercuribenzene sulfonic acid (PCMBS). pH variation was used to examine the environment of the sulfhydryl groups reactive to these drugs. PCMB reacted with at least two sulfhydryl groups which affect water permeability. This was shown by the double exponential character of the change in erythrocyte diffusional permeability with time after PCMB addition. However, only one inhibition rate process could be distinguished following PCMBS exposure, suggesting that one site bound by PCMB is not accessible to PCMBS. This site is postulated to be located in a hydrophobic region of the membrane, whereas the site reached by both drugs is located in the normal anion permeation channel. The effect of pH on the degree of inhibition due to each component and the inhibition rates is explained in terms of its effect on solubility of the reagents in the membrane and variation of the dissociated-to-undissociated ratio of PCMB.     

Mechanism of Maxi-K Channel Activation by Dehydrosoyasaponin-I

Dehydrosoyasaponin-I (DHS-I) is a potent activator of high-conductance, calcium-activated potassium (maxi-K) channels. Interaction of DHS-I with maxi-K channels from bovine aortic smooth muscle was studied after incorporating single channels into planar lipid bilayers. Nanomolar amounts of intracellular DHS-I caused the appearance of discrete episodes of high channel open probability interrupted by periods of apparently normal activity. Statistical analysis of these periods revealed two clearly separable gating modes that likely reflect binding and unbinding of DHS-I. Kinetic analysis of durations of DHS-I-modified modes suggested DHS-I activates maxi-K channels through a high-order reaction. Average durations of DHS-I-modified modes increased with DHS-I concentration, and distributions of these mode durations contained two or more exponential components. In addition, dose-dependent increases in channel open probability from low initial values were high order with average Hill slopes of 2.4–2.9 under different conditions, suggesting at least three to four DHS-I molecules bind to maximally activate the channel. Changes in membrane potential over a 60-mV range appeared to have little effect on DHS-I binding. DHS-I modified calcium- and voltage-dependent channel gating. 100 nM DHS-I caused a threefold decrease in concentration of calcium required to half maximally open channels. DHS-I shifted the midpoint voltage for channel opening to more hyperpolarized potentials with a maximum shift of −105 mV. 100 nM DHS-I had a larger effect on voltage-dependent compared with calcium-dependent channel gating, suggesting DHS-I may differentiate these gating mechanisms. A model specifying four identical, noninteracting binding sites, where DHS-I binds to open conformations with 10–20-fold higher affinity than to closed conformations, explained changes in voltage-dependent gating and DHS-I-induced modes. This model of channel activation by DHS-I may provide a framework for understanding protein structures underlying maxi-K channel gating, and may provide a basis for understanding ligand activation of other ion channels.     

Transport of large organic ions through syringomycin channels in membranes containing dipole modifiers

The effect of the membrane dipole potential (φ _d ) on conductance and the steady-state number of functioning channels formed by cyclic lipodepsipeptide syringomycin E (SRE) in bilayer lipid membranes made from phosphocholine and bathed in 0.4 M solution of sodium salts of aspartate, gluconate, and chloride was shown. The φ _d value varied with the introduction of phloretin to membrane bathing solutions, which reduces φ _d and RH 421, which increases φ _d . It was established that, in all studied systems, an increase in the membrane dipole potential caused a decrease in the steady-state number of open channels. In systems containing sodium salts of aspartate (Asp) or gluconate (Glc), changes in the number of functioning channels are one order lower than those of systems that contain sodium chloride. At the same time, the conductance (g) of single SRE channels in the membranes bathed in NaCl solution increases with increase in φ _d and in the systems containing NaAsp or NaGlc the conductance of single channels does not depend on the φ _d . The latter is due to the lack of cation/anion selectivity of the SRE channels in these systems. The different channel-forming activity of SRE in the experimental systems is determined by the gating charge of the channel and the partition coefficient of the dipole modifiers between the lipid and aqueous phases.     

Model investigations of the temperature dependence of demyelinated and reorganized axonal membrane

The temperature dependence (from 10° to 50°C) of the intracellular action potentials' parameters in a fiber with a simulated reorganization of the axonal membrane against the background of a systematic paranodal demyelination of the fiber was investigated. The temporal and spatial distribution of the potential as well as the ionic currents' kinetics have been represented. The reorganization of the axonal membrane was achieved by means of potassium channels blocking and increase of the sodium-channel permeability, while the demyelination was achieved by means of elongation of the nodes of Ranvier. In order to account for the temperature dependence of the rate constants and of the maximal sodium and potassium permeabilities, the temperature coefficients (Q ₁₀) have been used. It has been shown for the demyelinated and reorganized membrane that increased temperature blocks the conduction at temperatures much higher than the blocking temperature for the demyelinated fiber only. When temperature increases the amplitude of the potential decreases while the velocity increases up to temperatures approching the blocking temperature after which it abruptly drops. The dependence of the asymmetry and the wavelength of the potential on temperature is complex and nonmonotonic. For the reorganized membrane at the background of a given degree of demyelination with increasing temperature the ionic currents' flow and the membrane conduction respectively increase, but, at lower temperatures, when the temperature increase is combined with the increased degree of the fiber demyelination, the conduction is blocked.     

Resting potential-dependent regulation of the voltage sensitivity of sodium channel gating in rat skeletal muscle in vivo

Normal muscle has a resting potential of -85 mV, but in a number of situations there is depolarization of the resting potential that alters excitability. To better understand the effect of resting potential on muscle excitability we attempted to accurately simulate excitability at both normal and depolarized resting potentials. To accurately simulate excitability we found that it was necessary to include a resting potential-dependent shift in the voltage dependence of sodium channel activation and fast inactivation. We recorded sodium currents from muscle fibers in vivo and found that prolonged changes in holding potential cause shifts in the voltage dependence of both activation and fast inactivation of sodium currents. We also found that altering the amplitude of the prepulse or test pulse produced differences in the voltage dependence of activation and inactivation respectively. Since only the Nav1.4 sodium channel isoform is present in significant quantity in adult skeletal muscle, this suggests that either there are multiple states of Nav1.4 that differ in their voltage dependence of gating or there is a distribution in the voltage dependence of gating of Nav1.4. Taken together, our data suggest that changes in resting potential toward more positive potentials favor states of Nav1.4 with depolarized voltage dependence of gating and thus shift voltage dependence of the sodium current. We propose that resting potential-induced shifts in the voltage dependence of sodium channel gating are essential to properly regulate muscle excitability in vivo.