Phencyclidine (PCP) blocks glutamate-activated postsynaptic currents

Phencyclidine (PCP) was tested on the metathoracic tibialis muscles of Locusta migratoria. In physiological solution, the peak amplitude of the excitatory postsynaptic currents (EPSCs) evoked by nerve stimulation was linearly related to membrane potential between -50 and -150 mV. The decay time constant of the EPSC (tau EPSC) was exponentially dependent on voltage and decreased with hyperpolarization. The membrane potential change required to produce an e-fold change in tau EPSC was 315 mV. PCP (5-40 microM) produced a concentration-dependent depression of both EPSC peak amplitude and tau EPSC. A slight nonlinearity in the current-voltage relationship could be discerned at high concentrations of PCP. The shortening of the decay time constant of EPSC (tau EPSC) occurred without significant change in the voltage sensitivity observed under control conditions. Under all experimental conditions, the decay of the EPSCs remained a single exponential of time. Fluctuation analysis indicated that 5 microM PCP shortens the lifetime of the glutamate-activated channels by 25.7 +/- 3%. PCP (10-80 microM) did not induced desensitization of the glutamate receptors. These results suggest that PCP interacts with the open conformation of ion channels activated by the glutamate receptor.     

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

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

The voltage dependence of the decay of the excitatory postsynaptic current and the effect of concanavalin A at the crayfish neuromuscular junction

In voltage clamped crayfish muscle fibers the time constant tau of decay of the EPSC was measured at different clamp potentials E. At 6 degrees C, the average potential dependence of tau is described by tau = 2.3 ms.eE/328 mV. tau was shorter in fast fibers than in slow ones. Concanavalin A supressed the potential dependence by tau, resulting in an increase in tau compared with the control, especially at high negative potentials.     

Comparison of excitatory currents activated by different transmitters on crustacean muscle. I. Acetylcholine-activated channels

The properties of acetylcholine-activated excitatory currents on the gm1 muscle of three marine decapod crustaceans, the spiny lobsters Panulirus argus and interruptus, and the crab Cancer borealis, were examined using either noise analysis, analysis of synaptic current decays, or analysis of the voltage dependence of ionophoretically activated cholinergic conductance increases. The apparent mean channel open time (tau n) obtained from noise analysis at -80 mV and 12 degrees C was approximately 13 ms; tau n was prolonged e-fold for about every 100-mV hyperpolarization in membrane potential; tau n was prolonged e- fold for every 10 degrees C decrease in temperature. Gamma, the single- channel conductance, at 12 degrees C was approximately 18 pS and was not affected by voltage; gamma was increased approximately 2.5-fold for every 10 degrees C increase in temperature. Synaptic currents decayed with a single exponential time course, and at -80 mV and 12 degrees C, the time constant of decay of synaptic currents, tau ejc, was approximately 14-15 ms and was prolonged e-fold about every 140-mV hyperpolarization; tau ejc was prolonged about e-fold for every 10 degrees C decrease in temperature. The voltage dependence of the amplitude of steady-state cholinergic currents suggests that the total conductance increase produced by cholinergic agonists is increased with hyperpolarization. Compared with glutamate channels found on similar decapod muscles (see the following article), the acetylcholine channels stay open longer, conduct ions more slowly, and are more sensitive to changes in the membrane potential.     

Voltage dependence of proton pumping by bacteriorhodopsin is regulated by the voltage-sensitive ratio of M1 to M2.

