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.     

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)     

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.     

A Fast Transient Outward Monovalent Current in Rat Saphenous Myocytes Passing Through Ca2+ Channels

Transient outward currents in rat saphenous arterial myocytes were studied using the perforated configuration of the patch-clamp method. When myocytes were bathed in a Na-gluconate solution containing TEA to block large-conductance Ca2+-activated K+ (BK) currents, depolarizing pulses positive to +20 mV from a holding potential of -100 mV induced fast transient outward currents. The activation and inactivation time constants of the current were voltage dependent, and at +40 mV were 3.6 +/- 0.8 ms and 23.9 +/- 6.4 ms (n = 4), respectively. The steady-state inactivation of the transient outward current was steeply voltage dependent (z = 1.7), with 50% of the current inactivated at -55 mV. The current was insensitive to the A-type K+ channel blocker 4-AP (1-5 mM), and was modulated by external Ca, decreasing to approximately 0.85 of control values upon raising Ca2+ from 1 to 10 mM, and increasing approximately 3-fold upon lowering it to 0.1 mM. Transient outward currents were also recorded following replacement of internal K+ with either Na+ or Cs+, raising the possibility that the current was carried by monovalent ions passing through voltage-gated Ca2+ channels. This hypothesis was supported by the finding that the transient outward current had the same inactivation rate as the inward Ba2+ current, and that both currents were effectively blocked by the L-type Ca2+ channel blocker, nifedipine and enhanced by the agonist BAYK8644.     

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.     

T-type Ca2+ channel lowers the threshold of spike generation in the newt olfactory receptor cell

Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of -70 +/- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na+ concentration. After a complete removal of Na+, however, cells still showed action potentials which was abolished either by Ca2+ removal or by an application of Ca2+ channel blocker (Co2+ or Ni2+), indicating an involvement of Ca2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to -40 mV from the holding voltage of -100 mV induced a fast transient inward current, which consisted of Na+ (INa) and T-type Ca2+ (ICa.T) currents. The amplitude of ICa,T was about one fourth of that of INa. Depolarization to more positive voltages also induced L-type Ca2+ current (ICa,L). ICa,L was as small as a few pA in normal Ringer solution. The activating voltage of ICa,T was approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Ni2+ (a specific T-type Ca2+ channel blocker) even in the presence of Na+. These results suggest that ICa,T contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation.     

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.     

Steady-state availability of sodium channels. Interactions between activation and slow inactivation.

Changes in holding potential (Vh), affect both gating charge (the Q(Vh) curve) and peak ionic current (the F(Vh) curve) seen at positive test potentials. Careful comparison of the Q(Vh) and F(Vh) distributions indicates that these curves are similar, having two slopes (approximately 2.5e for Vh from -115 to -90 mV and approximately 4e for Vh from -90 to -65 mV) and very negative midpoints (approximately -86 mV). Thus, gating charge movement and channel availability appear closely coupled under fully-equilibrated conditions. The time course by which channels approach equilibration was explored using depolarizing prepulses of increasing duration. The high slope component seen in the F(Vh) and Q(Vh) curves is not evident following short depolarizing prepulses in which the prepulse duration approximately corresponds to the settling time for fast inactivation. Increasing the prepulse duration to 10 ms or longer reveals the high slope, and left-shifts the midpoint to more negative voltages, towards the F(Vh) and Q(Vh) distributions. These results indicate that a separate slow-moving voltage sensor affects the channels at prepulse durations greater than 10 ms. Charge movement and channel availability remain closely coupled as equilibrium is approached using depolarizing pulses of increasing durations. Both measures are 50% complete by 50 ms at a prepulse potential of -70 mV, with proportionately faster onset rates when the prepulse potential is more depolarized. By contrast, charge movement and channel availability dissociate during recovery from prolonged depolarizations. Recovery of gating charge is considerably faster than recovery of sodium ionic current after equilibration at depolarized potentials. Recovery of gating charge at -140 mV, is 65% complete within approximately 100 ms, whereas less than 30% of ionic current has recovered by this time. Thus, charge movement and channel availability appear to be uncoupled during recovery, although both rates remain voltage sensitive. These data suggest that channels remain inactivated due to a separate process operating in parallel with the fast gating charge. We demonstrate that this behavior can be simulated by a model in which the fast charge movement associated with channel activation is electrostatically-coupled to a separate slow voltage sensor responsible for the slow inactivation of channel conductance.     

