Depolarization increases the single-channel conductance and the open probability of crayfish glutamate channels.

We have studied the voltage sensitivity of glutamate receptors in outside-out patches taken from crayfish muscles. We found that single-channel conductance, measured directly at the single-channel level, increases as depolarization rises. At holding potentials from -90 mV to approximately 20 mV, the conductance is 109 pS. At holding potentials positive to 20 mV, the conductance is 213 pS. This increase in single-channel conductance was also observed in cell-attached patches. In addition, desensitization, rise time, and the dose-response curve were all affected by depolarization. To further clarify these multifaceted effects, we evaluated the kinetic properties of single-channel activity recorded from cell-attached patches in hyperpolarization (membrane potential around -75 mV) and depolarization (membrane potential approximately 105 mV). We found that the glutamate dissociation rate constant (k_) was affected most significantly by membrane potential; it declined 6.5-fold under depolarization. The rate constant of channel closing (k(c)) was also significantly affected; it declined 1.8-fold. The rate constant of channel opening (k(o)) declined only 1.2-fold. The possible physiological significance of the depolarization-mediated changes in the above rate constants is discussed.     

Gating characteristics of a steeply voltage-dependent gap junction channel in rat Schwann cells

The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, Gss was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary junctional currents was detected corresponding to an unitary channel conductance of approximately 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic Gss voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states.     

Single-channel acetylcholine receptor kinetics.

The temporal relationships among junctional acetylcholine receptor single-channel currents have been examined to probe the mechanism of channel activation. We have presented an analytical approach, termed single-channel ensemble analysis, that allows one to estimate the kinetic transition rate constants for channel-opening and closing as well as the rate of leaving the specific doubly-liganded, closed state from which opening occurs. This approach may be applied to data produced by any number of independent channels as long as the probability of channel opening is low, a condition that is experimentally verifiable. The method has been independently validated using simulated single-channel data generated by computer from one or 100 hypothetical channels. Typical experimental values for the transition rate constants estimated from acetylcholine-activated single channels at the garter snake neuromuscular junction were: opening = 1,200 s-1, closing = 455 s-1, back rate for leaving the doubly-liganded, closed state = 3,200 s-1 at a transmembrane potential of -92 mV at room temperature. Each of these three rate constants was voltage dependent, with the closing rate decreasing e-fold for 173 mV of hyperpolarization, the opening rate increasing e-fold for 78 mV, and the unbinding rate increasing e-fold for 105 mV. The channel-closing rate was agonist dependent, being greater at all potentials for channels activated with carbamylcholine than for channels activated with acetylcholine. However, the single-channel conductance and reversal potential were the same for these two agonists.     

Conductive properties and gating of channels formed by syringopeptin 25A, a bioactive lipodepsipeptide from Pseudomonas syringae pv. syringae, in planar lipid membranes

Syringopeptin 25A, a pseudomonad lipodepsipeptide, can form ion channels in planar lipid membranes. Pore conductance is around 40 pS in 0.1 M NaCl. Channel opening is strongly voltage dependent and requires a negative potential on the same side of the membrane where the toxin was added. These pores open and close with a lifetime of several seconds. At negative voltages, an additional pore state of around 10 pS and a lifetime of around 30 ms is also present. The voltage dependence of the rates of opening and closing of the stable pores is exponential. This allows estimation of the equivalent charge that is moved across the membrane during the process of opening at about 2.6 elementary charges. When NaCl is present, the pore is roughly 3 times more permeant for anions than for cations. The current voltage characteristic of the pore is nonlinear, i.e., pore conductance is larger at negative than at positive voltages. The maximal conductance of the pore depends on the concentration of the salt present, in a way that varies almost linearly with the conductivity of the solution. From this, an estimate of a minimal pore radius of 0.4 nm was derived.     

