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.     

Contraction threshold and the "hump" component of charge movement in frog skeletal muscle

The delayed component of intramembranous charge movement (hump, I gamma) was studied around the contraction threshold in cut skeletal muscle fibers of the frog (Rana esculenta) in a single Vaseline-gap voltage clamp. Charges (Q) were computed as 50-ms integrals of the ON (QON) and OFF (QOFF) of the asymmetric currents after subtracting a baseline. The hump appeared in parallel with an excess of QON over QOFF by approximately 2.5 nC/mu F. Caffeine (0.75 mM) not only shifted the contraction threshold but moved both the hump and the difference between the ON and OFF charges to more negative membrane potentials. When using 10-mV voltage steps on top of different prepulse levels, the delayed component, if present, was more readily observable. The voltage dependences of the ON and OFF charges measured with these pulses were clearly different: QON had a maximum at or slightly above the contraction threshold, while QOFF increased monotonically in the voltage range examined. Caffeine (0.75 mM) shifted this voltage dependence of QON toward more negative membrane potentials, while that of QOFF was hardly influenced. These results show that the delayed component of intramembranous charge movement either is much slower during the OFF than during the ON, or returns to the OFF position during the pulse. Tetracaine (25 microM) had similar effects on the charge movement currents, shifting the voltage dependence on the ON charge in parallel with the contraction threshold, but to more positive membrane potentials, and leaving QOFF essentially unchanged. The direct difference between the charge movement measured in the presence of caffeine and in control solution was either biphasic or resembled the component isolated by tetracaine, suggesting a common site of caffeine and tetracaine action. The results can be understood if the released Ca plays a direct role in the generation of the hump, as proposed in the first paper of this series (Csernoch et al. 1991. J. Gen. Physiol. 97:845-884).     

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

Sodium current and intramembrane gating charge movement (Q) were monitored in voltage-clamped frog node of Ranvier after modification of all sodium channels by batrachotoxin (BTX). Sodium current activation followed a single-exponential time course, provided a delay was interposed between the onset of the step ON depolarization and that of the current change. The delay decreased with increased ON depolarization and, for a constant ON depolarization, increased with prehyperpolarization. ON charge movement followed a single-exponential time course with time constants tau Q,ON slightly larger than tau Na, ON. For pulses between -70 and -50 mV, tau Q,ON/tau Na,ON = 1.14 +/- 0.08. The OFF charge movement and OFF sodium current tails after a depolarizing pulse followed single-exponential time courses, with tau Q, OFF larger than tau Na, OFF. tau Q,OFF/tau Na,OFF increased with OFF voltage from 1 near -100 mV to 2 near -160 mV. At a set OFF potential (-120 mV), both tau Q,OFF and tau Na,OFF increased with ON pulse duration. The delay in INa activation and the effect of ON pulse duration on tau Q,OFF and tau Na,OFF are inconsistent with a simple two-state, single-transition model for the gating of batrachotoxin-modified sodium channels.     

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)     

Gating currents from neuronal KV7.4 Channels: General features and correlation with the ionic conductance

The K_V7 (KCNQ) subfamily of voltage-gated K⁺ channels consists of five members (K_V7.1- K_V7.5) giving rise to non-inactivating, and slowly activating/deactivating currents mainly expressed in cardiac (K_V7.1) and neuronal (K_V7.2- K_V7.5) tissue. In the present study, using the cut-open oocyte voltage clamp, we studied the relation of the ionic currents from homomeric neuronal Kv7 channels (K_V7.2-K_V7.5) with the gating currents recorded after K⁺ conductance blockade from the same channels. Increasing the recording temperature from 18{degree sign}C to 28{degree sign}C accelerated activation/deactivation kinetics of the ionic currents in all homomeric K_V7 channels (activation Q10s at 0 mV were 3.8, 4.1, 8.3, and 2.8 for Kv7.2, Kv7.3, Kv7.4 and Kv7.5 channels, respectively), without large changes in currents voltage-dependence; moreover, at 28{degree sign}C, ionic currents carried by K_V7.4 channels also showed a significant increase in their maximal value. Gating currents were only resolved in K_V7.4 and K_V7.5 channels; the size of the ON gating charges at +40 mV was 1.34 ± 0.34 nC for K_V7.4, and 0.79 ± 0.20 nC for K_V7.5. At 28{degree sign}C, K_V7.4 gating currents had the following salient properties: 1) similar time integral of QON and QOFF, indicating no charge immobilization; 2) a left-shift in the V1/2 of the QON/V when compared to the G/V (≈ 50 mV in the presence of 2 mM extracellular Ba²⁺); 3) a QON decay faster than ionic current activation; and 4) a rising phase in the OFF gating charge after depolarizations larger than 0 mV. These observations suggest that, in K_V7.4 channels, VSD movement is followed by a slow and/or low bearing charge step linking to pore opening, a result which may help to clarify the molecular consequence of disease-causing mutations and drugs affecting channel gating.     

