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.     

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   

Sodium channel inactivation from closed states: evidence for an intrinsic voltage dependency.

The time course of Na channel inactivation from closed states was determined on inside-out excised patches from neuroblastoma N1E 115. Closed-state inactivation develops as a single exponential with mean time constants of 66.4 ms at -80 mV, 29.6 ms at -70 mV, 20.1 ms at -60 mV, and 15.1 ms at -50 mV. Corresponding mean steady-state values of the fitted exponentials were 0.321, 0.098, 0.035, and 0. Closed-state inactivation, in general, should develop either with a delay or as more than one exponential, depending on which closed state(s) directly inactivate. The absence of additional components cannot be attributed to a rate of exchange between closed states too rapid to detect. The time course is simply accounted for if all closed states directly inactivate and do so with the same rate constant for each closed state, suggesting that those conformational changes constituting the transitions between closed states have little effect on the structural components involved in inactivation. Closed to inactivated rate constants ranged from a mean of 0.0108 ms-1 at -80 mV to 0.0690 ms-1 at -50 mV. This voltage dependency is entirely intrinsic to closed-state inactivation with closed to inactivated rate constants similar for all closed states. Over the potential range studied nearly all the inactivation is from closed states.     

Inactivation of A currents and A channels on rat nodose neurons in culture

Cultured sensory neurons from nodose ganglia were investigated with whole-cell patch-clamp techniques and single-channel recordings to characterize the A current. Membrane depolarization from -40 mV holding potential activated the delayed rectifier current (IK) at potentials positive to -30 mV; this current had a sigmoidal time course and showed little or no inactivation. In most neurons, the A current was completely inactivated at the -40 mV holding potential and required hyperpolarization to remove the inactivation; the A current was isolated by subtracting the IK evoked by depolarizations from -40 mV from the total outward current evoked by depolarizations from -90 mV. The decay of the A current on several neurons had complex kinetics and was fit by the sum of three exponentials whose time constants were 10-40 ms, 100-350 ms, and 1-3 s. At the single-channel level we found that one class of channel underlies the A current. The conductance of A channels varied with the square root of the external K concentration: it was 22 pS when exposed to 5.4 mM K externally, the increased to 40 pS when exposed to 140 mM K externally. A channels activated rapidly upon depolarization and the latency to first opening decreased with depolarization. The open time distributions followed a single exponential and the mean open time increased with depolarization. A channels inactivate in three different modes: some A channels inactivated with little reopening and gave rise to ensemble averages that decayed in 10-40 ms; other A channels opened and closed three to four times before inactivating and gave rise to ensemble averages that decayed in 100-350 ms; still other A channels opened and closed several hundred times and required seconds to inactivate. Channels gating in all three modes contributed to the macroscopic A current from the whole cell, but their relative contribution differed among neurons. In addition, A channels could go directly from the closed, or resting, state to the inactivated state without opening, and the probability for channels inactivating in this way was greater at less depolarized voltages. In addition, a few A channels appeared to go reversibly from a mode where inactivation occurred rapidly to a slow mode of inactivation.     

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.     

A rapidly activating and slowly inactivating potassium channel cloned from human heart. Functional analysis after stable mammalian cell culture expression