The voltage dependence of light-induced proton pumping was studied with bacteriorhodopsin (bR) from Halobacterium salinarum, expressed in the plasma membrane of oocytes from Xenopus laevis in the range -160 mV to +60 mV at different light intensities. Depending on the applied field, the quenching effect by blue light, which bypasses the normal photo and transport cycle, is drastically increased at inhibiting (negative) potentials, and is diminished at pump current increasing (positive) potentials. At any potential, two processes with different time constants for the M --> bR decay of approximately 5 ms (tau1) and approximately 20 ms (tau2) are obtained. At pump-inhibiting potentials, a third, long-lasting process with tau3 approximately 300 ms at neutral pH is observed. The fast processes (tau1, tau2) can be assigned to the decay of M2 in the normal pump cycle, i.e., to the reprotonation of the Schiff base via the cytoplasmic side, whereas tau3 is due to the decay of M1 without net pumping, i.e., the reprotonation of the Schiff base via the extracellular side. The results are supported by determination of photocurrents induced by bR on planar lipid films. The pH dependence of the slow decay of M1 is fully in agreement with the interpretation that the reprotonation of the Schiff base occurs from the extracellular side. The results give strong evidence that an externally applied electrical field changes the ratio of the M1 and the M2 intermediate. As a consequence, the transport cycle branches into a nontransporting cycle at negative potentials. This interpretation explains the current-voltage behavior of bR on a new basis, but agrees with the isomerisation, switch, transfer model for vectorial transport.     

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.     

The voltage-dependent proton pumping in bacteriorhodopsin is characterized by optoelectric behavior

The light-driven proton pump bacteriorhodopsin (bR) was functionally expressed in Xenopus laevis oocytes and in HEK-293 cells. The latter expression system allowed high time resolution of light-induced current signals. A detailed voltage clamp and patch clamp study was performed to investigate the DeltapH versus Deltapsi dependence of the pump current. The following results were obtained. The current voltage behavior of bR is linear in the measurable range between -160 mV and +60 mV. The pH dependence is less than expected from thermodynamic principles, i.e., one DeltapH unit produces a shift of the apparent reversal potential of 34 mV (and not 58 mV). The M(2)-BR decay shows a significant voltage dependence with time constants changing from 20 ms at +60 mV to 80 ms at -160 mV. The linear I-V curve can be reconstructed by this behavior. However, the slope of the decay rate shows a weaker voltage dependence than the stationary photocurrent, indicating that an additional process must be involved in the voltage dependence of the pump. A slowly decaying M intermediate (decay time > 100 ms) could already be detected at zero voltage by electrical and spectroscopic means. In effect, bR shows optoelectric behavior. The long-lived M can be transferred into the active photocycle by depolarizing voltage pulses. This is experimentally demonstrated by a distinct charge displacement. From the results we conclude that the transport cycle of bR branches via a long-lived M(1)* in a voltage-dependent manner into a nontransporting cycle, where the proton release and uptake occur on the extracellular side.     

Spontaneous and GABA-induced single channel currents in cultured murine spinal cord neurons

Intracellular and patch clamp recordings were made from embryonic mouse spinal cord neurons growing in primary cell culture. Outside-out membrane patches obtained from these cells usually showed spontaneous single channel currents when studied at the resting potential (-56 +/- 1.5 mV). In 18 out of 30 patches tested, spontaneous single channel activity was abolished by making Tris+ the major cation on both sides of the membrane. The remaining patches continued to display spontaneous single channel currents under these conditions. These events reversed polarity at a patch potential of 0 mV and displayed a mean single channel conductance of 24 +/- 1.2 pS. Application of the putative inhibitory transmitter gamma-aminobutyric acid (0.5-10 microM) to outside-out patches of spinal cord cell membrane induced single channel currents in 10 out of 15 patches tested. These channels had a primary conductance of 29 +/- 2.8 pS in symmetrical 145 mM Cl- solutions. Frequency distributions for the open times of these channels were well fit by the sum of a fast exponential term ("of") with a time constant tau of = 4 +/- 1.3 ms and a slow exponential term ("os") with a time constant tau os = 24 +/- 8.1 ms. Frequency distributions for channel closed times were also well fit by a double exponential equation, with time constants tau cf = 2 +/- 0.2 ms and tau cs = 62 +/- 20.9 ms.     