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 current kinetics in Myxicola giant axons. Order of the back transition rate constants.

Gating current, Ig, was recorded in Myxicola axons with series resistance compensation and higher time resolution than in previous studies. Ig at ON decays as two exponentials with time constants, tau ON-F and tau ON-S, very similar to squid values. No indication of an additional very fast relaxation was detected, but could be still unresolved. Ig at OFF also displays two exponentials, neither reflecting recovery from charge immobilization. Deactivation of the two I(ON) components may proceed with well-separated exponentials at -100 mV. INa tail currents at OFF also display two exponentials plus a third very slow relaxation of 5-9% of the total tail current. The very slow component is probably deactivation of a very small subpopulation of TTX sensitive channels. A -100 mV, means for INa tail component time constants (four axons) are 76 microseconds (range: 53-89 microseconds) and 344 microseconds (range: 312-387 microseconds), and for IOFF (six axons) 62 microseconds (range: 34-87 microseconds) and 291 microseconds (range: 204-456 microseconds) in reasonable agreement. INa ON activation time constant, tau A, is clearly slower than tau ON-F at all potentials. Except for the interval -30 to -15 mV, tau A is clearly faster than tau ON-S, and has a different dependency on potential. tau ON-S is several fold smaller than tau h. Computations with a closed2----closed1----open activation model indicated Na tail currents are consistent with a closed1----open rate constant greater than the closed2----closed1.     

A damped oscillation in the intramembranous charge movement and calcium release flux of frog skeletal muscle fibers

Asymmetric membrane currents and calcium transients were recorded simultaneously from cut segments of frog skeletal muscle fibers voltage clamped in a double Vaseline-gap chamber in the presence of high concentration of EGTA intracellularly. An inward phase of asymmetric currents following the hump component was observed in all fibers during the depolarization pulse to selected voltages (congruent to -45 mV). The average value of the peak inward current was 0.1 A/F (SEM = 0.01, n = 18), and the time at which it occurred was 34 ms (SEM = 1.8, n = 18). A second delayed outward phase of asymmetric current was observed after the inward phase, in those experiments in which hump component and inward phase were large. It peaked at more variable time (between 60 and 130 ms) with amplitude 0.02 A/F (SEM = 0.003, n = 11). The transmembrane voltage during a pulse, measured with a glass microelectrode, reached its steady value in less than 10 ms and showed no oscillations. The potential was steady at the time when the delayed component of asymmetric current occurred. ON and OFF charge transfers were equal for all pulse durations. The inward phase moved 1.4 nC/microF charge (SEM = 0.8, n = 6), or about one third of the final value of charge mobilized by these small pulses, and the second outward phase moved 0.7 nC/microF (SEM = 0.8, n = 6), bringing back about half of the charge moved during the inward phase. When repolarization intersected the peak of the inward phase, the OFF charge transfer was independent of the repolarization voltage in the range -60 to -90 mV. When both pre- and post-pulse voltages were changed between -120 mV and -60 mV, the equality of ON and OFF transfers of charge persisted, although they changed from 113 to 81% of their value at -90 mV. The three delayed phases in asymmetric current were also observed in experiments in which the extracellular solution contained Cd2+, La3+ and no Ca2+. Large increases in intracellular [Cl-] were imposed, and had no major effect on the delayed components of the asymmetric current. The Ca2+ transients measured optically and the calculated Ca2+ release fluxes had three phases whenever a visible outward phase followed the inward phase in the asymmetric current. Several interventions intended to interfere with Ca release, reduced or eliminated the three delayed phases of the asymmetric current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Slow charge movement in mammalian skeletal muscle

