Fast activation of cardiac Ca++ channel gating charge by the dihydropyridine agonist, BAY K 8644.

Nonlinear charge movement (gating current) was studied by the whole-cell patch clamp method using cultured 17-d-old embryonic chick heart cells. Na+ and Ca++ currents were blocked by the addition of 10 microM TTX and 3 mM CoCl2; Cs+ replaced K+ both intra- and extracellularly. Linear capacitive and leakage currents were subtracted by a P/5 procedure. The small size (15 microns in diameter) and the lack of an organized internal membrane system in these myocytes permits a rapid voltage clamp of the surface membrane. Ca++ channel gating currents were activated positive to -60 mV; the rising phase was not distorted due to the system response time. The addition of BAY K 8644 (10(-6) M) caused a shortening of the time to peak of the Ca++ gating current, and a negative shift in the isochronal Qon vs. Vm curve. Qmax was unchanged by BAY K 8644. The voltage-dependent shift produced by BAY K 8644 is similar to that produced by isoproterenol (Josephson, I.R., and N. Sperelakis. 1990. Biophys. J. 57:305a. [Abstr.]). The results suggest that the binding of BAY K 8466 to one or more of the Ca++ channel subunits alters the kinetics and shifts the voltage dependence of gating. These changes in the gating currents can explain the parallel changes in the macroscopic Ca++ currents.     

Single-channel, macroscopic, and gating currents from sodium channels in the squid giant axon.

Single-channel, macroscopic ionic, and macroscopic gating currents were recorded from the voltage-dependent sodium channel using patch-clamp techniques on the cut-open squid giant axon. To obtain a complete set of physiological measurements of sodium channel gating under identical conditions, and to facilitate comparison with previous work, comparison was made between currents recorded in the absence of extracellular divalent cations and in the presence of physiological concentrations of extracellular Ca2+ (10 mM) and Mg2+ (50 mM). The single-channel currents were well resolved when divalent cations were not included in the extracellular solution, but were decreased in amplitude in the presence of Ca2+ and Mg2+ ions. The instantaneous current-voltage relationship obtained from macroscopic tail current measurements similarly was depressed by divalents, and showed a negative slope-conductance region for inward current at negative potentials. Voltage dependent parameters of channel gating were shifted 9-13 mV towards depolarized potentials by external divalent cations, including the peak fraction of channels open versus voltage, the time constant of tail current decline, the prepulse inactivation versus voltage relationship, and the charge-voltage relationship for gating currents. The effects of divalent cations are consistent with open channel block by Ca2+ and Mg2+ together with divalent screening of membrane charges.     

Permeation and gating in CaV3.1 (alpha1G) T-type calcium channels effects of Ca2+, Ba2+, Mg2+, and Na+

We examined the concentration dependence of currents through Ca(V)3.1 T-type calcium channels, varying Ca(2+) and Ba(2+) over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current-voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by P(Ca)/P(Na) = 87 and P(Ca)/P(Ba) = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent K(d) values were similar for Ca(2+) and Ba(2+), both for block of currents carried by Na(+) (3 muM for Ca(2+) vs. 4 muM for Ba(2+), at -30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca(2+) vs. 2.5 mM for Ba(2+); nearly voltage independent). Block by 3-10 muM Ca(2+) was time dependent, described by bimolecular kinetics with binding at approximately 3 x 10(8) M(-1)s(-1) and voltage-dependent exit. Ca(2+)(o), Ba(2+)(o), and Mg(2+)(o) also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e(-) per 98 A(2) from Gouy-Chapman theory. Additionally, inward currents inactivated approximately 35% faster in Ba(2+)(o) (vs. Ca(2+)(o) or Na(+)(o)). The accelerated inactivation in Ba(2+)(o) correlated with the transition from Na(+) to Ba(2+) permeation, suggesting that Ba(2+)(o) speeds inactivation by occupying the pore. We conclude that the selectivity of the "surface charge" among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca(2+) or Ba(2+).     

