Two classes of gating current from L-type Ca channels in guinea pig ventricular myocytes.

Intramembrane charge movement was recorded in guinea pig ventricular myocytes at 19-22 degrees C using the whole-cell patch clamp technique. From a holding potential of -110 mV, the dependence of intramembrane charge moved on test voltage (Q(V)) followed the sum of two Boltzmann components. One component had a transition voltage (V) of -48 mV and a total charge (Qmax) of congruent to 3 nC/microF. The other had a V of -18 mV and a Qmax of 11 nC/microF. Ba2+ currents through Ca channels began to activate at -45 mV and peaked at congruent to -15 mV. Na+ current peaked at -35 to -30 mV. Availability of charge (in pulses from -70 to +10 mV) depended on the voltage of conditioning depolarizations as two Boltzmann terms plus a constant. One term had a V of -88 mV and a Qmax of 2.5 nC/microF; the other had a V of -29 mV and a Qmax of 6.3 nC/microF. From the Q(V) dependence, the voltage dependence of the ionic currents, and the voltage dependence of the availability of charge, the low voltage term of Q(V) and availability was identified as Na gating charge, at a total of 3.5 nC/microF. The remainder, 11 nC/microF, was attributed to Ca channels. After pulses to -40 mV and above, the OFF charge movement had a slow exponentially decaying component. Its time constant had a bell-shaped dependence on OFF voltage peaking at 11 ms near -100 mV. Conditioning depolarizations above -40 mV increased the slow component exponentially with the conditioning duration (tau approximately equal to 480 ms). Its magnitude was reduced as the separation between conditioning and test pulses increased (tau approximately equal to 160 ms). The voltage distribution of the slow component of charge was measured after long (5 s) depolarizations. Its V was -100 mV, a shift of -80 mV from the value in normally polarized cells. This voltage was the same at which the time constant of the slow component peaked. Qmax and the steepness of the voltage distribution were unchanged by depolarization. This indicates that the same molecules that produce the charge movement in normally polarized cells also produce the slow component in depolarized cells. 100 microns D600 increased by 77% the slow charge movement after a 500-ms conditioning pulse. These results demonstrate two classes of charge movement associated with L-type Ca channels, with kinetics and voltage dependence similar to charge 1 and charge 2 of skeletal muscle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sodium and gating current time shifts resulting from changes in initial conditions

The sodium and gating currents of the squid giant axon elicited by a depolarizing pulse are delayed, with little change in shape, as a result of a hyperpolarizing prepulse. The delays are almost completely saturated, at approximately 45 microseconds, for prepulses to -140 mV. At 8 degrees C they develop with time constants of between 60 and 180 microseconds for prepulses in the -130- to -150-mV range. There is a correlation between the extra charge moved during the gating current and the increase in the time delay of the sodium current as the magnitude of the hyperpolarizing prepulse is increased. These results strengthen the conclusion that the gating current is indeed closely associated with the process of sodium channel opening and provide information concerning the kinetics of the early steps, which are hidden in ionic current measurements. The main features of the gating and sodium current time shifts and the correlation between charge movement and time shifts are duplicated by a sequential six-state model for sodium activation.     

External barium influences the gating charge movement of Shaker potassium channels.

External Ba2+ speeds the OFF gating currents (IgOFF) of Shaker K+ channels but only upon repolarization from potentials that are expected to open the channel pore. To study this effect we used a nonconducting and noninactivating mutant of the Shaker K+ channel, ShH4-IR (W434F). External Ba2+ slightly decreases the quantity of ON gating charge (QON) upon depolarization to potentials near -30 mV but has little effect on the quantity of charge upon stepping to more hyperpolarized or depolarized potentials. More strikingly, Ba2+ significantly increases the decay rate of IgOFF upon repolarization to -90 mV from potentials positive to approximately -55 mV. For Ba2+ to have this effect, the depolarizing command must be maintained for a duration that is dependent on the depolarizing potential (> 4 ms at -30 mV and > 1 ms at 0 mV). The actions of Ba2+ on the gating current are dose-dependent (EC50 approximately 0.2 mM) and are not produced by either Ca2+ or Mg2+ (2 mM). The results suggest that Ba2+ binds to a specific site on the Shaker K+ channel that destabilizes the open conformation and thus facilitates the return of gating charge upon repolarization.     

