Modulation of single cardiac sodium channels by DPI 201-106

Single sodium channel currents were analysed in cell attached patches from single ventricular cells of guinea pig hearts in the presence of a novel cardiotonic compound DPI 201-106. The mean single channel conductance of DPI-treated Na channels was not changed by DPI (20.8 +/- 4 pS, control, 3 patches; 21.3 +/- 1 pS with DPI, 5 mumol/1,3 patches). DPI voltage-dependently prolongs the cardiac sodium channel openings by removal of inactivation at potentials positive to -40 mV. At potentials negative to -40 mV a clustering of short openings at the very beginning of the depolarizing voltage steps can be observed causing a transient time course of the averaged currents. Long openings induced an extremely slow inactivation. Short openings, long openings and nulls appeared in groups referring to a modal gating behaviour of DPI-treated sodium channels. DPI-modified Na channels showed a monotonously prolonged mean open time with increased depolarizing voltage steps, e.g. the open state probability within a sweep was increased. However, the number of non-empty sweeps was decreased with the magnitude of the depolarizing steps, e.g. the probability of the channel being open as calculated from the averaged currents was voltage-dependently decreased by DPI (50% decrease at -50.7 +/- 9 9 mV, 3 patches). Short and long openings of DPI-modified channels could be separated by variation of the holding potential. The occurrence of long Na channel openings was much more suppressed by reducing the holding potential (half maximum inactivation at -112 +/- 8 mV, 4 patches) than that of short openings (half maximum inactivation at -88 +/- 8 mV, 4 patches). Otherwise, short living openings completely disappeared at potentials positive to -40 mV where the occurrence of long openings was favoured. The differential voltage dependence of blocking and activating effects of DPI on cardiac Na channels as well as the differential voltage dependence of the appearance of short and long openings refers to a modal gating behaviour of cardiac Na channels.     

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)     

Mechanism of inactivation gating of human T-type (low-voltage activated) calcium channels

Recovery from inactivation of T-type Ca channels is slow and saturates at moderate hyperpolarizing voltage steps compared with Na channels. To explore this unique kinetic pattern we measured gating and ionic currents in two closely related isoforms of T-type Ca channels. Gating current recovers from inactivation much faster than ionic current, and recovery from inactivation is much more voltage dependent for gating current than for ionic current. There is a lag in the onset of gating current recovery at -80 mV, but no lag is discernible at -120 mV. The delay in recovery from inactivation of ionic current is much more evident at all voltages. The time constant for the decay of off gating current is very similar to the time constant of deactivation of open channels (ionic tail current), and both are strongly voltage dependent over a wide voltage range. Apparently, the development of inactivation has little influence on the initial deactivation step. These results suggest that movement of gating charge occurs for inactivated states very quickly. In contrast, the transitions from inactivated to available states are orders of magnitude slower, not voltage dependent, and are rate limiting for ionic recovery. These findings support a deactivation-first path for T-type Ca channel recovery from inactivation. We have integrated these concepts into an eight-state kinetic model, which can account for the major characteristics of T-type Ca channel inactivation.     

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

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

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.     

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.     

Uncoupling of gating charge movement and closure of the ion pore during recovery from inactivation in the Kv1.5 channel

Both wild-type (WT) and nonconducting W472F mutant (NCM) Kv1.5 channels are able to conduct Na(+) in their inactivated states when K(+) is absent. Replacement of K(+) with Na(+) or NMG(+) allows rapid and complete inactivation in both WT and W472F mutant channels upon depolarization, and on return to negative potentials, transition of inactivated channels to closed-inactivated states is the first step in the recovery of the channels from inactivation. The time constant for immobilized gating charge recovery at -100 mV was 11.1 +/- 0.4 ms (n = 10) and increased to 19.0 +/- 1.6 ms (n = 3) when NMG(+)(o) was replaced by Na(+)(o). However, the decay of the Na(+) tail currents through inactivated channels at -100 mV had a time constant of 129 +/- 26 ms (n = 18), much slower than the time required for gating charge recovery. Further experiments revealed that the voltage-dependence of gating charge recovery and of the decay of Na(+) tail currents did not match over a 60 mV range of repolarization potentials. A faster recovery of gating charge than pore closure was also observed in WT Kv1.5 channels. These results provide evidence that the recovery of the gating elements is uncoupled from that of the pore in Na(+)-conducting inactivated channels. The dissociation of the gating charge movements and the pore closure could also be observed in the presence of symmetrical Na(+) but not symmetrical Cs(+). This difference probably stems from the difference in the respective abilities of the two ions to limit inactivation to the P-type state or prevent it altogether.     