Altered sodium and gating current kinetics in frog skeletal muscle caused by low external pH

The effect of low pH on the kinetics of Na channel ionic and gating currents was studied in frog skeletal muscle fibers. Lowering external pH from 7.4 to 5.0 slows the time course of Na current consistent with about a +25-mV shift in the voltage dependence of activation and inactivation time constants. Similar shifts in voltage dependence adequately describe the effects of low pH on the tail current time constant (+23.3 mV) and the gating charge vs. voltage relationship (+22.1 mV). A significantly smaller shift of +13.3 mV described the effect of pH 5.0 solution on the voltage dependence of steady state inactivation. Changes in the time course of gating current at low pH were complex and could not be described as a shift in voltage dependence. tau g, the time constant that describes the time course of the major component of gating charge movement, was slowed in pH 5.0 solution by a factor of approximately 3.5 for potentials from -60 to +45 mV. We conclude that the effects of low pH on Na channel gating cannot be attributed simply to a change in surface potential. Therefore, although it may be appropriate to describe the effect of low pH on some Na channel kinetic properties as a "shift" in voltage dependence, it is not appropriate to interpret such shifts as a measure of changes in surface potential. The maximum gating charge elicited from a holding potential of -150 mV was little affected by low pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Permeation and gating properties of the L-type calcium channel in mouse pancreatic beta cells

Ba2+ currents through L-type Ca2+ channels were recorded from cell- attached patches on mouse pancreatic beta cells. In 10 mM Ba2+, single- channel currents were recorded at -70 mV, the beta cell resting membrane potential. This suggests that Ca2+ influx at negative membrane potentials may contribute to the resting intracellular Ca2+ concentration and thus to basal insulin release. Increasing external Ba2+ increased the single-channel current amplitude and shifted the current-voltage relation to more positive potentials. This voltage shift could be modeled by assuming that divalent cations both screen and bind to surface charges located at the channel mouth. The single- channel conductance was related to the bulk Ba2+ concentration by a Langmuir isotherm with a dissociation constant (Kd(gamma)) of 5.5 mM and a maximum single-channel conductance (gamma max) of 22 pS. A closer fit to the data was obtained when the barium concentration at the membrane surface was used (Kd(gamma) = 200 mM and gamma max = 47 pS), which suggests that saturation of the concentration-conductance curve may be due to saturation of the surface Ba2+ concentration. Increasing external Ba2+ also shifted the voltage dependence of ensemble currents to positive potentials, consistent with Ba2+ screening and binding to membrane surface charge associated with gating. Ensemble currents recorded with 10 mM Ca2+ activated at more positive potentials than in 10 mM Ba2+, suggesting that external Ca2+ binds more tightly to membrane surface charge associated with gating. The perforated-patch technique was used to record whole-cell currents flowing through L-type Ca2+ channels. Inward currents in 10 mM Ba2+ had a similar voltage dependence to those recorded at a physiological Ca2+ concentration (2.6 mM). BAY-K 8644 (1 microM) increased the amplitude of the ensemble and whole-cell currents but did not alter their voltage dependence. Our results suggest that the high divalent cation solutions usually used to record single L-type Ca2+ channel activity produce a positive shift in the voltage dependence of activation (approximately 32 mV in 100 mM Ba2+).     

Interaction between calcium ions and surface charge as it relates to calcium currents

Summary Calcium ions affect the gating of Ca currents. Surface charge is involved but to what extent is unknown. We have examined this, using isolated nerve cell bodies ofHelix aspersa and the combined microelectrode-suction pipette method for voltage-clamp and internal perfusion. We found that Ba and Sr currents produced by substitution of these ions for extracellular Ca ions are activated at less positive potentials than Ca currents. Mg ions do not permeate the Ca channel and changes in [Mg]₀ produce shifts in the activation-potential curves that are comparable to the effects of changes in [Ba]₀ or [Sr]₀. Inactivation of Ba currents also occurs at less positive potentials. Perfusion intracellularly with EGTA reduced inactivation of Ca currents as a function of potential, but did not shift the inactivation-potential curve. Hence, Ca current-dependent inactivation which is blocked by intracellular EGTA probably does not involve a similar change of intracellular surface potential. The voltage shifts of activation and inactivation produced by extracellular divalent cations used singly or in mixtures can be described by the Gouy-Chapman theory for the diffuse double layer with binding (Gilbert & Ehrenstein, 1969; McLaughlin, Szabo & Eisenman, 1971). From the surface potential values and the Boltzman distribution, we have computed surface concentrations that predict the following experimental observations: 1) saturation of current-concentration relationships when surface potential is changing maximally; 2) the increase in peak current when Ca ions are replaced by Sr or Ba ions; and 3) the greater inhibitory effect of Mg onI _Ba thanI _Ca. Theory indicates that surface charge cannot be screened completely even at 1m [Mg]₀ and thus that Ca channel properties must be evaluated in the light of surface charge effects. For example, after correction for surface charge effects the relative permeabilities of Ca, Ba and Sr ions are equivalent. In the presence of Co ions, however, Ca ions are more permeable than Ba ions suggesting a channel binding site may be involved.     