Voltage-dependent charge movements were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Contraction was abolished with hypertonic sucrose. The standard (ON-OFF) protocol for eliciting charge movements was to depolarize the fiber from -90 mV to a variable test potential (V) and then repolarize the fiber to -90 mV. The quantity of charge moved saturated at test potentials of approximately 0 mV. The steady state dependence of the amount of charge that moves as a function of test potential could be well fitted by the Boltzmann relation: Q = Qmax/(1 + exp[-(V - V)/k]), where Qmax is the maximum charge that can be moved, V is the potential at which half the charge moves, and k is a constant. At 15 degrees C, these values were Qmax = 28.5 nC/microF, V = -34.2 mV, and k = 8.7 mV. Qmax, k, and V exhibited little temperature dependence over the range 7-25 degrees C. "Stepped OFF" charge movements were elicited by depolarizing the fiber from -90 mV to a fixed conditioning level that moved nearly all the mobile charge (0 mV), and then repolarizing the fiber to varying test potentials. The sum of the charge that moved when the fiber was depolarized directly from -90 mV to a given test potential and the stepped OFF charge that moved when the fiber was repolarized to the same test potential had at all test potentials a value close to Qmax for that fiber. In nearly all cases, the decay phase of ON, OFF, and stepped OFF charge movements could be well fitted with a single exponential. The time constant, tau decay, for an ON charge movement at a given test potential was comparable to tau decay for a stepped OFF charge movement at the same test potential. Tau decay had a bell-shaped dependence on membrane potential: it was slowest at a potential near V (the midpoint of the steady state charge distribution) and became symmetrically faster on either side of this potential. Raising the temperature from 7 to 15 degrees C caused tau decay to become faster by about the same proportion at all potentials, with a Q10 averaging 2.16. Raising the temperature from 15 to 25 degrees C caused tau decay to become faster at potentials near V, but not at potentials farther away.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Sodium channel gating currents in frog skeletal muscle

Charge movements similar to those attributed to the sodium channel gating mechanism in nerve have been measured in frog skeletal muscle using the vaseline-gap voltage-clamp technique. The time course of gating currents elicited by moderate to strong depolarizations could be well fitted by the sum of two exponentials. The gating charge exhibits immobilization: at a holding potential of -90 mV the proportion of charge that returns after a depolarizing prepulse (OFF charge) decreases with the duration of the prepulse with a time course similar to inactivation of sodium currents measured in the same fiber at the same potential. OFF charge movements elicited by a return to more negative holding potentials of -120 or -150 mV show distinct fast and slow phases. At these holding potentials the total charge moved during both phases of the gating current is equal to the ON charge moved during the preceding prepulse. It is suggested that the slow component of OFF charge movement represents the slower return of charge "immobilized" during the prepulse. A slow mechanism of charge immobilization is also evident: the maximum charge moved for a strong depolarization is approximately doubled by changing the holding potential from -90 to -150 mV. Although they are larger in magnitude for a -150-mV holding potential, the gating currents elicited by steps to a given potential have similar kinetics whether the holding potential is -90 or -150 mV.     

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.     