Role of calcium permeation in dihydropyridine receptor function. Insights into channel gating and excitation-contraction coupling.

The skeletal and cardiac muscle dihydropyridine receptors (DHPRs) differ with respect to their rates of channel activation and in the means by which they control Ca2+ release from the sarcoplasmic reticulum (Adams, B.A., and K.G. Beam. 1990. FASEB J. 4:2809-2816). We have examined the functional properties of skeletal (SkEIIIK) and cardiac (CEIIIK) DHPRs in which a highly conserved glutamate residue in the pore region of repeat III was mutated to a positively charged lysine residue. Using expression in dysgenic myotubes, we have characterized macroscopic ionic currents, intramembrane gating currents, and intracellular Ca2+ transients attributable to these two mutant DHPRs. CEIIIK supported very small inward Ca2+ currents at a few potentials (from -20 to +20 mV) and large outward cesium currents at potentials greater than +20 mV. SkEIIIK failed to support inward Ca2+ flux at any potential. However, large, slowly activating outward cesium currents were observed at all potentials greater than + 20 mV. The difference in skeletal and cardiac Ca2+ channel activation kinetics was conserved for outward currents through CEIIIK and SkEIIIK, even at very depolarized potentials (at +100 mV; SkEIIIK: tau(act) = 30.7 +/- 1.9 ms, n = 11; CEIIIK: tau(act) = 2.9 +/- 0.5 ms, n = 7). Expression of SkEIIIK in dysgenic myotubes restored both evoked contractions and depolarization-dependent intracellular Ca(2+) transients with parameters of voltage dependence (V(0.5) = 6.5 +/- 3.2 mV and k = 9.3 +/- 0.7 mV, n = 5) similar to those for the wild-type DHPR (Garcia, J., T. Tanabe, and K.G. Beam. 1994. J. Gen. Physiol. 103:125-147). However, CEIIIK-expressing myotubes never contracted and failed to exhibit depolarization-dependent intracellular Ca2+ transients at any potential. Thus, high Ca2+ permeation is required for cardiac-type excitation-contraction coupling reconstituted in dysgenic myotubes, but not skeletal-type. The strong rectification of the EIIIK channels made it possible to obtain measurements of gating currents upon repolarization to -50 mV (Qoff) following either brief (20 ms) or long (200 ms) depolarizing pulses to various test potentials. For SkEIIIK, and not CEIIK, Qoff was significantly (P < 0.001) larger after longer depolarizations to +60 mV (121.4 +/- 2.0%, n = 6). The increase in Qoff for long depolarizations exhibited a voltage dependence similar to that of channel activation. Thus, the increase in Q(off) may reflect a voltage sensor movement required for activation of L-type Ca2+ current and suggests that most DHPRs in skeletal muscle undergo this voltage-dependent transition.     

Voltage-activated currents in guinea pig pancreatic alpha 2 cells. Evidence for Ca2+-dependent action potentials

Glucagon-secreting alpha 2 cells were isolated from guinea pig pancreatic islets and used for electrophysiological studies of voltage- activated ionic conductances using the patch-clamp technique. The alpha 2 cells differed from beta cells in producing action potentials in the absence of glucose. The frequency of these potentials increased after addition of 10 mM arginine but remained unaffected in the presence of 5- 20 mM glucose. When studying the conductances underlying the action potentials, we identified a delayed rectifying K+ current, an Na+ current, and a Ca2+ current. The K+ current activated above -20 mV and then increased with the applied voltage. The Na+ current developed at potentials above -50 mV and reached a maximal peak amplitude of 550 pA during depolarizing pulses to -15 mV. The Na+ current inactivated rapidly (tau h approximately 0.7 ms at 0 mV). Half-maximal steady state inactivation was attained at -58 mV, and currents could no longer be elicited after conditioning pulses to potentials above -40 mV. The Ca2+ current first became detectable at -50 mV and reached a maximal amplitude of 90 pA (in extracellular [Ca2+] = 2.6 mM) at about -10 mV. Unlike the Na+ current, it inactivated little or not at all. Membrane potential measurements demonstrated that both the Ca2+ and Na+ currents contribute to the generation of the action potential. Whereas there was an absolute requirement of extracellular Ca2+ for action potentials to be elicited at all, suppression of the much larger Na+ current only reduced the upstroke velocity of the spikes. It is suggested that this behavior reflects the participation of a low-threshold Ca2+ conductance in the pacemaking of alpha 2 cells.     