Voltage-dependent modulation of L-type calcium currents by intracellular magnesium in rat ventricular myocytes

Effects of changing cytosolic free Mg(2+) concentration on L-type Ca(2+) (I(Ca)) and Ba(2+) currents (I(Ba)) were investigated in rat ventricular myocytes voltage-clamped with pipettes containing 0.2 or 1.8mM [Mg(2+)] ([Mg(2+)](p)) buffered with 30mM citrate and 10mM ATP. Increasing [Mg(2+)](p) from 0.2 to 1.8mM reduced current amplitude and accelerated its decay under a variety of experimental conditions. To investigate the mechanism for these effects, steady-state and instantaneous current-voltage relationships were studied with two-pulse and tail current (I(T)) protocols, respectively. Increasing [Mg(2+)](p) shifted the V(M) for half inactivation by -20mV but dramatically decreased I(Ca) amplitude at all potentials tested, consistent with a change in gating kinetics that decreases channel availability. This conclusion was supported by analysis of I(T) amplitude, but these latter experiments also suggested that, in the millimolar concentration range, [Mg(2+)](p) might also inhibit permeation through open Ca(2+) channels at positive V(M).     

Human ether-à-go-go-related gene K+ channel gating probed with extracellular ca2+. Evidence for two distinct voltage sensors

Human ether-à-go-go-related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 +/- 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-à-go-go-related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery.     

Modification of sodium and potassium channel gating kinetics by ether and halothane

The effect of ether and halothane on the kinetics of sodium and potassium currents were investigated in the crayfish giant axon. Both general anesthetics produced a reversible, dose-dependent speeding up of sodium current inactivation at all membrane potentials, with no change in the phase of the currents. Double-pulse inactivation experiments with ether also showed faster inactivation, but the rate of recovery from inactivation at negative potentials was not affected. Ether shifted the midpoint of the steady-state fast inactivation curve in the hyperpolarizing direction and made the curve steeper. The activation of potassium currents was faster with ether present, with no change in the voltage dependence of steady-state potassium currents. Ether and halothane are known to perturb the structure of lipid bilayer membranes; the alterations in sodium and potassium channel gating kinetics are consistent with the hypothesis that the rates of the gating processes of the channels can be affected by the state of the lipids surrounding the channels, but a direct effect of ether and halothane on the protein part of the channels cannot be ruled out. Ether did not affect the capacitance of the axon membrane.     

Effects of external protons on single cardiac sodium channels from guinea pig ventricular myocytes.

The effects of external protons on single sodium channel currents recorded from cell-attached patches on guinea pig ventricular myocytes were investigated. Extracellular protons reduce single channel current amplitude in a dose-dependent manner, consistent with a simple rapid channel block model where protons bind to a site within the channel with an apparent pKH of 5.10. The reduction in single channel current amplitude by protons is voltage independent between -70 and -20 mV. Increasing external proton concentration also shifts channel gating parameters to more positive voltages, consistent with previous macroscopic results. Similar voltage shifts are seen in the steady-state inactivation (h infinity) curve, the time constant for macroscopic current inactivation (tau h), and the first latency function describing channel activation. As pHo decreases from 7.4 to 5.5 the midpoint of the h infinity curve shifts from -107.6 +/- 2.6 mV (mean +/- SD, n = 16) to -94.3 +/- 1.9 mV (n = 3, P less than 0.001). These effects on channel gating are consistent with a reduction in negative surface potential due to titration of negative external surface charge. The Gouy-Chapman-Stern surface charge model incorporating specific proton binding provides an excellent fit to the dose-response curve for the shift in the midpoint of the h infinity curve with protons, yielding an estimate for total negative surface charge density of -1e/490 A2 and a pKH for proton binding of 5.16. By reducing external surface Na+ concentration, titration of negative surface charge can also quantitatively account for the reduction in single Na+ channel current amplitude, although we cannot rule out a potential role for channel block. Thus, titration by protons of a single class of negatively charged sites may account for effects on both single channel current amplitude and gating.     