Kinetic properties of intramembrane charge movement under depolarized conditions in frog skeletal muscle fibers

Intramembrane charge movement was measured on skeletal muscle fibers of the frog in a single Vaseline-gap voltage clamp. Charge movements determined both under polarized conditions (holding potential, VH = -100 mV; Qmax = 30.4 +/- 4.7 nC/micro(F), V = -44.4 mV, k = 14.1 mV; charge 1) and in depolarized states (VH = 0 mV; Qmax = 50.0 +/- 6.7 nC/micro(F), V = -109.1 mV, k = 26.6 mV; charge 2) had properties as reported earlier. Linear capacitance (LC) of the polarized fibers was increased by 8.8 +/- 4.0% compared with that of the depolarized fibers. Using control pulses measured under depolarized conditions to calculate charge 1, a minor change in the voltage dependence (to V = -44.6 mV and k = 14.5 mV) and a small increase in the maximal charge (to Qmax = 31.4 +/- 5.5 nC/micro(F] were observed. While in most cases charge 1 transients seemed to decay with a single exponential time course, charge 2 currents showed a characteristic biexponential behavior at membrane potentials between -90 and -180 mV. The voltage dependence of the rate constant of the slower component was fitted with a simple constant field diffusion model (alpha m = 28.7 s-1, V = -124.0 mV, and k = 15.6 mV). The midpoint voltage (V) was similar to that obtained from the Q-V fit of charge 2, while the steepness factor (k) resembled that of charge 1. This slow component could also be isolated using a stepped OFF protocol; that is, by hyperpolarizing the membrane to -190 mV for 200 ms and then coming back to 0 mV in two steps. The faster component was identified as an ionic current insensitive to 20 mM Co2+ but blocked by large hyperpolarizing pulses. These findings are consistent with the model implying that charge 1 and the slower component of charge 2 interconvert when the holding potential is changed. They also explain the difference previously found when comparing the steepness factors of the voltage dependence of charge 1 and charge 2.     

Effects of benzocaine on the kinetics of normal and batrachotoxin-modified Na channels in frog node of Ranvier.

The effects of benzocaine (0.5-1 mM) on normal Na currents, and on Na current and gating charge movement (Q) of batrachotoxin (BTX)-modified Na channels were analyzed in voltage-clamped frog node of Ranvier. Without BTX treatment the decay of Na current during pulses to between -40 and 0 mV could be decomposed into two exponential components both in the absence and in the presence of benzocaine. Benzocaine did not significantly alter the inactivation time constant of either component, but reduced both their amplitudes. The amplitude of the slow inactivating component was more decreased by benzocaine than the amplitude of the fast one, leading to an apparently faster decline of the overall Na current. After removal of Na inactivation and charge movement immobilization by BTX, benzocaine decreased the amplitude of INa with no change in time course. INa, QON, and QOFF were all reduced by the same factor. The results suggest that the rate of reaction of benzocaine with its receptor is slow compared to the rates of channel activation and inactivation. The differential effects of benzocaine on the two components of Na current inactivation in normal channels can be explained assuming two types of channel with different rates of inactivation and different affinities for the drug.     

Ca(2+)-dependent inactivation of cardiac L-type Ca2+ channels does not affect their voltage sensor

Inactivation of currents carried by Ba2+ and Ca2+, as well as intramembrane charge movement from L-type Ca2+ channels were studied in guinea pig ventricular myocytes using the whole-cell patch clamp technique. Prolonged (2 s) conditioning depolarization caused substantial reduction of charge movement between -70 and 10 mV (charge 1, or charge from noninactivated channels). In parallel, the charge mobile between -70 and -150 mV (charge 2, or charge from inactivated channels) was increased. The availability of charge 2 depended on the conditioning pulse voltage as the sum of two Boltzmann components. One component had a central voltage of -75 mV and a magnitude of 1.7 nC/microF. It presumably is the charge movement (charge 2) from Na+ channels. The other component, with a central voltage of approximately - 30 mV and a magnitude of 3.5 nC/microF, is the charge 2 of L-type Ca2+ channels. The sum of charge 1 and charge 2 was conserved after different conditioning pulses. The difference between the voltage dependence of the activation of L-type Ca2+ channels (half-activation voltage, V, of approximately -20 mV) and that of charge 2 (V of -100 mV) made it possible to record the ionic currents through Ca2+ channels and charge 2 in the same solution. In an external solution with Ba2+ as sole metal the maximum available charge 2 of L-type Ca2+ channels was 10-15% greater than that in a Ca(2+)-containing solution. External Cd2+ caused 20-30% reduction of charge 2 both from Na+ and L-type Ca2+ channels. Voltage- and Ca(2+)-dependent inactivation phenomena were compared with a double pulse protocol in cells perfused with an internal solution of low calcium buffering capacity. As the conditioning pulse voltage increased, inactivation monitored with the second pulse went through a minimum at about 0 mV, the voltage at which conditioning current had its maximum. Charge 2, recorded in parallel, did not show any increase associated with calcium entry. Two alternative interpretations of these observations are: (a) that Ca(2+)- dependent inactivation does not alter the voltage sensor, and (b) that inactivation affects the voltage sensor, but only in the small fraction of channels that open, and the effect goes undetected. A model of channel gating that assumes the first possibility is shown to account fully for the experimental results. Thus, extracellular divalent cations modulate voltage-dependent inactivation of the Ca2+ channel. Intracellular Ca2+ instead, appears to cause inactivation of the channel without affecting its voltage sensor.     