Rates and equilibria at the acetylcholine receptor of electrophorus electroplaques. A study of neurally evoked postsynaptic currents and of voltage-jump relaxations

Kinetic measurements are employed to reconstruct the steady-state activation of acetylcholine [Ach] receptor channels in electrophorus electroplaques. Neurally evoked postsynaptic currents (PSCs) decay exponentially; at 15 degrees C the rate constant, α, equals 1.2 ms(-1) at 0 mV and decreases e-fold for every 86 mV as the membrane voltage is made more negative. Voltage-jump relaxations have been measured with bath-applied ACh, decamethonium, carbachol, or suberylcholine. We interpret the reciprocal relaxation time 1/τ as the sum of the rate constant α for channel closing and a first-order rate constant for channel opening. Where measureable, the opening rate increases linearly with [agonist] and does not vary with voltage. The voltage sensitivity of small steady-state conductances (e- fold for 86 mV) equals that of the closing rate α, confirming that the opening rate has little or no additional voltage sensitivity. Exposure to α-bungarotoxin irreversibly decreases the agonist-induced conductance but does not affect the relaxation kinetics. Tubocurarine reversibly reduces both the conductance and the opening rate. In the simultaneous presence of two agonist species, voltage-jump relaxations have at least two exponential components. The data are fit by a model in which (a) the channel opens as the receptor binds the second in a sequence of two agonist molecules, with a forward rate constant to 10(7) to 2x10(8) M(-1)s(-1); and (b) the channel then closes as either agonist molecule dissociates, with a voltage-dependent rate constant of 10(2) to 3x10(3)s(-1).     

Thermodynamic and kinetic studies of the gating behavior of a K+- selective channel from the sarcoplasmic reticulum membrane

A voltage-dependent, K+-selective ionic channel from sarcoplasmic reticulum of rabbit skeletal muscle has been studied in a planar phospholipid bilayer membrane. The purpose [corrected] of this work is to study the mechanism by which the channel undergoes transitions between its conducting and nonconducting states. Thermodynamic studies show that the "open" and "closed" states of the channel exist in a voltage-dependent equilibrium, and that the channel displays only a single open state; the channel conductance is 120 pmho in 0.1 M K+. The channel's gating process follows single exponential kinetics at all voltages tested, and the individual opening and closing rate constants are exponentially dependent on voltage. The individual rate constants may also be determined from a stochastic analysis of channel fluctuations among multiple conductance levels. Neither the thermodynamic nor the kinetic parameters of gating depend on the absolute concentration of channels in the bilayer. The results are taken as evidence that the channel gates by an unusually simple two-state conformational mechanism in which the equivalent of 1.1 net charges are moved across the membrane during the formation of the open channel.     

Gating kinetics of batrachotoxin-modified sodium channels in neuroblastoma cells determined from single-channel measurements

We have observed the opening and closing of single batrachotoxin (BTX)-modified sodium channels in neuroblastoma cells using the patch-clamp method. The conductance of a single BTX-modified channel is approximately 10 pS. At a given membrane potential, the channels are open longer than are normal sodium channels. As is the case for normal sodium channels, the open dwell times become longer as the membrane is depolarized. For membrane potentials more negative than about -70 mV, histograms of both open-state dwell times and closed-state dwell times could be fit by single exponentials. For more depolarized potentials, although the open-state histograms could still be fit by single exponentials, the closed-state histograms required two exponentials. This data together with macroscopic voltage clamp data on the same system could be accounted for by a three-state closed-closed-open model with transition rates between these states that are exponential functions of membrane potential. One of the implications of this model, in agreement with experiment, is that there are always some closed BTX-modified sodium channels, regardless of membrane potential.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Nonselective cation channels in intact human T lymphocytes.

Nonselective cation channels were found in single channel recordings from cell-attached patches on human T lymphocytes. These channels were active under conditions that should lead to cell swelling (hypotonic bath solutions with NaCl or KCl); however, a definite dependence of activity on cell swelling has not been proven. Under these conditions similar channels were found in 20 of 23 patches from 11 different blood donors. The current-voltage relation was approximately linear for outward current (11-14 pS) and inwardly rectifying (to 23 pS) when the intact cells were depolarized with high KCl in the bath. The voltage dependence of channel activity is consistent with closing at hyperpolarized membrane potentials (Vm less than or equal to -50 mV) and block of open channels at strongly depolarized membrane potentials (Vm greater than 0 mV). Reversal potentials under all ionic gradients tested are consistent with a channel that is poorly selective between Na+ and K+ ions. Active channels in cell-attached patches were rapidly blocked by bath addition of the membrane-permeant inhibitor quinine. Channels that were active in cell-attached became quiescent after patch excision; however, two patches remained active long enough to obtain current-voltage relations. These were linear with a slope conductance for outward current of 8-11 pS. Because of the clustering of single-channel openings, detailed voltage dependence of kinetics and probability of opening were not studied.     