The electrophysiological properties of HK2 (Kv1.5), a K+ channel cloned from human ventricle, were investigated after stable expression in a mouse Ltk- cell line. Cell lines that expressed HK2 mRNA displayed a current with delayed rectifier properties at 23 degrees C, while sham transfected cell lines showed neither specific HK2 mRNA hybridization nor voltage-activated currents under whole cell conditions. The expression of the HK2 current has been stable for over two years. The dependence of the reversal potential of this current on the external K+ concentration (55 mV/decade) confirmed K+ selectivity, and the tail envelope test was satisfied, indicating expression of a single population of K+ channels. The activation time course was fast and sigmoidal (time constants declined from 10 ms to < 2 ms between 0 and +60 mV). The midpoint and slope factor of the activation curve were Eh = -14 +/- 5 mV and k = 5.9 +/- 0.9 (n = 31), respectively. Slow partial inactivation was observed especially at large depolarizations (20 +/- 2% after 250 ms at +60 mV, n = 32), and was incomplete in 5 s (69 +/- 3%, n = 14). This slow inactivation appeared to be a genuine gating process and not due to K+ accumulation, because it was present regardless of the size of the current and was observed even with 140 mM external K+ concentration. Slow inactivation had a biexponential time course with largely voltage-independent time constants of approximately 240 and 2,700 ms between -10 and +60 mV. The voltage dependence of slow inactivation overlapped with that of activation: Eh = -25 +/- 4 mV and k = 3.7 +/- 0.7 (n = 14). The fully activated current-voltage relationship displayed outward rectification in 4 mM external K+ concentration, but was more linear at higher external K+ concentrations, changes that could be explained in part on the basis of constant field (Goldman-Hodgkin-Katz) rectification. Activation and inactivation kinetics displayed a marked temperature dependence, resulting in faster activation and enhanced inactivation at higher temperature. The current was sensitive to low concentrations of 4- aminopyridine, but relatively insensitive to external TEA and to high concentrations of dendrotoxin. The expressed current did not resemble either the rapid or the slow components of delayed rectification described in guinea pig myocytes. However, this channel has many similarities to the rapidly activating delayed rectifying currents described in adult rat atrial and neonatal canine epicardial myocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Characterization of the isoform-specific differences in the gating of neuronal and muscle sodium channels

Human heart (hH1), human skeletal muscle (hSkM1), and rat brain (rIIA) Na channels were expressed in cultured cells and the activation and inactivation of the whole-cell Na currents measured using the patch clamp technique. hH1 Na channels were found to activate and inactivate at more hyperpolarized voltages than hSkM1 and rIIA. The conductance versus voltage and steady state inactivation relationships have midpoints of -48 and -92 mV (hH1), -28 and -72 mV (hSkM1), and -22 and -61 mV (rIIA). At depolarized voltages, where Na channels predominately inactivate from the open state, the inactivation of hH1 is 2-fold slower than that of hSkM1 and rIIA. The recovery from fast inactivation of all three isoforms is well described by a single rapid component with time constants at -100 mV of 44 ms (hH1), 4.7 ms (hSkM1), and 7.6 ms (rIIA). After accounting for differences in voltage dependence, the kinetics of activation, inactivation, and recovery of hH1 were found to be generally slower than those of hSkM1 and rIIA. Modeling of Na channel gating at hyperpolarized voltages where the channel does not open suggests that the slow rate of recovery from inactivation of hH1 accounts for most of the differences in the steady-state inactivation of these Na channels.     

Calcium-dependent inactivation of light-sensitive channels in Drosophila photoreceptors