Gating of Shaker K+ channels: II. The components of gating currents and a model of channel activation.

Steady-state and kinetic properties of gating currents of the Shaker K+ channels were studied in channels expressed in Xenopus oocytes and recorded with the cut-open oocyte voltage clamp. The charge versus potential (Q-V) curve reveals at least two components of charge, the first moving in the hyperpolarized region (V1/2 = -63 mV) and the second, with a larger apparent valence, moving in the more depolarized region (V1/2 = -44 mV). The kinetic analysis of gating currents revealed also two exponential decaying components that corresponded in their voltage dependence with the charge components described in the steady-state. The first component was found to correlate with the effects of prepulses that produce the Cole-Moore shift of the ionic and gating currents and seems to be occurring completely within closed conformations of the channel. The second component seems to be related to the events occurring between the closed states just preceding, but not including, the transition to the open state. The ON and OFF gating currents exhibit a pronounced rising phase at potentials at which the second component becomes important, and this region corresponds to the potential range where the channel opens. The results could not be explained with simple parallel models, but the data can be fitted to a sequential model that could be related to a first rearrangement of the putative four subunits in cooperative fashion, followed by a concerted charge movement that leads to the open channel. The first series of charge movements are produced by transitions between several closed states carrying less than two electronic charges per step, while a step carrying about 3.5 electronic charges can explain the second component. This step is followed by the transition to the open state carrying less than 0.5 electronic charges. This model is able to reproduce all the kinetic and steady-state properties of the gating currents and predicts many of the properties of the ionic currents.     

The steady-state distribution of gating charge in crayfish giant axons.

Progressive shifts of holding potential (Vh) in crayfish giant axons, from -140 to -70 mV, reduce gating currents seen in depolarizing steps (to 0 mV test potential) while proportionately increasing gating currents in hyperpolarizing steps (to -240 mV). The resulting sigmoid equilibrium charge distribution (Q-Vh curve) shows an effective valence of 1.9e and a midpoint of -100 mV. By contrast, Q-V curves obtained using hyperpolarizing and/or depolarizing steps from a single holding potential, change their "shape" depending on the chosen holding potential. For holding potentials at the negative end of the Q-Vh distribution (e.g., -140 mV), negligible charge moves in hyperpolarizing pulses and the Q-V curve can be characterized entirely from depolarizing voltage steps. The slope of the resulting simple sigmoid Q-V curve also indicates an effective valence of 1.9e. When the axon is held at less negative potentials significant charge moves in hyperpolarizing voltage steps. The component of the Q-V curve collected using hyperpolarizing pulses shows a significantly reduced slope (approximately 0.75e) by comparison with the 1.9e slope found using depolarizing pulses or from the Q-Vh curve. As holding potential is shifted in the depolarizing direction along the Q-Vh curve, an increasing fraction of total charge movement must be assessed in hyperpolarizing voltage steps. Thus charge moving in the low slope component of the Q-V curve increases as holding potential is depolarized, while charge moving with high apparent valence decreases proportionately. Additional results, together with simulations based on a simple kinetic model, suggest that the reduced apparent valence of the low slope component of the Q-V curve results from gating charge immobilization occurring at holding potential. Immobilization selectively retards that fraction of total charge moving in hyperpolarizing pulses. Misleading conclusions, as to the number and valence of the gating particles, may therefore be derived from Q-V curves obtained by other than depolarizing pulses from negative saturated holding potentials.     