Q beta and Q gamma components of intramembranous charge movement in frog cut twitch fibers

Intramembranous charge movement was measured in frog cut twitch fibers mounted in a double Vaseline-gap chamber with a TEA.Cl solution at 13-14 degrees C in the central pool. When a fiber was depolarized from a holding potential of -90 mV to a potential near -60 mV, the current from intramembranous charge movement was outward in direction and had an early, rapid component and a late, more slowly developing component, referred to as I beta and I gamma, respectively (1979. J. Physiol. [Lond.]. 289:83-97). When the pulse to -60 mV was preceded by a 100-600-ms pulse to -40 mV, early I beta and late I gamma components were also observed, but in the inward direction. The shape of the Q gamma vs. voltage curve can be estimated with this two-pulse protocol. The first pulse to voltage V allows the amounts of Q beta and Q gamma charge in the active state to change from their respective resting levels, Q beta (-90) and Q gamma (-90), to new steady levels, Q beta (V) and Q gamma (V). A second 100-120-ms pulse, usually to -60 mV, allows the amount of Q beta charge in the active state to change from Q beta (V) to Q beta (-60) but is not sufficiently long for the amount of Q gamma charge to change completely from Q gamma (V) to Q gamma (-60). The difference between the amount of Q gamma charge at the end of the second pulse and Q gamma (-60) is estimated from the OFF charge that is observed on repolarization to -90 mV. The OFF charge vs. voltage data were fitted, with gap corrections, with a Boltzmann distribution function plus a constant. The mean values of V (the potential at which, in the steady state, charge is distributed equally between the resting and active states) and k (the voltage dependence factor) were -59.2 mV (SEM, 1.1 mV) and 1.2 mV (SEM, 0.6 mV), respectively. The one-pulse charge vs. voltage data from the same fibers were fitted with a sum of two Boltzmann functions (1990. J. Gen. Physiol. 96:257-297). The mean values of V and k for the steeply voltage-dependent Boltzmann function, which is likely to be associated with the Q gamma component of charge, were -55.3 mV (SEM, 1.3 mV) and 3.3 mV (SEM, 0.6 mV), respectively, similar to the corresponding values obtained with the two-pulse protocol.(ABSTRACT TRUNCATED AT 400 WORDS)     

Charge movement in a fast twitch skeletal muscle from rat

Voltage-dependent charge movement in the rat omohyoid muscle was investigated using the three microelectrode voltage clamp technique. The charge that moved during a depolarization from the holding potential (-90 mV) to the test potential, V, increased with increasing V, saturating around 0 mV. The charge vs. voltage relationship was well fitted by Q = Q_max/{1 + exp[-(V - V)/k]}, with Q_max = 28.5 nC/μF, V = -34.2 mV, and k = 8.7 mV. Repolarization of the fiber from the test potential back to the holding potential caused an equal but opposite amount of charge to move. The kinetics of ON charge movement could be well described by a model developed for frog muscle by Horowicz and Schneider (1981b), which suggests that rat and frog charge movements are similar. This model failed to describe the kinetics of OFF charge movement for steps in potential from 0 mV to test potentials of -10 to -90 mV. OFF-charge movement rose to a peak more slowly and decayed more slowly than predicted by the theory.     