Whole-cell voltage clamp recordings were made from photoreceptors of dissociated Drosophila ommatidia under conditions when the light- sensitive channels activate spontaneously, generating a "rundown current" (RDC). The Ca2+ and voltage dependence of the RDC was investigated by applying voltage steps (+80 to -100 mV) at a variety of extracellular Ca2+ concentrations (0-10 mM). In Ca(2+)-free Ringer large currents are maintained tonically throughout 50-ms-long voltage steps. In the presence of external Ca2+, hyperpolarizing steps elicit transient currents which inactivate increasingly rapidly as Ca2+ is raised. On depolarization inactivation is removed with a time constant of approximately 10 ms at +80 mV. The Ca(2+)-dependent inactivation is suppressed by 10 mM internal BAPTA, suggesting it requires Ca2+ influx. The inactivation is absent in the trp mutant, which lacks one class of Ca(2+)-selective, light-sensitive channel, but appears unaffected by the inaC mutant which lacks an eye-specific protein kinase C. Hyperpolarizing voltage steps applied during light responses in wild- type (WT) flies before rundown induce a rapid transient facilitation followed by slower inhibition. Both processes accelerate as Ca2+ is raised, but the time constant of inhibition (12 ms with 1.5 mM external Ca2+ at -60 mV) is approximately 10 times slower than that of the RDC inactivation. The Ca(2+)-mediated inhibition of the light response recovers in approximately 50-100 ms on depolarization, recovery being accelerated with higher external Ca2+. The Ca2+ and voltage dependence of the light-induced current is virtually eliminated in the trp mutant. In inaC, hyperpolarizing voltage steps induced transient currents which appeared similar to those in WT during early phases of the light response. However, 200 ms after the onset of light, the currents induced by voltage steps inactivated more rapidly with time constants similar to those of the RDC. It is suggested that the Ca(2+)-dependent inactivation of the light-sensitive channels first occurs at some concentration of Ca2+ not normally reached during the moderate illumination regimes used, but that the defect in inaC allows this level to be reached.     

Two types of Ca2+ currents with different sensitivities to organic Ca2+ channel antagonists in guinea pig pancreatic alpha 2 cells

The possibility that guinea pig pancreatic alpha 2 cells are equipped with more than one type of Ca2+ channel was explored using the patch-electrode voltage-clamp technique. At a holding potential of -100 mV, a slowly developing (tau m approximately 5 ms at -40 mV assuming m2 kinetics) Ca2+ current appeared. This conductance first became detectable at potentials of about -60 mV and reached a maximum amplitude of 50-100 pA between -30 and -20 mV. During long depolarizations, it inactivated completely (tau h approximately 100 ms at -40 mV). Half-maximal steady state inactivation was observed at about -60 mV. A second, more rapidly developing (tau m approximately 2 ms at 0 mV) Ca2+ current was observed during pulses to -40 mV and above. It had a peak amplitude of 150-200 pA between 0 and 10 mV, was less dependent on the holding potential, and inactivated very little, even during long pulses. Both conductances were blocked by Co2+ but were unaffected by tetrodotoxin. The rapidly developing current differed from the slowly developing one in being sensitive to the antagonists D-600 and nifedipine, conducting Ba2+ better than Ca2+, increasing upon exposure to forskolin, and showing time-dependent decay (rundown). These findings indicate that the alpha 2 cells are equipped with two kinds of Ca2+ channels.     

Structural elements in domain IV that influence biophysical and pharmacological properties of human alpha1A-containing high-voltage-activated calcium channels

We have cloned two splice variants of the human homolog of the alpha1A subunit of voltage-gated Ca2+ channels. The sequences of human alpha1A-1 and alpha1A-2 code for proteins of 2510 and 2662 amino acids, respectively. Human alpha1A-2alpha2bdeltabeta1b Ca2+ channels expressed in HEK293 cells activate rapidly (tau+10mV = 2.2 ms), deactivate rapidly (tau-90mV = 148 micros), inactivate slowly (tau+10mV = 690 ms), and have peak currents at a potential of +10 mV with 15 mM Ba2+ as charge carrier. In HEK293 cells transient expression of Ca2+ channels containing alpha1A/B(f), an alpha1A subunit containing a 112 amino acid segment of alpha1B-1 sequence in the IVS3-IVSS1 region, resulted in Ba2+ currents that were 30-fold larger compared to wild-type (wt) alpha1A-2-containing Ca2+ channels, and had inactivation kinetics similar to those of alpha1B-1-containing Ca2+ channels. Cells transiently transfected with alpha1A/B(f)alpha2bdeltabeta1b expressed higher levels of the alpha1, alpha2bdelta, and beta1b subunit polypeptides as detected by immunoblot analysis. By mutation analysis we identified two locations in domain IV within the extracellular loops S3-S4 (N1655P1656) and S5-SS1 (E1740) that influence the biophysical properties of alpha1A. alpha1AE1740R resulted in a threefold increase in current magnitude, a -10 mV shift in steady-state inactivation, and an altered Ba2+ current inactivation, but did not affect ion selectivity. The deletion mutant alpha1ADeltaNP shifted steady-state inactivation by -20 mV and increased the fast component of current inactivation twofold. The potency and rate of block by omega-Aga IVA was increased with alpha1ADeltaNP. These results demonstrate that the IVS3-S4 and IVS5-SS1 linkers play an essential role in determining multiple biophysical and pharmacological properties of alpha1A-containing Ca2+ channels.     