Nonlinear charge movement in mammalian cardiac ventricular cells. Components from Na and Ca channel gating [published erratum appears in J Gen Physiol 1989 Aug;94(2):401]

Intramembrane charge movement was recorded in rat and rabbit ventricular cells using the whole-cell voltage clamp technique. Na and K currents were eliminated by using tetraethylammonium as the main cation internally and externally, and Ca channel current was blocked by Cd and La. With steps in the range of -110 to -150 used to define linear capacitance, extra charge moves during steps positive to approximately -70 mV. With holding potentials near -100 mV, the extra charge moving outward on depolarization (ON charge) is roughly equal to the extra charge moving inward on repolarization (OFF charge) after 50-100 ms. Both ON and OFF charge saturate above approximately +20 mV; saturating charge movement is approximately 1,100 fC (approximately 11 nC/muF of linear capacitance). When the holding potential is depolarized to -50 mV, ON charge is reduced by approximately 40%, with little change in OFF charge. The reduction of ON charge by holding potential in this range matches inactivation of Na current measured in the same cells, suggesting that this component might arise from Na channel gating. The ON charge remaining at a holding potential of -50 mV has properties expected of Ca channel gating current: it is greatly reduced by application of 10 muM D600 when accompanied by long depolarizations and it is reduced at more positive holding potentials with a voltage dependence similar to that of Ca channel inactivation. However, the D600-sensitive charge movement is much larger than the Ca channel gating current that would be expected if the movement of channel gating charge were always accompanied by complete opening of the channel.     

Gating currents in Shaker K+ channels. Implications for activation and inactivation models.

We have studied ionic and gating currents in mutant and wild-type Shaker K+ channels to investigate the mechanisms of channel activation and the relationship between the voltage sensor of the channel and its inactivation particle. The turn on of the gating current shows a rising phase, indicating that the hypothetical identical activation subunits are not independent. Hyperpolarizing prepulses indicate that most of the voltage-dependence occurs in the transitions between closed states. The open-to-closed transition is voltage independent, as suggested by the presence of a rising phase in the off gating currents. In Shaker channels showing fast inactivation, the off gating charge is partially immobilized as a result of depolarizing pulses that elicit inactivation. In mutant channels lacking inactivation, the charge is recovered quickly at the end of the pulse. Internal TEA mimics the inactivation particle in its behavior but the charge immobilization is established faster and is complete. We conclude that the activation mechanism cannot be due to the movement of identical independent gating subunits, each undergoing first order transitions, and that the inactivation particle is responsible for charge immobilization in this channel.     

Gating currents in th intact crayfish giant axon.

Both single-sweep and signal-averaged asymmetry current are measured from intact crayfish axons after ionic currents are blocked with tetrodotoxin and 4-aminopyridine. The ON asymmetry charge saturates at about 0 mV and no ON charge movement is detectable at voltages negative to -140 mV. The areas of ON and OFF asymmetry charge are equal for short depolarizations but the ratio QOFF/QON decreases for longer depolarizing pulses. Sodium and asymmetry current magnitudes can be changed in parallel by lowering the hold potential or by imposing conditioning prepulses. Our results are consistent with the concept that asymmetry current in generated by movement of trapped charge in association with Na channel gating.     

Neutralization of gating charges in domain II of the sodium channel alpha subunit enhances voltage-sensor trapping by a beta-scorpion toxin

beta-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3-S4 loop at the extracellular end of the S4 voltage sensor in domain II of the alpha subunit. Here, we probe the role of gating charges in the IIS4 segment in beta-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances beta-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the beta-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from -80 to -140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor-trapping model in which the beta-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances toxin-induced channel activation.     

Cloning and expression of the human T-type channel Ca(v)3.3: insights into prepulse facilitation

The full-length human Ca(v)3.3 (alpha(1I)) T-type channel was cloned, and found to be longer than previously reported. Comparison of the cDNA sequence to the human genomic sequence indicates the presence of an additional 4-kb exon that adds 214 amino acids to the carboxyl terminus and encodes the 3' untranslated region. The electrophysiological properties of the full-length channel were studied after transient transfection into 293 human embryonic kidney cells using 5 mM Ca(2+) as charge carrier. From a holding potential of -100 mV, step depolarizations elicited inward currents with an apparent threshold of -70 mV, a peak of -30 mV, and reversed at +40 mV. The kinetics of channel activation, inactivation, deactivation, and recovery from inactivation were very similar to those reported previously for rat Ca(v)3.3. Similar voltage-dependent gating and kinetics were found for truncated versions of human Ca(v)3.3, which lack either 118 or 288 of the 490 amino acids that compose the carboxyl terminus. A major difference between these constructs was that the full-length isoform generated twofold more current. These results suggest that sequences in the distal portion of Ca(v)3.3 play a role in channel expression. Studies on the voltage-dependence of activation revealed that a fraction of channels did not gate as low voltage-activated channels, requiring stronger depolarizations to open. A strong depolarizing prepulse (+100 mV, 200 ms) increased the fraction of channels that gated at low voltages. In contrast, human Ca(v)3.3 isoforms with shorter carboxyl termini were less affected by a prepulse. Therefore, Ca(v)3.3 is similar to high voltage-activated Ca(2+) channels in that depolarizing prepulses can regulate their activity, and their carboxy termini play a role in modulating channel activity.     