Pharmacological and kinetic analysis of K channel gating currents

We have measured gating currents from the squid giant axon using solutions that preserve functional K channels and with experimental conditions that minimize Na channel contributions to these currents. Two pharmacological agents were used to identify a component of gating current that is associated with K channels. Low concentrations of internal Zn2+ that considerably slow K channel ionic currents with no effect on Na channel currents altered the component of gating current associated with K channels. At low concentrations (10-50 microM) the small, organic, dipolar molecule phloretin has several reported specific effects on K channels: it reduces K channel conductance, shifts the relationship between channel conductance and membrane voltage (Vm) to more positive potentials, and reduces the voltage dependence of the conductance-Vm relation. The K channel gating charge movements were altered in an analogous manner by 10 microM phloretin. We also measured the dominant time constants of the K channel ionic and gating currents. These time constants were similar over part of the accessible voltage range, but at potentials between -40 and 0 mV the gating current time constants were two to three times faster than the corresponding ionic current values. These features of K channel function can be reproduced by a simple kinetic model in which the channel is considered to consist of two, two-state, nonidentical subunits.     

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.     

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)     

Properties of an early outward current in single cells of the mouse ventricle

Single ventricular myocytes of adult mice were prepared by enzymatic dissociation for voltage clamp experiments with the one suction pipette dialysis method. After blocking the Na current by 10(-4) mol/l TTX early outward currents (IEO) with incomplete inactivation could be elicited by clamping from -50 mV to test potentials (VT) positive to -30 mV. Interfering Ca currents were very small (less than 0.6 nA at VT = 0 mV). The approximation of IEO by the q4r-model showed a pronounced decrease in the time constant of activation (tau q) to more positive potentials. At 50 ms test pulses the time course of the incomplete inactivation could be described by two exponentials and a constant. The time constant of the fast exponential (tau r1) showed a slight decline towards more positive test potentials (8.1 +/- 1.0 ms at -10 mV; 5.8 +/- 1.2 ms at +50 mV, mean +/- SD, n = 5) whereas the time constant of the slow exponential (tau r2) was voltage independent (41.1 +/- 7.9 ms, mean +/- SD, n = 5). The contributions of the fast exponential and the pedestal increased towards positive test potentials. The Q10 value for the time constants of activation and fast inactivation was 2.36 +/- 0.19 and 2.51 +/- 0.09 (mean +/- SD, n = 3), respectively. After an initial delay the recovery of IEO at a recovery potential of -50 mV could be fitted monoexponentially with a time constant of 16.3 +/- 2.9 ms (mean +/- SD, n = 3). The time course of the onset of inactivation determined with the double pulse protocol was slower than the decay at the same potential, and could be described as sum of a fast (tau = 18.4 +/- 6.0 ms) and a slow (tau = 62.1 +/- 19.9ms, mean +/- SD, n = 3) exponential. IEO could be blocked completely by 1 mmol/l 4-aminopyridine at potentials up to +20 mV. Stronger depolarizations had an unblocking effect.     

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.     

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.     

Ca2+-dependent membrane currents in vascular smooth muscle cells of the rabbit.

The membrane potential in vascular smooth muscle cells contributes to the regulation of cytosolic [Ca2+], which in turn regulates membrane potential by means of Ca2+i-dependent ionic currents. We investigated the characteristics of Ca2+i-dependent currents in rabbit coronary and pulmonary arterial smooth muscle cells. Ca2+i-dependent currents were recorded using the whole-cell patch-clamp technique while cytosolic [Ca2+] was increased by caffeine. The reversal potentials of caffeine-induced currents were between -80 and -10 mV under normal ionic conditions, whereas they were about 0 mV when K+-free NaCl solutions were used both in pipette and bath. The total substitution of extracellular Na+ with membrane-impermeable cation N-Methyl-D-glucamine did not affect caffeine-induced currents, implying no significant contribution of Na+ as a permeant ion to the currents. The substitution of extracellular NaCl with sucrose reduced outward component of the currents and shifted the reversal potentials according to the change in Cl- equilibrium potential. Upon application of the niflumic acid under K+-free conditions, most of the current induced by caffeine was inhibited. Taken together, the results of the present study indicate that K+ and Cl- currents are major components of Ca2+i-dependent currents in vascular smooth muscles isolated from coronary and pulmonary arteries of the rabbit, and the relative contribution of each type of current to total currents are not different between the two arteries.     

