Na channel kinetics: developing models from non-stationary current fluctuations by analytic methods

In a previous study, we analyzed Na current fluctuations in myelinated axons from Xenopus laevis under voltage clamp conditions. The statistical properties were analyzed in terms of covariance functions for consecutive time intervals of varying duration during the pulse step. The underlying channel kinetics was analyzed by performing stochastic simulations of published Na channel models and calculating corresponding covariance functions. None of the models explained the fluctuation results. We therefore developed a novel minimal Na channel model that satisfactorily described the results. In the present paper, we extend the analysis and specify the possible models explaining the experimental data by using analytical methods. We derive general relations between the experimental data, including the covariance functions, and the rate constants of specific one-open-state models. A general feature of these models is that they comprise an inactivation step from the first closed state and a relatively low backward rate from the open state. This is in accordance the minimal model inferred from numerical stochastic calculations in the previous study.     

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.     

Tail currents in the myelinated axon of Xenopus laevis suggest a two-open-state Na channel.

Na tail currents in the myelinated axon of Xenopus laevis were measured at -70 mV after steps to -10 mV. The tail currents were biexponential, comprising a fast and a slow component. The time constant of the slow tail component, analyzed in the time window 0.35-0.50 ms, was independent of step duration, and had a value of 0.23 ms. The amplitude, extrapolated back to time 0, varied, however, with step duration. It reached a peak after 0.7 ms and inactivated relatively slowly (at 2.1 ms the absolute value was reduced by approximately 30%). The amplitude of the fast component, estimated by subtracting the amplitude of the slow component from the calculated total tail current amplitude, reached a peak (three times larger than that of the slow component) after 0.5 ms and inactivated relatively fast (at 2.1 ms it was reduced by approximately 65%). The results were explained by a novel Na channel model, comprising two open states bifurcating from a common closed state and with separate inactivating pathways. A voltage-regulated use of the two pathways explains a number of findings reported in the literature.     

Two modes of gating during late Na+ channel currents in frog sartorius muscle

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.     

Alteration of the conductance of Na+ channels in the nodal membrane of frog nerve by holding potential and tetrodotoxin

(1) Na+ currents and Na+-current fluctuations were measured in myelinated frog nerve fibres at 15 degrees C during 7.7 ms depolarizations to V = 40, 60 and 80 mV. (2) The conductance gamma of a single Na+ channel and the number No of channels per node were calculated from ensemble average values of the mean Na+ current and the variance of Na+-current fluctuations. (3) For a hyperpolarizing holding potential of VH = -28 mV the mean values of the channel conductance and number were gamma = 9.8 pS and No = 74000. (4) After changing the holding potential to the resting potential (VH = 0) the conductance gamma increased by a factor of 1.37 whereas the number No decreased by a factor of 0.60. (5) Addition of 8 nM tetrodotoxin at a holding potential of VH = -28 mV increased gamma by a factor of 1.55 and reduced No by a factor of 0.25. (6) The increase of the channel conductance at reduced channel numbers suggests negative cooperativity between Na+ channels in the nodal membrane.     

Voltage-dependent properties of three different gating modes in single cardiac Na+ channels.

Three different modes of Na+ channel action, the F mode (fast inactivating), the S mode (slowly inactivating), and the P mode (persistent), were studied at different potentials in exceptionally small cell-attached patches containing one and only one channel. Switching between the modes was independent of voltage. In the F mode, the mean open time (tau o) at -30 and -40 mV was 0.14 and 0.16 ms, respectively, which was significantly larger than at -60 and 0 mV, where the values were 0.07 and 0.08 ms, respectively. The time before which half of the first channel openings occurred (t 0.5), decreased from 0.58 ms at -60 mV to 0.14 ms at 0 mV. The fit of steady-state activation with a Boltzmann function yielded a half-maximum value (V 0.5) at -48.1 mV and a slope (k) of 5.6 mV. The mean open time in the S mode increased steadily from 0.12 ms at -80 mV to 1.09 ms at -30 mV, but was not prolonged further at -20 mV (1.07 ms). Concomitantly, t 0.5 decreased from 1.61 ms at -80 mV to 0.22 ms at -20mV. Here the midpoint of steady-state activation was found at -61.2 mV, and the slope was 8.7 mV. The mean open time in the P mode increased from 0.07 ms at -60 mV to 0.45 ms at 0 mV and t 0.5 declined from 2.14 ms at -60 mV to 0.19 ms at +20 mV. Steady-state activation had its midpoint at -14.7 mV, and the slope was 10.9 mV. It is concluded that a single Na+ channel may switch among the F, S, and P mode and that the three modes differ by a characteristic pattern of voltage dependence of tau 0, t 0.5, and steady-state activation.     

Post-repolarization block of cardiac sodium channels by saxitoxin.