Modulation of voltage-dependent sodium and potassium currents by charged amphiphiles in cardiac ventricular myocytes. Effects via modification of surface potential

Modulation of voltage-dependent sodium and potassium currents by charged amphiphiles was investigated in cardiac ventricular myocytes using the patch-clamp technique. Negatively charged sodium dodecylsulfate (SDS) increased amplitude of INa, whereas positively charged dodecyltrimethylammonium (DDTMA) decreased INa. Furthermore, SDS shifted the steady-state activation and inactivation of INa in the negative direction, whereas DDTMA shifted the curves in the opposite direction. These shifts provided an explanation for the changes in current amplitude. Activation and inactivation kinetics of INa were accelerated by SDS but slowed by DDTMA. These changes in both steady- state gating and kinetics of INa are consistent with a decrease of the intramembrane field by SDS and an increase of the field by DDTMA due to an alteration of surface potential after their insertion into the outer monolayer of the sarcolemma. The effect of SDS on the steady-state inactivation of INa was concentration dependent and partially reversed by screening surface charges with increased extracellular [Ca2+]. These amphiphiles also altered the activation of the delayed rectifier K+ current (IK,del), producing a shift in the negative direction by SDS but in the positive direction by DDTMA. These results suggest that the insertion of charged amphiphiles into the cell membrane alters the behavior of voltage-dependent INa and IK,del by changing the surface charge density, and consequently the surface potential and implies, although indirectly, that the lipid surface charges are important to the voltage-dependent gating of these channels.     

Modulation of the conductance of unitary cardiac L-type Ca(2+) channels by conditioning voltage and divalent ions

The accompanying paper (Josephson, I. R., A. Guia, E. G. Lakatta, and M. D. Stern. 2002. Biophys. J. 83:2575-2586) examined the effects of conditioning prepulses on the kinetics of unitary L-type Ca(2+) channel currents using Ca(2+) and Ba(2+) ions to determine the ionic-dependence of gating mechanisms responsible for channel inactivation and facilitation. Here we demonstrate that in addition to alterations in gating kinetics, the conductance of single L-type Ca(2+) channels was also dependent on the prior conditioning voltage and permeant ions. All recordings were made in the absence of any Ca(2+) channel agonists. Strongly depolarizing prepulses produced an increased frequency of long-duration (mode 2) openings during the test voltage steps. Mode 2 openings also displayed >25% larger single channel current amplitude (at 0 mV) than briefer (but well-resolved) mode 1 openings. The conductance of mode 2 openings was 26 pS for 105 mM Ba(2+), 18 pS for 5 mM Ba(2+), and 6 pS for 5 mM Ca(2+) ions; these values were 70% greater than the conductance of Ca(2+) channel openings of all durations (mode 1 and mode 2). Thus, the prepulse-driven shift into mode 2 gating results in a longer-lived Ca(2+) channel conformation that, in addition, displays altered permeation properties. These results, and those in the accompanying paper, support the hypothesis that multiple aspects of single L-type Ca(2+) channel behavior (gating kinetics, modal transitions, and ion permeation) are interrelated and are modulated by the magnitude of the conditioning depolarization and the nature and concentration of the ions permeating the channel.     