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.     

Fast activation of dihydropyridine-sensitive calcium channels of skeletal muscle. Multiple pathways of channel gating

Dihydropyridine (DHP) receptors of the transverse tubule membrane play two roles in excitation-contraction coupling in skeletal muscle: (a) they function as the voltage sensor which undergoes fast transition to control release of calcium from sarcoplasmic reticulum, and (b) they provide the conducting unit of a slowly activating L-type calcium channel. To understand this dual function of the DHP receptor, we studied the effect of depolarizing conditioning pulse on the activation kinetics of the skeletal muscle DHP-sensitive calcium channels reconstituted into lipid bilayer membranes. Activation of the incorporated calcium channel was imposed by depolarizing test pulses from a holding potential of -80 mV. The gating kinetics of the channel was studied with ensemble averages of repeated episodes. Based on a first latency analysis, two distinct classes of channel openings occurred after depolarization: most had delayed latencies, distributed with a mode of 70 ms (slow gating); a small number of openings had short first latencies, < 12 ms (fast gating). A depolarizing conditioning pulse to +20 mV placed 200 ms before the test pulse (-10 mV), led to a significant increase in the activation rate of the ensemble averaged-current; the time constant of activation went from tau m = 110 ms (reference) to tau m = 45 ms after conditioning. This enhanced activation by the conditioning pulse was due to the increase in frequency of fast open events, which was a steep function of the intermediate voltage and the interval between the conditioning pulse and the test pulse. Additional analysis demonstrated that fast gating is the property of the same individual channels that normally gate slowly and that the channels adopt this property after a sojourn in the open state. The rapid secondary activation seen after depolarizing prepulses is not compatible with a linear activation model for the calcium channel, but is highly consistent with a cyclical model. A six- state cyclical model is proposed for the DHP-sensitive Ca channel, which pictures the normal pathway of activation of the calcium channel as two voltage-dependent steps in sequence, plus a voltage-independent step which is rate limiting. The model reproduced well the fast and slow gating models of the calcium channel, and the effects of conditioning pulses. It is possible that the voltage-sensitive gating transitions of the DHP receptor, which occur early in the calcium channel activation sequence, could underlie the role of the voltage sensor and yield the rapid excitation-contraction coupling in skeletal muscle, through either electrostatic or allosteric linkage to the ryanodine receptors/calcium release channels.     

Sequence of Gating Charge Movement and Pore Gating in hERG Activation and Deactivation Pathways

K_V11.1 voltage-gated K⁺ channels are noted for unusually slow activation, fast inactivation, and slow deactivation kinetics, which tune channel activity to provide vital repolarizing current during later stages of the cardiac action potential. The bulk of charge movement in human ether-a-go-go-related gene (hERG) is slow, as is return of charge upon repolarization, suggesting that the rates of hERG channel opening and, critically, that of deactivation might be determined by slow voltage sensor movement, and also by a mode-shift after activation. To test these ideas, we compared the kinetics and voltage dependence of ionic activation and deactivation with gating charge movement. At 0 mV, gating charge moved ∼threefold faster than ionic current, which suggests the presence of additional slow transitions downstream of charge movement in the physiological activation pathway. A significant voltage sensor mode-shift was apparent by 24 ms at +60 mV in gating currents, and return of charge closely tracked pore closure after pulses of 100 and 300 ms duration. A deletion of the N-terminus PAS domain, mutation R4AR5A or the LQT2-causing mutation R56Q gave faster-deactivating channels that displayed an attenuated mode-shift of charge. This indicates that charge movement is perturbed by N- and C-terminus interactions, and that these domain interactions stabilize the open state and limit the rate of charge return. We conclude that slow on-gating charge movement can only partly account for slow hERG ionic activation, and that the rate of pore closure has a limiting role in the slow return of gating charges.     