Concentration-jump analysis of voltage-dependent conductances activated by glutamate and kainate in neurons of the avian cochlear nucleus.

We have examined the mechanisms underlying the voltage sensitivity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors in voltage-clamped outside-out patches and whole cells taken from the nucleus magnocellularis of the chick. Responses to either glutamate or kainate had outwardly rectifying current-voltage relations. The rate and extent of desensitization during prolonged exposure to agonist, and the rate of deactivation after brief exposure to agonist, decreased at positive potentials, suggesting that a kinetic transition was sensitive to membrane potential. Voltage dependence of the peak conductance and of the deactivation kinetics persisted when desensitization was reduced with aniracetam or blocked with cyclothiazide. Furthermore, the rate of recovery from desensitization to glutamate was not voltage dependent. Upon reduction of extracellular divalent cation concentration, kainate-evoked currents increased but preserved rectifying current-voltage relations. Rectification was strongest at lower kainate concentrations. Surprisingly, nonstationary variance analysis of desensitizing responses to glutamate or of the current deactivation after kainate removal revealed an increase in the mean single-channel conductance with more positive membrane potentials. These data indicate that the rectification of the peak response to a high agonist concentration reflects an increase in channel conductance, whereas rectification of steady-state current is dominated by voltage-sensitive channel kinetics.     

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.     

Ionic channels induced by surfactin in planar lipid bilayer membranes.

Surfactin is a lipopeptide produced by certain strains of Bacillus subtilis and has potent surface activity. Here, we present the first results showing that ion-conducting pores can be formed by surfactin in artificial lipid membranes. With a low aqueous concentration of surfactin (1 microM) and a restricted membrane area (5.10(-5) cm2) we observed conductance jumps that indicate the formation of individual ionic channels in the presence of K+, Rb+, Cs+, Na+ or Li+ chlorides. Although for every salt concentration (Ci), the distribution in amplitude of the conductance steps (lambda i) may be rather broad, there is always a step amplitude which is more frequent than the others. In addition, the channels corresponding to this most frequent step amplitude are the longest in duration. For Ci = 1 M, the cationic selectivity sequence deduced from these most frequent events is K+ greater than Rb+ greater than Na+ greater than Cs+ = Li+ with respective values for lambda Mi: 130, 110, 80 and 30 pS. In KCl solutions lambda MKCl increases as a function of Ci for low Ci, and shows a plateau for Ci greater than 0.5 M. When measured on larger area membranes (10(-2)cm2) with 1 M solutions of the monovalent salts KCl, NaCl, RbCl and CsCl or the divalent salt CaCl2, the macroscopic low voltage conductance (G0) increases with a slope of 2 on a log-log plot as a function of surfactin concentration. These results demonstrate that surfactin produces selective cationic channels in lipid bilayer membranes and suggest that at higher salt concentration, a dimer is involved in this functional channel-forming process.     

Characterization of a Light-Controlled Anion Channel in the Plasma Membrane of Mesophyll Cells of Pea

In leaf mesophyll cells of pea (Pisum sativum) light induces a transient depolarization that is at least partly due to an increased plasma membrane conductance for anions. Several channel types were identified in the plasma membrane of protoplasts from mesophyll cells using the patch-clamp technique. One of these was an anion channel with a single-channel conductance of 32 picasiemens in symmetrical 100/100 KCl solutions. In asymmetrical solutions the reversal potential indicates a high selectivity for Cl- over K+ at high cytoplasmic Cl-. At negative membrane voltages the channel openings were interrupted by very short closures. In the open channel conductance several substrates were identified. At a cytoplasmic negative logarithm of Ca concentration higher than 6.3, no channel openings were observed. When the protoplast was illuminated in the cell-attached configuration, at least one channel type had a higher opening probability. This channel can tentatively be identified as the above-described anion channel based on conductance and the characteristic short closures at negative membrane potentials. This light activation of the 32-picasiemen anion channel is a strong indication that this channel conducts the light-induced depolarizing current. Because channel activity is strongly Ca2+-dependent, a role of cytoplasmic Ca2+ concentration changes in the light activation of the conductance is discussed.     