The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. I. Basic characterization and kinetic analysis

Enzymatically isolated myocytes from ferret right ventricles (12-16 wk, male) were studied using the whole cell patch clamp technique. The macroscopic properties of a transient outward K+ current I(to) were quantified. I(to) is selective for K+, with a PNa/PK of 0.082. Activation of I(to) is a voltage-dependent process, with both activation and inactivation being independent of Na+ or Ca2+ influx. Steady-state inactivation is well described by a single Boltzmann relationship (V1/2 = -13.5 mV; k = 5.6 mV). Substantial inactivation can occur during a subthreshold depolarization without any measurable macroscopic current. Both development of and recovery from inactivation are well described by single exponential processes. Ensemble averages of single I(to) channel currents recorded in cell-attached patches reproduce macroscopic I(to) and indicate that inactivation is complete at depolarized potentials. The overall inactivation/recovery time constant curve has a bell-shaped potential dependence that peaks between -10 and -20 mV, with time constants (22 degrees C) ranging from 23 ms (-90 mV) to 304 ms (-10 mV). Steady-state activation displays a sigmoidal dependence on membrane potential, with a net aggregate half- activation potential of +22.5 mV. Activation kinetics (0 to +70 mV, 22 degrees C) are rapid, with I(to) peaking in approximately 5-15 ms at +50 mV. Experiments conducted at reduced temperatures (12 degrees C) demonstrate that activation occurs with a time delay. A nonlinear least- squares analysis indicates that three closed kinetic states are necessary and sufficient to model activation. Derived time constants of activation (22 degrees C) ranged from 10 ms (+10 mV) to 2 ms (+70 mV). Within the framework of Hodgkin-Huxley formalism, Ito gating can be described using an a3i formulation.     

A tyrosine substitution in the cavity wall of a k channel induces an inverted inactivation

Ion permeation and gating kinetics of voltage-gated K channels critically depend on the amino-acid composition of the cavity wall. Residue 470 in the Shaker K channel is an isoleucine, making the cavity volume in a closed channel insufficiently large for a hydrated K(+) ion. In the cardiac human ether-a-go-go-related gene channel, which exhibits slow activation and fast inactivation, the corresponding residue is tyrosine. To explore the role of a tyrosine at this position in the Shaker channel, we studied I470Y. The activation became slower, and the inactivation faster and more complex. At +60 mV the channel inactivated with two distinct rates (tau(1) = 20 ms, tau(2) = 400 ms). Experiments with tetraethylammonium and high K(+) concentrations suggest that the slower component was of the P/C-type. In addition, an inactivation component with inverted voltage dependence was introduced. A step to -40 mV inactivates the channel with a time constant of 500 ms. Negative voltage steps do not cause the channel to recover from this inactivated state (tau > 10 min), whereas positive voltage steps quickly do (tau = 2 ms at +60 mV). The experimental findings can be explained by a simple branched kinetic model with two inactivation pathways from the open state.     

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.     