Modulation of Kir1.1 inactivation by extracellular Ca and Mg

Kir1.1 inactivation, associated with transient internal acidification, is strongly dependent on external K, Ca, and Mg. Here, we show that in 1 mM K, a 15 min internal acidification (pH 6.3) followed by a 30 min recovery (pH 8.0) produced 84 ± 3% inactivation in 2 mM Ca but only 18 ± 4% inactivation in the absence of external Ca and Mg. In 100 mM external K, the same acidification protocol produced 29 ± 4% inactivation in 10 mM external Ca but no inactivation when extracellular Ca was reduced below 2 mM (with 0 Mg). However, chelation of external K with 15 mM of 18-Crown-6 (a crown ether) restored inactivation even in the absence of external divalents. External Ca was more effective than external Mg at producing inactivation, but Mg caused a greater degree of open channel block than Ca, making it unlikely that Kir1.1 inactivation arises from divalent block per se. Because the Ca sensitivity of inactivation persisted in 100 mM external K, it is also unlikely that Ca enhanced Kir1.1 inactivation by reducing the local K concentration at the outer mouth of the channel. The removal of four surface, negative side chains at E92, D97, E104, and E132 (Kir1.1b) increased the sensitivity of inactivation to external Ca (and Mg), whereas addition of a negative surface charge (N105E-Kir1.1b) decreased the sensitivity of inactivation to Ca and Mg. This result is consistent with the notion that negative surface charges stabilize external K in the selectivity filter or at the S₀-K binding site just outside the filter. Extracellular Ca and Mg probably potentiate the slow, K-dependent inactivation of Kir1.1 by decreasing the affinity of the channel for external K independently of divalent block. The removal of external Ca and Mg largely eliminated both Kir1.1 inactivation and the K-dependence of pH gating, thereby uncoupling the selectivity filter gate from the cytoplasmic-side bundle-crossing gate.     

Patch recordings from the electrocytes of electrophorus. Na channel gating currents

Gating currents were recorded at 11 degrees C in cell-attached and inside-out patches from the innervated membrane of Electrophorus main organ electrocytes. With pipette tip diameters of 3-8 microns, maximal charge measured in patches ranged from 0.74 to 7.19 fC. The general features of the gating currents are similar to those from the squid giant axon. The steady-state voltage dependence of the ON gating charge was characterized by an effective valence of 1.3 +/- 0.4 and a midpoint voltage of -56 +/- 7 mV. The charge vs. voltage relation lies approximately 30 mV negative to the channel open probability curve. The ratio of the time constants of the OFF gating current and the Na current was 2.3 at -120 mV and equal at -80 mV. Charge immobilization and Na current inactivation develop with comparable time courses and have very similar voltage dependences. Between 60 and 80% of the charge is temporarily immobilized by inactivation.     

Hysteresis in the voltage dependence of HCN channels: conversion between two modes affects pacemaker properties

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels are important for rhythmic activity in the brain and in the heart. In this study, using ionic and gating current measurements, we show that cloned spHCN channels undergo a hysteresis in their voltage dependence during normal gating. For example, both the gating charge versus voltage curve, Q(V), and the conductance versus voltage curve, G(V), are shifted by about +60 mV when measured from a hyperpolarized holding potential compared with a depolarized holding potential. In addition, the kinetics of the tail current and the activation current change in parallel to the voltage shifts of the Q(V) and G(V) curves. Mammalian HCN1 channels display similar effects in their ionic currents, suggesting that the mammalian HCN channels also undergo voltage hysteresis. We propose a model in which HCN channels transit between two modes. The voltage dependence in the two modes is shifted relative to each other, and the occupancy of the two modes depends on the previous activation of the channel. The shifts in the voltage dependence are fast (tau approximately 100 ms) and are not accompanied by any apparent inactivation. In HCN1 channels, the shift in voltage dependence is slower in a 100 mM K extracellular solution compared with a 1 mM K solution. Based on these findings, we suggest that molecular conformations similar to slow (C-type) inactivation of K channels underlie voltage hysteresis in HCN channels. The voltage hysteresis results in HCN channels displaying different voltage dependences during different phases in the pacemaker cycle. Computer simulations suggest that voltage hysteresis in HCN channels decreases the risk of arrhythmia in pacemaker cells.     