Effects of the Enantiomers of BayK 8644 on the Charge Movement of L-type Ca Channels in Guinea-pig Ventricular Myocytes

The effects of the agonist enantiomer S(-)Bay K 8644 on gating charge of L-type Ca channels were studied in single ventricular myocytes. From a holding potential (Vh) of -40 mV, saturating (250 nm) S(-)Bay K shifted the half-distribution voltage of the activation charge (Q1) vs. V curve -7.5 +/- 0.8 mV, almost identical to the shift produced in the Ba conductance vs. V curve (-7.7 +/- 2 mV). The maximum Q1 was reduced by 1.7 +/- 0.2 nC/microF, whereas Q2 (charge moved in inactivated channels) was increased in a similar amount (1.4 +/- 0.4 nC/microF). The steady-state availability curves for Q1, Q2, and Ba current showed almost identical negative shifts of -14.8 +/- 1.7 mV, -18.6 +/- 5.8 mV, and -15.2 +/- 2.7 mV, respectively. The effects of the antagonist enantiomer R(+)BayK 8644 were also studied, the Q1 vs. V curve was not significantly shifted, but Q1max (Vh = -40 mV) was reduced and the Q1 availability curve shifted by -24.6 +/- 1.2 mV. We concluded that: a) the left shift in the Q1 vs. V activation curve produced by S(-)BayK is a purely agonistic effect; b) S(-)BayK induced a significantly larger negative shift in the availability curve than in the Q1 vs. V relation, consistent with a direct promotion of inactivation; c) as expected for a more potent antagonist, R(+)Bay K induced a significantly larger negative shift in the availability curve than did S(-)Bay K.     

Shaker potassium channel gating. II: Transitions in the activation pathway

Voltage-dependent gating behavior of Shaker potassium channels without N-type inactivation (ShB delta 6-46) expressed in Xenopus oocytes was studied. The voltage dependence of the steady-state open probability indicated that the activation process involves the movement of the equivalent of 12-16 electronic charges across the membrane. The sigmoidal kinetics of the activation process, which is maintained at depolarized voltages up to at least +100 mV indicate the presence of at least five sequential conformational changes before opening. The voltage dependence of the gating charge movement suggested that each elementary transition involves 3.5 electronic charges. The voltage dependence of the forward opening rate, as estimated by the single- channel first latency distribution, the final phase of the macroscopic ionic current activation, the ionic current reactivation and the ON gating current time course, showed movement of the equivalent of 0.3 to 0.5 electronic charges were associated with a large number of the activation transitions. The equivalent charge movement of 1.1 electronic charges was associated with the closing conformational change. The results were generally consistent with models involving a number of independent and identical transitions with a major exception that the first closing transition is slower than expected as indicated by tail current and OFF gating charge measurements.     

Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+).

Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.     

Distribution and kinetics of membrane dielectric polarization. 1. Long- term inactivation of gating currents

Gating currents were measured by subtracting the linear component of the capacitative current recorded at very positive or very negative potentials. When the membrane is depolarized for a few minutes, repolarized to the usual holding potential (HP) of --70 mV for 1 ms, and then pulsed to 0 mV, the charge transferred in 2--4 ms is approximately 50% of that which was transferred during the same pulse holding at --70 mV. This charge decrease, called slow inactivation of the gating current, was found to be consistent with a shift of the charge vs. potential (Q-V) curve to more hyperpolarized potentials. When the HP is 0 mV, the total charge available to move is the same as the total charge available when the HP is --70 mV. The time constants of the fast component of the ON gating current are smaller at depolarized holding potentials than at --70 mV. When the HP is --70 mV and a prepulse of 50 ms duration is given to 0 mV, the Q-V curve is also shifted to more hyperpolarized potentials (charge immobilization), but the effect is not as pronounced as the one obtained by holding at 0 mV. When the HP is 0 mV, a prepulse to --70 mV for 50 ms partially shifts back the Q-V curve, indicating that fast inactivation of the gating charge may be recovered in the presence of slow inactivation. A physical model consisting of a gating particle that interacts with a fast inactivating particle, and a slow inactivating particle, reproduces most of the experimental results.     