Phasic block of rat cardiac Na+ current by saxitoxin was assessed using pulse trains and two-pulse voltage clamp protocols, and the results were fit to several kinetic models. For brief depolarizations (5 to 50 ms) the depolarization duration did not affect the rate of development or the amplitude of phasic block for pulse trains. The pulse train data were well described by a recurrence relation based upon the guarded receptor model, and it provided rate constants that accurately predicted first-pulse block as well as recovery time constants in response to two-pulse protocols. However, the amplitudes and rates of phasic block development at rapid rates (> 5 Hz) were less than the model predicted. For two pulse protocols with a short (10 ms) conditioning step to -30 mV, block developed only after repolarization to -150 mV and then recovered as the interpulse interval was increased. This suggested that phasic block under these conditions was caused by binding with increased affinity to a state that exists transiently after repolarization to -150 mV. This "post-repolarization block" was fit to a three-state model consisting of a transient state with high affinity for the toxin, the toxin bound state, and the ultimate resting state of the channel. This model accounted for the biphasic post-repolarization block development and recovery observed in two-pulse protocols, and it more accurately described phasic block in pulse trains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of batrachotoxin-modified Na+ channels in GH3 cells. Characterization and pharmacological modification

Batrachotoxin (BTX)-modified Na+ currents were characterized in GH3 cells with a reversed Na+ gradient under whole-cell voltage clamp conditions. BTX shifts the threshold of Na+ channel activation by approximately 40 mV in the hyperpolarizing direction and nearly eliminates the declining phase of Na+ currents at all voltages, suggesting that Na+ channel inactivation is removed. Paradoxically, the steady-state inactivation (h infinity) of BTX-modified Na+ channels as determined by a two-pulse protocol shows that inactivation is still present and occurs maximally near -70 mV. About 45% of BTX-modified Na+ channels are inactivated at this voltage. The development of inactivation follows a sum of two exponential functions with tau d(fast) = 10 ms and tau d(slow) = 125 ms at -70 mV. Recovery from inactivation can be achieved after hyperpolarizing the membrane to voltages more negative than -120 mV. The time course of recovery is best described by a sum of two exponentials with tau r(fast) = 6.0 ms and tau r(slow) = 240 ms at -170 mV. After reaching a minimum at -70 mV, the h infinity curve of BTX-modified Na+ channels turns upward to reach a constant plateau value of approximately 0.9 at voltages above 0 mV. Evidently, the inactivated, BTX-modified Na+ channels can be forced open at more positive potentials. The reopening kinetics of the inactivated channels follows a single exponential with a time constant of 160 ms at +50 mV. Both chloramine-T (at 0.5 mM) and alpha-scorpion toxin (at 200 nM) diminish the inactivation of BTX-modified Na+ channels. In contrast, benzocaine at 1 mM drastically enhances the inactivation of BTX-modified Na+ channels. The h infinity curve reaches minimum of less than 0.1 at -70 mV, indicating that benzocaine binds preferentially with inactivated, BTX-modified Na+ channels. Together, these results imply that BTX-modified Na+ channels are governed by an inactivation process.     

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.     

Batrachotoxin-activated Na+ channels in planar lipid bilayers. Competition of tetrodotoxin block by Na+

Single Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers formed from neutral phospholipids and were observed in the presence of batrachotoxin. The batrachotoxin-modified channel activates in the voltage range -120 to - 80 mV and remains open almost all the time at voltages positive to -60 mV. Low levels of tetrodotoxin (TTX) induce slow fluctuations of channel current, which represent the binding and dissociation of single TTX molecules to single channels. The rates of association and dissociation of TTX are both voltage dependent, and the association rate is competitively inhibited by Na+. This inhibition is observed only when Na+ is increased on the TTX binding side of the channel. The results suggest that the TTX receptor site is located at the channel's outer mouth, and that the Na+ competition site is not located deeply within the channel's conduction pathway.     

Kinetic properties of single sodium channels in rat heart and rat brain

Single Na channel currents were compared in ventricular myocytes and cortical neurons of neonatal rats using the gigaseal patch-clamp method to determine whether tissue-specific differences in gating can be detected at the single-channel level. Single-channel currents were recorded in cell-attached and excised membrane patches at test potentials of -70 to -20 mV and at 9-11 degrees C. In both cell-attached and excised patches brain Na channel mean open time progressively increased from less than 1 ms at -70 mV to approximately 2 ms at -20 mV. Near threshold, single openings with dispersed latencies were observed. By contrast, in cell-attached patches, heart Na channel mean open time peaked near -50 mV, was three times brain Na channel mean open time, and declined continuously to approximately 2 ms at -20 mV. Near threshold, openings occurred frequently usually as brief bursts lasting several milliseconds and rarely as prolonged bursts lasting tens of milliseconds. Unlike what occurs in brain tissue where excision did not change gating, in excised heart patches both the frequency of prolonged bursting and the mean open time of single units increased markedly. Brain and cardiac Na channels can therefore be distinguished on the basis of their mean open times and bursting characteristics.     