Voltage- and time-dependent K+ channel currents in the basolateral membrane of villus enterocytes isolated from guinea pig small intestine

Patch-clamp studies were carried out in villus enterocytes isolated from the guinea pig proximal small intestine. In the whole-cell mode, outward K+ currents were found to be activated by depolarizing command pulses to -45 mV. The activation followed fourth order kinetics. The time constant of K+ current activation was voltage-dependent, decreasing from approximately 3 ms at -10 mV to 1 ms at +50 mV. The K+ current inactivated during maintained depolarizations by a voltage- independent, monoexponential process with a time constant of approximately 470 ms. If the interpulse interval was shorter than 30 s, cumulative inactivation was observed upon repeated stimulations. The steady state inactivation was voltage-dependent over the voltage range from -70 to -30 mV with a half inactivation voltage of -46 mV. The steady state activation was also voltage-dependent with a half- activation voltage of -22 mV. The K+ current profiles were not affected by chelation of cytosolic Ca2+. The K+ current induced by a depolarizing pulse was suppressed by extracellular application of TEA+, Ba2+, 4-aminopyridine or quinine with half-maximal inhibitory concentrations of 8.9 mM, 4.6 mM, 86 microM and 26 microM, respectively. The inactivation time course was accelerated by quinine but decelerated by TEA+, when applied to the extracellular (but not the intracellular) solution. Extracellular (but not intracellular) applications of verapamil and nifedipine also quickened the inactivation time course with 50% effective concentrations of 3 and 17 microM, respectively. Quinine, verapamil and nifedipine shifted the steady state inactivation curve towards more negative potentials. Outward single K+ channel events with a unitary conductance of approximately 8.4 pS were observed in excised inside-out patches of the basolateral membrane, when the patch was depolarized to -40 mV. The ensemble current rapidly activated and thereafter slowly inactivated with similar time constants to those of whole-cell K+ currents. It is concluded that the basolateral membrane of guinea pig villus enterocytes has a voltage-gated, time-dependent, Ca(2+)-insensitive, small-conductance K+ channel. Quinine, verapamil, and nifedipine accelerate the inactivation time course by affecting the inactivation gate from the external side of the cell membrane.     

Low molecular weight poly(A)+ mRNA species encode factors that modulate gating of a non-Shaker A-type K+ channel

Voltage-dependent K+ channels control repolarization of action potentials and help establish firing patterns in nerve cells. To determine the nature and role of molecular components that modulate K+ channel function in vivo, we coinjected Xenopus oocytes with cRNA encoding a cloned subthreshold A-type K+ channel (mShal1, also referred to as mKv4.1) and a low molecular weight (LMW) fraction (2-4 kb) of poly(A)+ mRNA (both from rodent brain). Coinjected oocytes exhibited a significant (fourfold) increase in the surface expression of mShal1 K+ channels with no change in the open-channel conductance. Coexpression also modified the gating kinetics of mShal1 current in several respects. Macroscopic inactivation of whole oocyte currents was fitted with the sum of two exponential components. Both fast and slow time constants of inactivation were accelerated at all membrane potentials in coinjected oocytes (tau f = 47.2 ms vs 56.5 ms at 0 mV and tau s = 157 ms vs 225 ms at 0 mV), and the corresponding ratios of amplitude terms were shifted toward domination by the fast component (Af/As = 2.71 vs 1.17 at 0 mV). Macroscopic activation was characterized in terms of the time-to-peak current, and it was found to be more rapid at all membrane potentials in coinjected oocytes (9.9 ms vs 13.5 ms at 0 mV). Coexpression also leads to more rapid recovery from inactivation (approximately 2.4-fold faster at -100 mV). The coexpressed K+ currents in oocytes resemble currents expressed in mouse fibroblasts (NIH3T3) transfected only with mShal1 cDNA. These results indicate that mammalian regulatory subunits or enzymes encoded by LMW mRNA species, which are apparently missing or expressed at low levels in Xenopus oocytes, may modulate gating in some native subthreshold A-type K+ channels.     

A delayed rectifier potassium current in Xenopus oocytes.