Voltage dependence of intramembrane charge movement and conductance activation of batrachotoxin-modified sodium channels in frog node of Ranvier

Sodium current and sodium channel intramembrane gating charge movement (Q) were monitored in voltage-clamped frog node of Ranvier after modification of all sodium channels by batrachotoxin (BTX). BTX caused an approximately threefold increase in steepness of the Q vs. voltage relationship and a 50-mV negative shift in its midpoint. The maximum amount of intramembrane charge was virtually identical before and after BTX treatment. BTX treatment eliminated the charge immobilization observed in untreated nodes after relatively long depolarizing pulses and slowed the rate of OFF charge movement after a pulse. After BTX treatment, the voltage dependence of charge movement was the same as the steady-state voltage dependence of sodium conductance activation. The observations are consistent with the hypothesis that BTX induces an aggregation of the charged gating particles associated with each channel and causes them to move as a unit having approximately three times the average valence of the individual particles. Movement of this single aggregated unit would open the BTX-modified sodium channel.     

Molecular action of lidocaine on the voltage sensors of sodium channels

Block of sodium ionic current by lidocaine is associated with alteration of the gating charge-voltage (Q-V) relationship characterized by a 38% reduction in maximal gating charge (Q(max)) and by the appearance of additional gating charge at negative test potentials. We investigated the molecular basis of the lidocaine-induced reduction in cardiac Na channel-gating charge by sequentially neutralizing basic residues in each of the voltage sensors (S4 segments) in the four domains of the human heart Na channel (hH1a). By determining the relative reduction in the Q(max) of each mutant channel modified by lidocaine we identified those S4 segments that contributed to a reduction in gating charge. No interaction of lidocaine was found with the voltage sensors in domains I or II. The largest inhibition of charge movement was found for the S4 of domain III consistent with lidocaine completely inhibiting its movement. Protection experiments with intracellular MTSET (a charged sulfhydryl reagent) in a Na channel with the fourth outermost arginine in the S4 of domain III mutated to a cysteine demonstrated that lidocaine stabilized the S4 in domain III in a depolarized configuration. Lidocaine also partially inhibited movement of the S4 in domain IV, but lidocaine's most dramatic effect was to alter the voltage-dependent charge movement of the S4 in domain IV such that it accounted for the appearance of additional gating charge at potentials near -100 mV. These findings suggest that lidocaine's actions on Na channel gating charge result from allosteric coupling of the binding site(s) of lidocaine to the voltage sensors formed by the S4 segments in domains III and IV.     

Properties of L-type calcium channel gating current in isolated guinea pig ventricular myocytes.

Nonlinear capacitative current (charge movement) was compared to the Ca current (ICa) in single guinea pig ventricular myocytes. It was concluded that the charge movement seen with depolarizing test steps from -50 mV is dominated by L-type Ca channel gating current, because of the following observations. (a) Ca channel inactivation and the immobilization of the gating current had similar voltage and time dependencies. The degree of channel inactivation was directly proportional to the amount of charge immobilization, unlike what has been reported for Na channels. (b) The degree of Ca channel activation was closely correlated with the amount of charge moved at all test potentials between -40 and +60 mV. (c) D600 was found to reduce the gating current in a voltage- and use-dependent manner. D600 was also found to induce "extra" charge movement at negative potentials. (d) Nitrendipine reduced the gating current in a voltage-dependent manner (KD = 200 nM at -40 mV). However, nitrendipine did not increase charge movement at negative test potentials. Although contamination of the Ca channel gating current from other sources cannot be fully excluded, it was not evident in the data and would appear to be small. However, it was noted that the amount of Ca channel gating charge was quite large compared with the magnitude of the Ca current. Indeed, the gating current was found to be a significant contaminant (19 +/- 7%) of the Ca tail currents in these cells. In addition, it was found that Ca channel rundown did not diminish the gating current. These results suggest that Ca channels can be "inactivated" by means that do not affect the voltage sensor.