A delayed rectifier current is modulated by the circadian pacemaker in Bulla

Basal retinal neurons of the marine mollusc Bulla gouldiana continue to express a circadian modulation of their membrane conductance for at least two cycles in cell culture. Voltage-dependent currents of these pacemaker cells were recorded using the whole-cell perforated patch-clamp technique to characterize outward currents and investigate their putative circadian modulation. Three components of the outward potassium current were identified. A transient outward current (IA) was activated after depolarization from holding potentials greater than -30 mV, inactivated with a time constant of 50 ms, and partially blocked by 4-aminopyridine (1-5 mM). A Ca(2+)-dependent potassium current (IK(Ca)) was activated by depolarization to potentials more positive than -10 mV and was blocked by removing Ca2+ from the bath or by applying the Ca2+ channel blockers Cd2+ (0.1-0.2 mM) and Ni2+ (1-5 mM). A sustained Ca(2+)-independent current component including the delayed rectifier current (IK) was recorded at potentials positive to -20 mV in the absence of extracellular Na+ and Ca2+ and was partially blocked by tetraethylammonium chloride (TEA, 30mM). Whole-cell currents recorded before and after the projected dawn and normalized to the cell capacitance revealed a circadian modulation of the delayed rectifier current (IK). However, the IA and IK(Ca) currents were not affected by the circadian pacemaker.     

Gating of Shaker K+ channels: I. Ionic and gating currents.

Ionic and gating currents from noninactivating Shaker B K+ channels were studied with the cut-open oocyte voltage clamp technique and compared with the macropatch clamp technique. The performance of the cut-open oocyte voltage clamp technique was evaluated from the electrical properties of the clamped upper domus membrane, K+ tail current measurements, and the time course of K+ currents after partial blockade. It was concluded that membrane currents less than 20 microA were spatially clamped with a time resolution of at least 50 microseconds. Subtracted, unsubtracted gating currents with the cut-open oocyte voltage clamp technique and gating currents recorded in cell attached macropatches had similar properties and time course, and the charge movement properties directly obtained from capacity measurements agreed with measurements of charge movement from subtracted records. An accurate estimate of the normalized open probability Po(V) was obtained from tail current measurements as a function of the prepulse V in high external K+. The Po(V) was zero at potentials more negative than -40 mV and increased sharply at this potential, then increased continuously until -20 mV, and finally slowly increased with voltages more positive than 0 mV. Deactivation tail currents decayed with two time constants and external potassium slowed down the faster component without affecting the slower component that is probably associated with the return between two of the closed states near the open state. In correlating gating currents and channel opening, Cole-Moore type experiments showed that charge moving in the negative region of voltage (-100 to -40 mV) is involved in the delay of the conductance activation but not in channel opening. The charge moving in the more positive voltage range (-40 to -10 mV) has a similar voltage dependence to the open probability of the channel, but it does not show the gradual increase with voltage seen in the Po(V).     

State-dependent inactivation of the alpha1G T-type calcium channel.

We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the alpha1G channel, with symmetrical Na(+)(i) and Na(+)(o) and 2 mM Ca(2+)(o). After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential -100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from -120 to -70 mV (e-fold for 31 mV; tau = 2.5 ms at -100 mV), but tau = 12-17 ms from-40 to +60 mV. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100-300 ms, inactivation was strong but incomplete (approximately 98%). Inactivation was also produced by long, weak depolarizations (tau = 220 ms at -80 mV; V(1/2) = -82 mV), which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast (tau = 85 ms at -100 mV), but weakly voltage dependent. Recovery was similar after 60-ms steps to -20 mV or 600-ms steps to -70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at -100 mV during recovery from inactivation, consistent with 相似文献