Global parameter optimization for cardiac potassium channel gating models.

Quantitative ion channel model evaluation requires the estimation of voltage dependent rate constants. We have tested whether a unique set of rate constants can be reliably extracted from nonstationary macroscopic voltage clamp potassium current data. For many models, the rate constants derived independently at different membrane potentials are not unique. Therefore, our approach has been to use the exponential voltage dependence predicted from reaction rate theory (Stevens, C. F. 1978. Biophys. J. 22:295-306; Eyring, H., S. H. Lin, and S. M. Lin. 1980. Basic Chemical Kinetics. Wiley and Sons, New York) to couple the rate constants derived at different membrane potentials. This constrained the solution set of rate constants to only those that also obeyed this additional set of equations, which was sufficient to obtain a unique solution. We have tested this approach with data obtained from macroscopic delayed rectifier potassium channel currents in voltage-clamped guinea pig ventricular myocyte membranes. This potassium channel has relatively simple kinetics without an inactivation process and provided a convenient system to determine a globally optimized set of voltage-dependent rate constants for a Markov kinetic model. The ability of the fitting algorithm to extract rate constants from the macroscopic current data was tested using "data" synthesized from known rate constants. The simulated data sets were analyzed with the global fitting procedure and the fitted rate constants were compared with the rate constants used to generate the data. Monte Carlo methods were used to examine the accuracy of the estimated kinetic parameters. This global fitting approach provided a useful and convenient method for reliably extracting Markov rate constants from macroscopic voltage clamp data over a broad range of membrane potentials. The limitations of the method and the dependence on initial guesses are described.     

Properties of Voltage-gated Ion Channels Formed by Syringomycin E in Planar Lipid Bilayers

Using the planar lipid bilayer technique we demonstrate that the lipodepsipeptide antibiotic, syringomycin E, forms voltage-sensitive ion channels of weak anion selectivity. The formation of channels in bilayers made from dioleoylglycerophosphatidylserine doped with syringomycin E at one side (1–40 μg/ml) was greatly affected by cis-positive voltage. A change of voltage from a positive to a negative value resulted in (i) an abrupt increase in the single channel conductance (the rate of increase was voltage dependent) simultaneous with (ii) a closing of these channels and an exponential decrease in macroscopic conductance over time. The strong voltage dependence of multichannel steady state conductance, the single channel conductance, the rate of opening of channels at positive voltages and closing them at negative voltages, as well as the observed abrupt increase of single channel conductance after voltage sign reversal suggest that the change of the transmembrane field induces a significant rearrangement of syringomycin E channels, including a change in the spacing of charged groups that function as voltage sensors. The conductance induced by syringomycin E increased with the sixth power of syringomycin E concentration suggesting that at least six monomers are required for channel formation. Received: 3 April 1995/Revised: 24 August 1995     

Single acetylcholine-activated channel currents in developing muscle cells

The properties of single acetylcholine-activated ion channels in developing rat myoblasts and myotubes in tissue culture have been investigated using the gigaohm seal patch clamp technique. Two classes of ACh-activated channels were identified. The major class of channels (accounting for >95% of all channel openings) has a conductance of 35 pS and a mean open time of 15 msec (at room temperature and ?80 mV). The minor class of channels has a larger conductance (55 pS) and a briefer mean open time (2–3 msec). Functional ACh-activated channels are present in undifferentiated mononucleated myoblasts 1–2 days in culture, although the channel density on such cells is low. Over the next week in culture, as the myoblasts fuse to form multinucleate myotubes, there is a marked increase in channel density and an increase in the proportion of large conductance channels. No significant change, however, occurs in channel conductance or open time (within a given class of channels) during this period. At high concentrations of ACh, channels desensitize and channel openings occur in groups, similar to what has been previously described in adult muscle. The rate of channel opening within a group of openings increases with increasing agonist concentration while mean open time is independent of agonist concentration, as expected from simple models of drug action. During a group of openings, the channel is open for half the time (i.e., channel opening rate is equal to channel closing rate) at a concentration of approximately 6 μm ACh.     