Influence of external pH on two types of low-voltage-activated calcium currents in primary sensory neurons of rats

The influence of extracellular pH (pH(o)) on low-voltage-activated calcium channels of acutely isolated DRG neurons of rats was examined using the whole cell patch-clamp technique. It has been found that in the neurons of middle size with capacitance C=60+/-4.8 pF (mean+/-S.E., n=8) extracellular acidification from pH(o) 7.35 to pH(o) 6.0 significantly and reversibly decreased LVA calcium current densities by 75+/-3.7%, shifted potential for half-maximal activation to more positive voltages by 18.7+/-0.6 mV with significant reduction of its voltage dependence. The half-maximal potential of steady-state inactivation shifted to more positive voltages by 12.1+/-1.7 mV (n=8) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of middle size have midpoint pK(a)=6.6+/-0.02 and hill coefficient h=0.94+/-0.04 (n=5). In small cells with capacitance C=26+/-3.6 pF (n=5), acidosis decreased LVA calcium current densities only by 15.3+/-1.3% and shifted potential for half-maximal activation by 5.5+/-1.0 mV with reduction of its voltage dependence. Half-maximal potential of steady-state inactivation shifted to more positive voltages by 10+/-1.6 mV (n=4) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of small size have midpoint pK(a)=7.9+/-0.04 and hill coefficient h=0.25+/-0.1 (n=4). These two identified types of LVA currents besides different pH sensitivity demonstrated different kinetic properties. The deactivation of LVA currents with weak pH sensitivity after switching off depolarization to -30 mV had substantially longer decay time than do currents with strong pH sensitivity (tau(d) approximately 5 ms vs. 2 ms respectively). It was found that the prolongation of depolarization steps slows the subsequent deactivation of T-type currents in small DRG neurons. Deactivation traces in these neurons were better described by the sum of two exponentials. Thus, we suppose that T-type channels in small DRG neurons are presented mostly by alpha1I subunit. We suggest that these two types of LVA calcium channels with different sensitivity to external pH can be differently involved in the origin of neuropathic changes.     

The initial inward current in spherical clusters of chick embryonic heart cells

The rapid inward sodium current in spherical clusters of 11-d-old embryonic chick heart cells, ranging in size between 65 and 90 micron diameter, was studied using the two-microelectrode voltage-clamp technique. Using these preparations, it was possible to resolve the activation phase of the rapid inward current for potentials negative to -25 mV at 37 degrees C. The rapid inward current exhibited a voltage and time dependence similar to that observed in other excitable tissues. It was initiated at potential steps more positive than -45 mV. The magnitude of the current reached its maximum value at a potential of approximately -20 mV. The measured reversal potential was that predicted by the Nernst equation for sodium ions. The falling phase of the current followed a single exponential time-course with a time constant of inactivation, tau h, ranging between 2.14 ms at -40 mV and 0.18 ms at -5 mV. The time constant of inactivation, tau h, determined by a single voltage-step protocol was compared to the constant, tau c, determined by a double voltage-step protocol and no significant different between the two constants of inactivation was found. Furthermore, the time constants of inactivation and reactivation at the same potential in the same preparation were similar. The results of this study demonstrate that the sodium current of heart cells recorded at 37 degrees C can be described by Hodgkin-Huxley kinetics with speeds approximately four times faster than the squid giant axon at 15 degrees C.     