Sodium and calcium currents in dispersed mammalian septal neurons

Voltage-gated Na+ and Ca2+ conductances of freshly dissociated septal neurons were studied in the whole-cell configuration of the patch-clamp technique. All cells exhibited a large Na+ current with characteristic fast activation and inactivation time courses. Half-time to peak current at -20 mV was 0.44 +/- 0.18 ms and maximal activation of Na+ conductance occurred at 0 mV or more positive membrane potentials. The average value was 91 +/- 32 nS (approximately 11 mS cm-2). At all membrane voltages inactivation was well fitted by a single exponential that had a time constant of 0.44 +/- 0.09 ms at 0 mV. Recovery from inactivation was complete in approximately 900 ms at -80 mV but in only 50 ms at -120 mV. The decay of Na+ tail currents had a single time constant that at -80 mV was faster than 100 microseconds. Depolarization of septal neurons also elicited a Ca2+ current that peaked in approximately 6-8 ms. Maximal peak Ca2+ current was obtained at 20 mV, and with 10 mM external Ca2+ the amplitude was 0.35 +/- 0.22 nA. During a maintained depolarization this current partially inactivated in the course of 200-300 ms. The Ca2+ current was due to the activity of two types of conductances with different deactivation kinetics. At -80 mV the closing time constants of slow (SD) and fast (FD) deactivating channels were, respectively, 1.99 +/- 0.2 and 0.11 +/- 0.03 ms (25 degrees C). The two kinds of channels also differed in their activation voltage, inactivation time course, slope of the conductance-voltage curve, and resistance to intracellular dialysis. The proportion of SD and FD channels varied from cell to cell, which may explain the differential electrophysiological responses of intracellularly recorded septal neurons.     

Simple shifts in the voltage dependence of sodium channel gating caused by divalent cations

The effect of elevated divalent cation concentration on the kinetics of sodium ionic and gating currents was studied in voltage-clamped frog skeletal muscle fibers. Raising the Ca concentration from 2 to 40 mM resulted in nearly identical 30-mV shifts in the time courses of activation, inactivation, tail current decay, and ON and OFF gating currents, and in the steady state levels of inactivation, charge immobilization, and charge vs. voltage. Adding 38 mM Mg to the 2 mM Ca bathing a fiber produced a smaller shift of approximately 20 mV in gating current kinetics and the charge vs. voltage relationship. The results with both Ca and Mg are consistent with the hypothesis that elevated concentrations of these alkali earth cations alter Na channel gating by changing the membrane surface potential. The different shifts produced by Ca and Mg are consistent with the hypothesis that the two ions bind to fixed membrane surface charges with different affinities, in addition to possible screening.     

Modal gating behavior of cardiac sodium channels in cell-free membrane patches.

Single voltage-activated Na+ channel currents were obtained from membrane patches of isolated ventricular cells of guinea pig hearts. The currents were compared when measured from cell-attached patches and from the same patch but at least 20 minutes after manual excision. The averaged currents showed a distinctly delayed decay in the excised patches due to the appearance of long lasting openings or bursts of openings. In contrast to control patches, the open time distribution in excised patches requires at least two exponentials. A short mean open time was voltage independent for cell-attached patches (0.38 ms +/- 0.07 ms between -60 and -20 mV, 6 cell-attached patches; and 0.41 +/- 0.1 ms, 7 excised patches). The long mean open time found in excised patches was clearly voltage dependent and increased from 0.48 +/- 0.14 ms (-80 mV) to 2.87 +/- 0.35 ms (-20 mV, regression coefficient +0.88, 7 patches). Sweeps with long openings appeared in clusters. The clustering of records with long openings, short openings, or without openings (nulls) was quantified by a runs analysis which showed a highly significant nonrandom ordering. The results show that in excised patches inactivation is temporally hibernating.     

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).     

Batrachotoxin uncouples gating charge immobilization from fast Na inactivation in squid giant axons.

The fast inactivation of sodium currents and the immobolization of sodium gating charge are thought to be closely coupled to each other. This notion was tested in the squid axon in which kinetics and steady-state properties of the gating charge movement were compared before and after removal of the Na inactivation by batrachotoxin (BTX), pronase, or chloramine-T. The immobilization of gating charge was determined by measuring the total charge movement (QON) obtained by integrating the ON gating current (Ig,ON) using a double pulse protocol. After removal of the fast inactivation with pronase or chloramine-T, the gating charge movement was no longer immobilized. In contrast, after BTX modification, the channels still exhibited an immobilization of the gating charge (QON) with an onset time course and voltage dependence similar to that for the activation process. These results show that BTX can uncouple the charge immobilization from the fast Na inactivation mechanism, suggesting that the Na gating charge movement can be immobilized independently of the inactivation of the channel.     