A delayed voltage-dependent K+ current endogenous to Xenopus oocytes has been investigated by the voltage-clamp technique. Both activation and inactivation of the K+ current are voltage-dependent processes. The K+ currents were activated when membrane potential was depolarized from a holding potential of -90 to -50 mV. The peak current was reached within 150 ms at membrane potential of +30 mV. Voltage-dependent inactivation of the current was observed by depolarizing the membrane potential from -50 to 0 mV at 10-mV increments. Voltage-dependent inactivation was a slow process with a time constant of 16.5 s at -10 mV. Removal of Ca2+ from the bath has no effect on current amplitudes, which indicates that the current is Ca2+)-insensitive. Tail current analysis showed that reversal potentials were shifted by changing external K+ concentration, as would be expected for a K(+)-selective channel. The current was sensitive to quinine, a K+ channel blocker, with a Ki of 35 microM. The blockade of quinine is voltage-independent in the range of -20 to +60 mV. Whereas oocytes from the same animal have a relatively homogeneous current distribution, average amplitude of the K+ current varied among oocytes from different animals from 30 to 400 nA at membrane potential of +30 mV. Our results indicate the presence of the endogenous K+ current in Xenopus oocytes with characteristics of the delayed rectifier found in some nerve and muscle cells.     

A slow outward current and a hypoosmolality induced anion conductance in embryonic chicken osteoclasts

In this paper we report on a hypoosmolality induced current, I(osmo), in embryonic chicken osteoclasts, which could only be studied when blocking a simultaneously active, unidentified slow outward current, I(slo). I(slo) was observed in all of the examined cells when both the intracellular and extracellular solutions contained sodium as the major cation and no potassium. The current was outwardly rectifying and activated at membrane potentials more positive than -44 +/- 12 mV (n = 31). The time to half activation of the current was also voltage dependent and was 350 ms at Vm = +80 mV, and 78 ms at Vm = +120 mV. The current did not inactivate during periods up to 5 s. Extracellular 4-AP (5 mM), TEA (5 mM) and Ba2+ (1 mM), blockers of K+ conductances in chicken osteoclasts, did not influence I(slo). However, I(slo) was inhibited by 50 microM extracellular verapamil, which allowed us to study I(osmo) in isolation. Exposure of the osteoclasts to hypotonic solution resulted in the development of a depolarization activated I(osmo). It developed after a 1-min delay and reached its maximum within 10 minutes. Half-maximal activation occurred after 4.4 +/- 0.9 min (n = 9). The current activated within a few ms upon depolarization and did not inactivate during at least 5 sec. I(osmo) reversed around the calculated Nernst potential for Cl- (E(Cl) = +7.3 mV and V(rev) = +5.4 +/- 3.6 mV, n = 9). The underlying conductance, G(osmo) exhibited moderate outward rectification around 0 mV in symmetrical Cl- solutions. Ion substitution experiments showed that G(osmo) is an anion conductance with P(Cl) approximately = P(F) > P(gluc) > P(Na). I(osmo) was blocked by 0.5 mM SITS but 50 microM verapamil, 5 mM TEA, 5 mM 4-AP, 1 mM Ba2+, 50 microM cytochalasin D and 0.5 mM alendronate did not have any effect on the current. Cl- currents have been implicated in charge neutralization during osteoclastic acid secretion for bone resorption. The present results imply that osmolality may be a factor controlling this charge neutralization.     

Inactivation of N-type calcium current in chick sensory neurons: calcium and voltage dependence