Nonselective ionic channels inAplysia neurones

Summary Single-channel recordings from outside-out patches ofAplysia neurones in K-free solutions revealed the presence in most membrane patches of ionic channels showing surprising selectivity properties, as deduced from reversal potential measurements. After complete substitution of external NaCl by mannitol (in the presence of internal CsCl), these channels are more permeable to Cl than to Cs, but are also slightly permeable to Cs:P _Cl/P _Cs=4. Furthermore, in the presence of external NaCl, their ability to discriminate cations from anions seems lower than in external mannitol. Substitutions of external Cl by various anions showed that the channels are more permeable to NO₃ than to Cl, and that they are appreciably permeable to isethionate, SO₄ and methanesulfonate. Their elementary conductance is about 100 pS in 600mm symmetrical Cl. However, different conductance states (usually 2 or 3) can often be detected in the same membrane patch. By using voltage ramps, we established theI–V curves corresponding to each of these states and found small but significant differences between the reversal potentials of each state.     

Conductance increases produced by bath application of cholinergic agonists to Electrophorus electroplaques

When solutions containing agonists are applied to the innervated face of an Electrophorus electroplaque, the membrane's conductance increases. The agonist-induced conductance is increased at more negative membrane potentials. The "instantaneous" current-voltage curve for agonist-induced currents is linear and shows a reversal potential near zero mV; chord conductances, calculated on the basis of this reversal potential, change epsilon-fold for every 62-mV change in potential when the conductance is small. Conductance depends non-linearly on small agonist concentrations; at all potentials, the dose-response curve has a Hill coefficient of 1.45 for decamethonium (Deca) and 1.90 for carbamylcholine (Carb). With agonist concentrations greater than 10(-4) M Carb or 10(-%) M Deca, the conductance rises to a peak 0.5-1.5 min after introduction of agonist, then declines with time; this effect resembles the "desensitization" reported for myoneural junctions. Elapid alpha-toxin, tubocurarine, and desensitization reduce the conductance without changing the effects of potential; the apparent dissociation constant for tubocurarine is 2 X 10(-7) M. By contrast, procaine effects a greater fractional inhibition of the conductance at high negative potentials.     

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.     

Slowing of sodium channel opening kinetics in squid axon by extracellular zinc

The interaction of Zn ion on Na channels was studied in squid giant axons. At a concentration of 30 mM Zn2+ slows opening kinetics of Na channels with almost no alteration of closing kinetics. The effects of Zn2+ can be expressed as a "shift" of the gating parameters along the voltage axis, i.e., the amount of additional depolarization required to overcome the Zn2+ effect. In these terms the mean shifts caused by 30 mM Zn2+ were +29.5 mV for Na channel opening (on) kinetics (t1/2 on), +2 mV for closing (off) kinetics (tau off), and +8.4 mV for the gNa-V curve. Zn2+ does not change the shape of the instantaneous I-V curve for inward current, but reduces it in amplitude by a factor of or approximately 0.67. Outward current is unaffected. Effects of Zn2+ on gating current (measured in the absence of TTX) closely parallel its actions on gNa. On gating current kinetics are shifted by +27.5 mV, off kinetics by +6 mV, and the Q-V distribution by +6.5 mV. Kinetic modeling shows that Zn2+ slows the forward rate constants in activation without affecting backward rate constants. More than one of the several steps in activation must be affected. The results are not compatible with the usual simple theory of uniform fixed surface charge. They suggest instead that Zn2+ is attracted by a negatively charged element of the gating apparatus that is present at the outer membrane surface at rest, and migrates inward on activation.