Ionic currents and ion channels of lobster olfactory receptor neurons

The role of the soma of spiny lobster olfactory receptor cells in generating odor-evoked electrical signals was investigated by studying the ion channels and macroscopic currents of the soma. Four ionic currents; a tetrodotoxin-sensitive Na+ current, a Ca++ current, a Ca(++)-activated K+ current, and a delayed rectifier K+ current, were isolated by application of specific blocking agents. The Na+ and Ca++ currents began to activate at -40 to -30 mV, while the K+ currents began to activate at -30 to -20 mV. The size of the Na+ current was related to the presence of a remnant of a neurite, presumably an axon, and not to the size of the soma. No voltage-dependent inward currents were observed at potentials below those activating the Na+ current, suggesting that receptor potentials spread passively through the soma to generate action potentials in the axon of this cell. Steady-state inactivation of the Na+ current was half-maximal at -40 mV. Recovery from inactivation was a single exponential function that was half-maximal at 1.7 ms at room temperature. The K+ currents were much larger than the inward currents and probably underlie the outward rectification observed in this cell. The delayed rectifier K+ current was reduced by GTP-gamma-S and AIF-4, agents which activate GTP-binding proteins. The channels described were a 215-pS Ca(++)-activated K+ channel, a 9.7-pS delayed rectifier K+ channel, and a 35-pS voltage-independent Cl- channel. The Cl- channel provides a constant leak conductance that may be important in stabilizing the membrane potential of the cell.     

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.     

Estimation of Na+ dwell time in the gramicidin A channel. Na+ ions as blockers of H+ currents

The permeation of Na+ through gramicidin A channels shows a simple saturation with increasing Na+ concentration that can be described by two different models. The first model assumes that one Na+ binds to the channel with high affinity (approximately 30 M-1) and that conduction occurs by a 'knock-on' mechanism requiring double occupancy of the channel; the other model assumes that Na+ binding is of low affinity (less than 1 M-1), and that double occupancy of the channel is rare. NMR measurements have shown tight Na+ binding, favoring the first model, but measurements of flux ratios and water transport support the second model. We present here a relatively model-independent measurement of the dwell time of Na+ inside the channel, in which we characterize the fluctuations in H+ current through the channel induced by 'block' from the more slowly permeating Na+ ions. The mean Na+ dwell time inside the channel is estimated to be approximately 10 ns at a membrane potential of 200 mV. This result is inconsistent with tight Na+ binding, thus favoring the second model.     

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.     

Simultaneous measurements of Ca2+ currents and intracellular Ca2+ concentrations in single skeletal muscle fibers of the frog

The relationship between Ca2+ current amplitudes and myoplasmic Ca2+ transients was studied in single muscle fibers. Segments of muscle fibers were voltage-clamped in a double Vaseline gap chamber. Ca2+ transients were measured as an optical signal derived from the interaction between Ca2+ and the dye antipyrylazo III. The cells were maintained at -90 mV. Ca2+ currents were detected at pulse potentials to -50 mV, reached a maximum value at 0 mV, were reduced in size for larger depolarizations, and reversed at about 40 mV. Ca2+ transients were also detected at -50 Mv and progressively increased in size with larger pulse potentials up to 10 mV. Depolarizations to voltages greater than 10 mV did not further increase the size of the transient. The magnitude and time course of transients from 10 to 70 mV were almost identical Ca2+ fluxes into the myoplasm (Ca2+ input fluxes) were calculated from the Ca2+ transients applying a removal model. The size of the input fluxes increased with depolarization up to 0 mV. Between 0 and 70 mV the peak input flux slightly increased, while the flux measured at 200 ms remained unchanged. In conclusion, Ca2+ transients and input fluxes were not reduced during pulses to large positive potentials, even though a drastic reduction of Ca2+ current occurred at these potentials. These observations make it very unlikely that a voltage-dependent Ca2+ entry is the triggering signal for contraction.     

Cardiac Na currents and the inactivating, reopening, and waiting properties of single cardiac Na channels