Effect of ajmaline on transient outward current in rat ventricular myocytes

The mechanism of ajmaline-induced inhibition of the transient outward current (I(to)) has been investigated in right ventricular myocytes of rat using the whole cell patch clamp technique. Ajmaline decreased the amplitude and the time integral of I(to) in a concentration-dependent, but frequency- and use-independent manner. In contrast to the single exponential time course of I(to)-inactivation in control conditions (tau(i) = 37.1 +/- 2.7 ms), the apparent inactivation was fitted by a sum of two exponentials under the effect of ajmaline with concentration-dependent fast and slow components (tau(f) = 11.7 +/- 0.8 ms, tau(s) = 57.6 +/- 2.7 ms at 10 micromol/l) suggesting block development primarily in the open channel state. An improved expression enabling to calculate the association and dissociation rate constants from the concentration dependence of tau(f) and tau(s) was derived and resulted in k(on) = 4.57 x 10(6) +/- 0.32 x 10(6) mol(-1).l.s(-1) and k(off) = 20.12 +/- 5.99 s(-1). The value of K(d) = 4.4 micromol/l calculated as k(off) / k(on) was considerably lower than IC(50) = 25.9 +/- 2.9 micromol/l evaluated from the concentration dependence of the integrals of I(to). Simulations on a simple model combining Hodgkin-Huxley type gating kinetics and drug-channel interaction entirely in open channel state agreed well with the experimental data including the difference between the K(d) and IC(50). According to the model, the fraction of blocked channels increases upon depolarization and declines if depolarization is prolonged. The repolarizing step induces recovery from block with time constant of 52 ms. We conclude that in the rat right ventricular myocytes, ajmaline is an open channel blocker with fast recovery from the block at resting voltage.     

Acute thyroid hormone promotes slow inactivation of sodium current in neonatal cardiac myocytes.

Sodium current (INa) inactivation kinetics in neonatal cardiac myocytes were analyzed using whole cell voltage clamp before and after acute treatments with thyroid hormone (3,5,3'-triiodo-L-thyronine, T3). In untreated neonatal myocytes, INa inactivation was predominantly mono-exponential, with 93 +/- 3% (S.D.; n = 9) of the peak amplitude decaying with a time constant, tau h1, of 1.8 +/- 0.5 ms at -30 mV. The remaining 7% of control INa decayed more slowly, with a time constant, tau h2, of 9.3 +/- 3.0 ms at -30 mV. The contribution of slowly-inactivating channels to peak current was increased from 7% to 43 +/- 27% within 5 min of exposure to 5-20 nM T3 (nine cells; P less than 0.005). The time constants for both the fast- and slow-inactivating components of peak current (tau h1 and tau h2) were not significantly changed by acute T3 treatment, nor was steady-state INa inactivation (h infinity) affected. Thyroid hormone action on sodium inactivation was partially reversible by lidocaine. These findings indicate that T3 acts at the neonatal cardiac cell membrane to promote slow inactivation kinetics in sodium channels.     

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

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

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.     

Fast voltage clamp discloses a new component of presteady-state currents from the Na(+)-glucose cotransporter.

The human Na(+)-glucose cotransporter (hSGLT1) has been shown to generate, in the absence of sugar, presteady-state currents in response to a change in potential, which could be fitted with single exponentials once the voltage had reached a new constant value. By the cut-open oocyte technique (voltage rising-speed approximately 1 mV/microsecond), phlorizin-sensitive transient currents could be detected with a higher time resolution during continuous intracellular perfusion. In the absence of sugar and internal Na+, and with 90 mM external Na+ concentration ([Na+]o), phlorizin-sensitive currents exhibited two relaxation time-constants: tau 1 increased from 2 to 10 ms when Vm decreased from +60 mV to -80 mV and remained at 10 ms for more negative Vm; tau 2 ranged from 0.4 to 0.8 ms in a weakly voltage-dependent manner. According to a previously proposed model, these two time constants could be accounted for by 1) Na+ crossing a fraction of the membrane electrical field to reach its binding site on the carrier and 2) conformational change of the free carrier. To test this hypothesis, the time constants were measured as [Na+]o was progressively reduced to 0 mM. At 30 and 10 mM external Na+, tau 1 reached the same plateau value of 10 ms but at more negative potentials (-120 and -160 mV, respectively). Contrary to the prediction of the model, two time constants continued to be detected in the bilateral absence of Na+ (at pH 8.0). Under these conditions, tau 1 continuously increased through the whole voltage range and did not reach the 10 ms level even when Vm had attained -200 mV while tau 2 remained in the range of 0.4-0.8 ms. These results indicate that 1) conformational change of the free carrier across the membrane must occur in more than one step and 2) Na+ binding/debinding is not responsible for either of the two observed exponential components of transient currents. By use of the simplest kinetic model accounting for the portion of the hSGLT1 transport cycle involving extracellular Na+ binding/debinding and the dual-step conformational change of the free carrier, tau 1 and tau 2 were fitted throughout the voltage range, and a few sets of parameters were found to reproduce the data satisfactorily. This study shows that 1) tau 1 and tau 2 correspond to two steps in the conformational change of the free carrier, 2) Na+ binding/debinding modulates the slow time constant (tau 1) and 3) a voltage-independent slow conformational change of the free carrier accounts for the observed plateau value of 10 ms.     