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.     

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.     

Functional association of the beta 1 subunit with human cardiac (hH1) and rat skeletal muscle (mu 1) sodium channel alpha subunits expressed in Xenopus oocytes

Native cardiac and skeletal muscle Na channels are complexes of alpha and beta 1 subunits. While structural correlates for activation, inactivation, and permeation have been identified in the alpha subunit and the expression of alpha alone produces functional channels, beta 1- deficient rat skeletal muscle (mu 1) and brain Na channels expressed in Xenopus oocytes do not gate normally. In contrast, the requirement of a beta 1 subunit for normal function of Na channels cloned from rat heart or human heart (hH1) has been disputed. Coinjection of rat brain beta 1 subunit cRNA with hH1 (or mu 1) alpha subunit cRNA into oocytes increased peak Na currents recorded 2 d after injection by 240% (225%) without altering the voltage dependence of activation. In mu 1 channels, steady state inactivation was shifted to more negative potentials (by 6 mV, p < 0.01), but the shift of 2 mV was not significant for hH1 channels. Nevertheless, coexpression with beta 1 subunit speeded the decay of macroscopic current of both isoforms. Ensemble average hH1 currents from cell-attached patches revealed that coexpression of beta 1 increases the rate of inactivation (quantified by time to 75% decay of current; p < 0.01 at -30, -40, and -50 mV). Use- dependent decay of hH1 Na current during repeated pulsing to -20 mV (1 s, 0.5 Hz) after a long rest was reduced to 16 +/- 2% of the first pulse current in oocytes coexpressing alpha and beta 1 subunits compared to 35 +/- 8% use-dependent decay for oocytes expressing the alpha subunit alone. Recovery from inactivation of mu 1 and hH1 Na currents after 1-s pulses to -20 mV is multiexponential with three time constants; coexpression of beta 1 subunit decreased all three recovery time constants. We conclude that the beta 1 subunit importantly influences the function of Na channels produced by coexpression with either the hH1 or mu 1 alpha subunits.     

Isoform-specific lidocaine block of sodium channels explained by differences in gating

When depolarized from typical resting membrane potentials (V(rest) approximately -90 mV), cardiac sodium (Na) currents are more sensitive to local anesthetics than brain or skeletal muscle Na currents. When expressed in Xenopus oocytes, lidocaine block of hH1 (human cardiac) Na current greatly exceeded that of mu1 (rat skeletal muscle) at membrane potentials near V(rest), whereas hyperpolarization to -140 mV equalized block of the two isoforms. Because the isoform-specific tonic block roughly parallels the drug-free voltage dependence of channel availability, isoform differences in the voltage dependence of fast inactivation could underlie the differences in block. However, after a brief (50 ms) depolarizing pulse, recovery from lidocaine block is similar for the two isoforms despite marked kinetic differences in drug-free recovery, suggesting that differences in fast inactivation cannot entirely explain the isoform difference in lidocaine action. Given the strong coupling between fast inactivation and other gating processes linked to depolarization (activation, slow inactivation), we considered the possibility that isoform differences in lidocaine block are explained by differences in these other gating processes. In whole-cell recordings from HEK-293 cells, the voltage dependence of hH1 current activation was approximately 20 mV more negative than that of mu1. Because activation and closed-state inactivation are positively coupled, these differences in activation were sufficient to shift hH1 availability to more negative membrane potentials. A mutant channel with enhanced closed-state inactivation gating (mu1-R1441C) exhibited increased lidocaine sensitivity, emphasizing the importance of closed-state inactivation in lidocaine action. Moreover, when the depolarization was prolonged to 1 s, recovery from a "slow" inactivated state with intermediate kinetics (I(M)) was fourfold longer in hH1 than in mu1, and recovery from lidocaine block in hH1 was similarly delayed relative to mu1. We propose that gating processes coupled to fast inactivation (activation and slow inactivation) are the key determinants of isoform-specific local anesthetic action.     