Activation of squid axon K+ channels. Ionic and gating current studies

We have used data obtained from measurements of ionic and gating currents to study the process of K+ channel activation in squid giant axons. A marked improvement in the recording of K+ channel gating currents (IKg) was obtained by total replacement of Cl- in the external solution by NO-3, which eliminates approximately 50% of the Na+ channel gating current with no effect on IKg. The midpoint of the steady state charge-voltage (Qrel - V) relationship is approximately 40 mV hyperpolarized to that of the steady state activation (fo - V) curve, which is an indication that the channel has many nonconducting states. Ionic and gating currents have similar time constants for both ON and OFF pulses. This eliminates any Hodgkin-Huxley nx scheme for K+ channel activation. An interrupted pulse paradigm shows that the last step in the activation process is not rate limiting. IKg shows a nonartifactual rising phase, which indicates that the first step is either the slowest step in the activation sequence or is voltage independent. These data are consistent with the following general scheme for K+ channel activation: (formula; see text)     

A slow component of intramembranous charge movement during sarcoplasmic reticulum calcium release in frog cut muscle fibers

Cut muscle fibers from Rana temporaria were mounted in a double Vaseline-gap chamber and equilibrated with an end-pool solution that contained 20 mM EGTA and 1.76 mM Ca (sarcomere length, 3.3-3.8 microns; temperature, 14-16 degrees C). Sarcoplasmic reticulum (SR) Ca release, delta[CaT], was estimated from changes in myoplasmic pH (Pape, P.C., D.- S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259-336). The maximal value of delta[CaT] obtained during a depleting depolarization was assumed to equal the SR Ca content before stimulation, [CaSR]R (expressed as myoplasmic concentration). After a depolarization to -55 to -40 mV in fibers with [CaSR]R = 1,000-3,000 microM, currents from intramembranous charge movement, Icm, showed an early I beta component. This was followed by an I gamma hump, which decayed within 50 ms to a small current that was maintained for as long as 500 ms. This slow current was probably a component of Icm because the amount of OFF charge, measured after depolarizations of different durations, increased according to the amount of ON charge. Icm was also measured after the SR had been depleted of most of its Ca, either by a depleting conditioning depolarization or by Ca removal from the end pools followed by a series of depleting depolarizations. The early I beta component was essentially unchanged by Ca depletion, the I gamma hump was increased (for [CaSR]R > 200 microM), the slow component was eliminated, and the total amount of OFF charge was essentially unchanged. These results suggest that the slow component of ON Icm is not movement of a new species of charge but is probably movement of Q gamma that is slowed by SR Ca release or some associated event such as the accompanying increase in myoplasmic free [Ca] that is expected to occur near the Ca release sites. The peak value of the apparent rate constant associated with this current, 2-4%/ms at pulse potentials between -48 and -40 mV, is decreased by half when [CaSR]R approximately equal to 500-1,000 microM, which gives a peak rate of SR Ca release of approximately 5-10 microM/ms.     

Inactivation of the sodium channel. II. Gating current experiments

Gating current (Ig) has been studied in relation to inactivation of Na channels. No component of Ig has the time course of inactivation; apparently little or no charge movement is associated with this step. Inactivation nonetheless affects Ig by immobilizing about two-thirds of gating charge. Immobilization can be followed by measuring ON charge movement during a pulse and comparing it to OFF charge after the pulse. The OFF:ON ratio is near 1 for a pulse so short that no inactivation occurs, and the ratio drops to about one-third with a time course that parallels inactivation. Other correlations between inactivation and immobilization are that: (a) they have the same voltage dependence; (b) charge movement recovers with the time coures of recovery from inactivation. We interpret this to mean that the immobilized charge returns slowly to "off" position with the time course of recovery from inactivation, and that the small current generated is lost in base-line noise. At -150 mV recover is very rapid, and the immobilized charge forms a distinct slow component of current as it returns to off position. After destruction of inactivation by pronase, there is no immobilization of charge. A model is presented in which inactivation gains its voltage dependence by coupling to the activation gate.