Protons and calcium alter gating of the hyperpolarization-activated cation current (I(h)) in rod photoreceptors

We investigated the effects of protons and calcium ions on the voltage-dependent gating of the hyperpolarization-activated, nonselective cation channel current, I(h), in rod photoreceptors. I(h) is a cesium-sensitive current responsible for the peak-plateau sag during the rod response to bright light. The voltage dependence of I(h) activation shifted about 5 mV per pH unit, with external acidification producing positive shifts and alkalinization producing negative shifts. Increasing external [Ca(2+)] from 3 to 20 mM resulted in a large (approximately 17 mV) positive shift in I(h) activation. External [Ca(2+)] (20 mM) blocked pH-induced shifts in activation. Cytoplasmic acidification produced by 25 mM sodium acetate led to a negative shift in inactivation (-9 mV) and internal alkalinization produced with 20 mM ammonium chloride resulted in a positive shift (+6 mV). Surface charge binding and screening theory (Gouy-Chapman-Stern) accounted for the observed shifts in I(h) activation, with the best fit achieved when protons and calcium ions were assumed to bind to distinct sites on the membrane. Since light induces changes in the retinal ionic environment, these results permit us to gauge the degree to which rod light responses could be modified via alterations in I(h) activation.     

Mechanism of effect of extracellular pH on L-type Ca(2+) channel currents in human mesenteric arterial cells

Extracellular pH (pH(o)) influences vasoconstriction partly by modulating Ca(2+) influx through voltage-gated Ca(2+) channels in the vasculature. The mechanism of this effect of pH(o) is, however, controversial. Using the whole cell voltage-clamp technique, we examined the influence of pH(o) on L-type Ca(2+) channel currents in isolated human mesenteric arterial myocytes. Acidification to pH 6.2 and alkalinization to 8.2 from 7.2 decreased by approximately 50% and increased by 25-30%, respectively, the peak amplitude of Ca(2+) and Ba(2+) currents (1.5 and 10 mM), with an apparent pK(a) of 6.8. Activation and inactivation of Ca(2+) and Ba(2+) currents were shifted toward positive membrane voltages during acidification and in the opposite direction during alkalinization. The relationship between the current amplitude and shifts in the gating parameters in solutions of different pH(o) conformed closely to that predicted by the Gouy-Chapman model, in which the divalent cation concentration at the outer surface of the membrane varies with the extent to which protons neutralize the membrane surface potential.     

Effects of permeant ion concentrations on the gating of L-type Ca2+ channels in hair cells

We determined the gating and permeation properties of single L-type Ca(2+) channels, using hair cells and varying concentrations (5-70 mM) of the charge carriers Ba(2+) and Ca(2+). The channels showed distinct gating modes with high- and low-open probability. The half-activation voltage (V(1/2)) shifted in the hyperpolarizing direction from high to low permeant ion concentrations consistent with charge screening effects. However, the differences in the slope of the voltage shifts (in VM(-1)) between Ca(2+) (0.23) and Ba(2+) (0.13), suggest that channel-ion interaction may also contribute to the gating of the channel. We examined the effect of mixtures of Ba(2+) and Ca(2+) on the activation curve. In 5 mM Ca(2+), the V(1/2) was, -26.4 +/- 2.0 mV compared to Ba(2+), -34.7 +/- 2.9 mV, as the charge carrier. However, addition of 1 mM Ba(2+) in 4 mM Ca(2+), a molar ratio, which yielded an anomalous-mole fraction effect, was sufficient to shift the V(1/2) to -34.7 +/- 1.5 mV. Although Ca(2+)-dependent inactivation of the L-type channels in hair cells can yield the present findings, we provide evidence that the anomalous gating of the channel may stem from the closed interaction between ion permeation and gating.     

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.     

Kinetic properties and inactivation of the gating currents of sodium channels in squid axon.

Associated with the opening and closing of the sodium channels of nerve membrane is a small component of capacitative current, the gating current. After termination of a depolarizing step the gating current and sodium current decay with similar time courses. Both currents decay more rapidly at relatively negative membrane voltages than at positive ones. The gating current that flows during a depolarizing step is diminished by a pre-pulse that inactivates the sodium permeability. A pre-pulse has no effect after inactivation has been destroyed by internal perfusion with the proteolytic enzyme pronase. Gating charge (considered as positive charge) moves outward during a positive voltage step, with voltage dependent kinetics. The time constant of the outward gating current is a maximum at about minus 10 mV, and has a smaller value at voltages either more positive or negative than this value.