Patch recordings from the electrocytes of Electrophorus electricus. Na currents and PNa/PK variability

Sodium currents were recorded in cell-attached and inside-out patches from the innervated membrane of Electrophorus electrocytes. Electrocytes from Sachs and main electric organs were prepared as described by Pasquale et al. (1986. J. Membr. Biol. 93:195.). Maximal currents in the Sachs organ, measured with 1-2 microns diameter patch pipettes and at room temperature, were in the range of 20 to 300 pA (27 patches) and were obtained near +10 mV. This range of current corresponds to approximately 70 to 1,300 channels in a patch. Maximal current in main organ cells also occurred near +10 mV and were in the range of 100 to 400 pA. Delayed K current was observed in a few patches. The inactivation phase of the currents during maintained depolarizations appears to be a single-exponential relaxation. The time constant decreases from 1 ms near -55 mV to a minimum of 0.3 ms near 0 mV, and then gradually increases with stronger depolarization. The mean currents are half inactivated near -90 mV with an apparent voltage dependence of e-fold per 6 mV. No apparent differences were observed in the decay time course or steady-state inactivation of the currents in the same patch before and after excision. From ensemble fluctuation analysis the peak open probability was found to be approximately 0.5 at +25 mV and increased only gradually with larger depolarizations. The single channel conductances were approximately 20 pS with 200 mM Na outside and 200 mM K inside, and 40 pS in 400 mM solutions. Reversal potentials in the 200 Na parallel 200 K solutions ranged from +51 to +94 mV in multichannel patches, corresponding to selectivity ratios PNa/PK from 8 to 43. Large differences in reversal potentials were seen even among patches from the same cell. Several controls rule out obvious sources of error in the reversal potential measurements. It is concluded that there is heterogeneity in the selectivity properties of the Na 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)     

Voltage-dependent open-state inactivation of cardiac sodium channels: gating current studies with Anthopleurin-A toxin

The gating charge and voltage dependence of the open state to the inactivated state (O-->I) transition was measured for the voltage- dependent mammalian cardiac Na channel. Using the site 3 toxin, Anthopleurin-A (Ap-A), which selectively modifies the O-->I transition (see Hanck, D. A., and M. F. Sheets. 1995. Journal of General Physiology. 106:601-616), we studied Na channel gating currents (Ig) in voltage-clamped single canine cardiac Purkinje cells at approximately 12 degrees C. Comparison of Ig recorded in response to step depolarizations before and after modification by Ap-A toxin showed that toxin-modified gating currents decayed faster and had decreased initial amplitudes. The predominate change in the charge-voltage (Q-V) relationship was a reduction in gating charge at positive potentials such that Qmax was reduced by 33%, and the difference between charge measured in Ap-A toxin and in control represented the gating charge associated with Na channels undergoing inactivation by O-->I. By comparing the time course of channel activation (represented by the gating charge measured in Ap-A toxin) and gating charge associated with the O-->I transition (difference between control and Ap-A charge), the influence of activation on the time course of inactivation could be accounted for and the inherent voltage dependence of the O-->I transition determined. The O-->I transition for cardiac Na channels had a valence of 0.75 e-. The total charge of the cardiac voltage-gated Na channel was estimated to be 5 e-. Because charge is concentrated near the opening transition for this isoform of the channel, the time constant of the O-->I transition at 0 mV could also be estimated (0.53 ms, approximately 12 degrees C). Prediction of the mean channel open time-voltage relationship based upon the magnitude and valence of the O- ->C and O-->I rate constants from INa and Ig data matched data previously reported from single Na channel studies in heart at the same temperature.     

Slow currents through single sodium channels of the adult rat heart

The currents through single Na+ channels from the sarcolemma of ventricular cells dissociated from adult rat hearts were studied using the patch-clamp technique. All patches had several Na+ channels; most had 5-10, while some had up to 50 channels. At 10 degrees C, the conductance of the channel was 9.8 pS. The mean current for sets of many identical pulses inactivated exponentially with a time constant of 1.7 +/- 0.6 ms at -40 mV. Careful examination of the mean currents revealed a small, slow component of inactivation at pulse potentials ranging from -60 to -30 mV. The time constant of the slow component was between 8 and 14 ms. The channels that caused the slow component had the same conductance and reversal potential as the fast Na+ currents and were blocked by tetrodotoxin. The slow currents appear to have been caused by repeated openings of one or more channels. The holding potential influenced the frequency with which such channel reopening occurred. The slow component was prominent during pulses from a holding potential of -100 mV, while it was very small during pulses from -140 mV. Ultraslow currents through the Na+ channel were observed occasionally in patches that had large numbers of channels. They consisted of bursts of 10 or more sequential openings of a single channel and lasted for up to 150 ms. We conclude that the single channel data cannot be explained by standard models, even those that have two inactivated states or two open states of the channel. Our results suggest that Na+ channels can function in several different "modes," each with a different inactivation rate.