Tetrodotoxin (TTX)-sensitive Na currents were examined in single dissociated ventricular myocytes from neonatal rats. Single channel and whole cell currents were measured using the patch-clamp method. The channel density was calculated as 2/micron 2, which agreed with our usual finding of four channels per membrane patch. At 20 degrees C, the single channel conductance was 20 pS. The open time distributions were fit by a single-exponential function with a mean open time of approximately 1.0 ms at membrane potentials from -60 to -40 mV. Averaged single channel and whole cell currents were similar when scaled and showed both fast and slow rates of inactivation. The inactivation and activation gating shifted quickly to hyperpolarized potentials for channels in cell-attached as well as excised patches, whereas a much slower shift occurred in whole cells. Slowly inactivating currents were present in both whole cell and single channel current measurements at potentials as positive as -40 mV. In whole cell measurements, the potential range could be extended, and slow inactivation was present at potentials as positive as -10 mV. The curves relating steady state activation and inactivation to membrane potential had very little overlap, and slow inactivation occurred at potentials that were positive to the overlap. Slow inactivation is in this way distinguishable from the overlap or window current, and the slowly inactivating current may contribute to the plateau of the rat cardiac action potential. On rare occasions, a second set of Na channels having a smaller unit conductance and briefer duration was observed. However, a separate set of threshold channels, as described by Gilly and Armstrong (1984. Nature [Lond.]. 309:448), was not found. For the commonly observed Na channels, the number of openings in some samples far exceeded the number of channels per patch and the latencies to first opening or waiting times were not sufficiently dispersed to account for the slowly inactivating currents: the slow inactivation was produced by channel reopening. A general model was developed to predict the number of openings in each sample. Models in which the number of openings per sample was due to a dispersion of waiting times combined with a rapid transition from an open to an absorbing inactivated state were unsatisfactory and a model that was more consistent with the results was identified.     

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   

Modification of sodium inactivation in myelinated nerve by Anemonia toxin II and iodate. Analysis of current fluctuations and current relaxations

(1) Na+ currents and Na+ current fluctuations were measured in single myelinated nerve fibres of Rana esculenta under voltage-clamp conditions. The process of Na+ inactivation was modified by external treatment with 7 microM Anemonia Toxin II or by internal application of 20 or 40 mM IO3(-). (2) At depolarization of 24 and 32 mV the spectral density of Na+ current fluctuations could be described as the sum of two contributions, Sh(f) and Sm(f), representing the spectrum from fluctuations of the inactivation (h) and activation (m) gates, respectively. At higher depolarizations of 40 and 48 mV the low frequency (h) fluctuations could be better fitted by the sum, Sh1(f)+Sh2(f), of two separate Lorentzian functions. (3) The Na+ current and the variance of Na+ current fluctuations between 150 and 450 ms after depolarization are increased by one order of magnitude after application of Anemonia Toxin II or IO3(-). (4) The kinetics of Na+ current inactivation were described as A1 x exp(-t/tau h1) + A2 x exp(-t/tau h2) + B. The constant, tau h1, of fast Na+ inactivation was the same in normal and modified nerve fibres. The slow inactivation time constant, tau h2, increased with increasing depolarizations in modified fibres but decreased under control conditions. In all cases tau h2 showed a similar voltage dependence as the time constant found by fitting the low frequency fluctuations of Na+ current with one Lorentzian function, Sh(f). (5) It is concluded that Anemonia Toxin II and IO3(-) modify a fraction of Na+ channels in an all-or-none manner. A lower limit of the number of modified Na+ channels is estimated from the Na+ current and the variance Na+ current fluctuations. 7 microM external Anemonia Toxin II modifies more than 17% and 20 or 40 mM internal IO3(-) more than 8% of all Na+ channels. The inactivation gates in modified channels experience an electric field different from that in normal fibres.     

Inward current generated by Na-Ca exchange during the action potential in single atrial cells of the rabbit.

To investigate the underlying ionic mechanism of the late plateau phase of the action potential in rabbit atrium the whole-cell patch-clamp technique with intracellular perfusion was used. We recorded the inward current during repolarizations following a brief 2 ms depolarizing pulse to +40 mV from a holding potential of between -70 and -80 mV. The development of this current coincides with the onset of the late plateau phase of the action potential. Peak activation of the current occurs about 10 ms from the beginning of the depolarizing pulse, and it decays spontaneously with a slow timecourse. Its voltage dependency from -40 mV to +40 mV shows very steep activation (-40 to -20 mV) and shows almost the same maximum magnitude between -10 mV and +40 mV. This behaviour is quite different from that of the calcium current. The inward current and the late plateau phase of the action potential were both abolished by the application of 5 mM EGTA, 1 microM ryanodine and by reducing the Na+ gradient. The fully activated current-voltage relation of the inward current was plotted as the difference current before and after treatment with Ryanodine, Diltiazem, 20 mM Na+ inside or 30% Na+ outside and shows an exponential voltage dependence with the largest magnitude of the current occurring at negative potentials. The current-voltage (I-V) curve was well fitted by the Na-Ca exchange equation, i = A exp (-(1 - r)EF/RT). The results suggest that the inward current contributes to the generation of the late plateau phase of the rabbit atrial action potential, and is activated by intracellular calcium released from the sarcoplasmic reticulum. Sarcoplasmic reticulum calcium release appears to be triggered both by the membrane voltage and by the calcium current. It is concluded that the inward current is generated by Na-Ca exchange.