We have studied the inactivation of high-voltage-activated (HVA), omega- conotoxin-sensitive, N-type Ca2+ current in embryonic chick dorsal root ganglion (DRG) neurons. Voltage steps from -80 to 0 mV produced inward Ca2+ currents that inactivated in a biphasic manner and were fit well with the sum of two exponentials (with time constants of approximately 100 ms and > 1 s). As reported previously, upon depolarization of the holding potential to -40 mV, N current amplitude was significantly reduced and the rapid phase of inactivation all but eliminated (Nowycky, M. C., A. P. Fox, and R. W. Tsien. 1985. Nature. 316:440-443; Fox, A. P., M. C. Nowycky, and R. W. Tsien. 1987a. Journal of Physiology. 394:149-172; Swandulla, D., and C. M. Armstrong. 1988. Journal of General Physiology. 92:197-218; Plummer, M. R., D. E. Logothetis, and P. Hess. 1989. Neuron. 2:1453-1463; Regan, L. J., D. W. Sah, and B. P. Bean. 1991. Neuron. 6:269-280; Cox, D. H., and K. Dunlap. 1992. Journal of Neuroscience. 12:906-914). Such kinetic properties might be explained by a model in which N channels inactivate by both fast and slow voltage-dependent processes. Alternatively, kinetic models of Ca-dependent inactivation suggest that the biphasic kinetics and holding-potential-dependence of N current inactivation could be due to a combination of Ca-dependent and slow voltage- dependent inactivation mechanisms. To distinguish between these possibilities we have performed several experiments to test for the presence of Ca-dependent inactivation. Three lines of evidence suggest that N channels inactivate in a Ca-dependent manner. (a) The total extent of inactivation increased 50%, and the ratio of rapid to slow inactivation increased approximately twofold when the concentration of the Ca2+ buffer, EGTA, in the patch pipette was reduced from 10 to 0.1 mM. (b) With low intracellular EGTA concentrations (0.1 mM), the ratio of rapid to slow inactivation was additionally increased when the extracellular Ca2+ concentration was raised from 0.5 to 5 mM. (c) Substituting Na+ for Ca2+ as the permeant ion eliminated the rapid phase of inactivation. Other results do not support the notion of current-dependent inactivation, however. Although high intracellular EGTA (10 mM) or BAPTA (5 mM) concentrations suppressed the rapid phase inactivation, they did not eliminate it. Increasing the extracellular Ca2+ from 0.5 to 5 mM had little effect on this residual fast inactivation, indicating that it is not appreciably sensitive to Ca2+ influx under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of Ca2+ agonist and depolarization on Ca2+ channel current in guinea pig detrusor cells [published erratum appears in J Gen Physiiol 1996 Jun;107(6):755]

The interaction of large depolarization and dihydropyridine Ca2+ agonists, both of which are known to enhance L-type Ca2+ channel current, was examined using a conventional whole-cell clamp technique. In guinea pig detrusor cells, only L-type Ca2+ channels occur. A second open state (long open state: O2) of the Ca2+ channels develops during large depolarization (at +80 mV, without Ca2+ agonists). This was judged from lack of inactivation of the Ca2+ channel current during the large depolarizing steps (5 s) and slowly deactivating inward tail currents (= 10-15 ms) upon repolarization of the cell membrane to the holding potential (-60 mV). Application of Bay K 8644 (in 2.4 mM Ca(2+)- containing solutions) increased the amplitude of the Ca2+ currents evoked by simple depolarizations, and made it possible to observe inward tail currents (= 2.5-5 ms at -60 mV). The open state induced by large depolarization (O2*) in the Bay K 8644 also seemed hardly to inactivate. After preconditioning with large depolarizing steps, the decay time course of the inward tail currents upon repolarization to the holding potential (-60 mV) was significantly slowed, and could be fitted reasonably with two exponentials. The fast and slow time constants were 10 and 45 ms, respectively, after 2 s preconditioning depolarizations. Qualitatively the same results were obtained using Ba2+ as a charge carrier. Although the amplitudes of the inward currents observed in the test step and the subsequent repolarization to the holding potential were decreased in the same manner by additional application of nifedipine (in the presence of Bay K 8644), the very slow deactivation time course of the tail current was little changed. The additive enhancement by large depolarization and Ca2+ agonists of the inward tail current implies that two mechanisms separately induce long opening of the Ca2+ channels: i.e., that there are four open states.