Gating of mammalian cardiac gap junction channels by transjunctional voltage.

Numerous two-cell voltage-clamp studies have concluded that the electrical conductance of mammalian cardiac gap junctions is not modulated by the transjunctional voltage (Vj) profile, although gap junction channels between low conductance pairs of neonatal rat ventricular myocytes are reported to exhibit Vj-dependent behavior. In this study, the dependence of macroscopic gap junctional conductance (gj) on transjunctional voltage was quantitatively examined in paired 3-d neonatal hamster ventricular myocytes using the double whole-cell patch-clamp technique. Immunolocalization with a site-specific antiserum directed against amino acids 252-271 of rat connexin43, a 43-kD gap junction protein as predicted from its cDNA sequence, specifically stained zones of contact between cultured myocytes. Instantaneous current-voltage (Ij-Vj) relationships of neonatal hamster myocyte pairs were linear over the entire voltage range examined (0 less than or equal to Vj less than or equal to +/- 100 mV). However, the steady-state Ij-Vj relationship was nonlinear for Vj greater than +/- 50 mV. Both inactivation and recovery processes followed single exponential time courses (tau inactivation = 100-1,000 ms, tau recovery approximately equal to 300 ms). However, Ij recovered rapidly upon polarity reversal. The normalized steady-state junctional conductance-voltage relationship (Gss-Vj) was a bell-shaped curve that could be adequately described by a two-state Boltzmann equation with a minimum Gj of 0.32-0.34, a half-inactivation voltage of -69 and +61 mV and an effective valence of 2.4-2.8. Recordings of gap junction channel currents (ij) yielded linear ij-Vj relationships with slope conductances of approximately 20-30 and 45-50 pS. A kinetic model, based on the Boltzmann relationship and the polarity reversal data, suggests that the opening (alpha) and closing (beta) rate constants have nearly identical voltage sensitivities with a Vo of +/- 62 mV. The data presented in this study are not consistent with the contingent gating scheme (for two identical gates in series) proposed for other more Vj-dependent gap junctions and alternatively suggest that each gate responds to the applied Vj independently of the state (open or closed) of the other gate.     

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   

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)     

Decrease of inward rectification as a mechanism for arachidonic acid-induced potentiation of hKir2.3

Previously, we showed that arachidonic acid (AA) potentiates currents flowing through a cloned human inwardly rectifying K(+) channel, hKir2.3. The mechanism by which this potentiation occurs is not understood. Here, we report that this potentiation is mediated by multiple mechanisms and that one of them, which we studied in more detail, is consistent with AA-induced decrease of inward rectification. AA (10 micro M) potentiation of hKir2.3 whole-cell current increased with depolarization (40% greater at -47 mV than at -127 mV) and decreased with elevated extracellular [K(+)] (158+/-21%, 56+/-8% and 38+/-9% in 5.4, 70 and 135 mM K(+), respectively). Hyperpolarization elicited inward currents consisting of an instantaneous and two time-dependent components with time constants (at -97 mV) of 6.4+/-1.1 ms and 27.8+/-4.1 ms, respectively. AA (10 microM) significantly decreased the slow time constant (14.1+/-0.7 ms). Consistent with the kinetic changes, AA (10 microM) right-shifted the voltage dependence of the chord conductance (mid-point shifted by +9 mV). In inside-out patches where inward rectification was minimal, AA potentiation (38+/-3%) was smaller than in whole-cell recording and was not voltage dependent. These results are consistent with the idea that AA potentiates hKir2.3 in part by decreasing inward rectification of the channel.