Sodium currents in the giant axon of the crab Carcinus maenas

Measurements were made of the kinetics and steady-state properties of the sodium conductance changes in the giant axon of the crab Carcinus maenas. The conductance measurements were made in the presence of small concentrations of tetrodotoxin and as much electrical compensation as possible in order to minimize errors caused by the series resistance. After an initial delay of 10-150 microsec, the conductance increase during depolarizing voltage clamp pulses followed the Hodgkin-Huxley kinetics. Values of the time constant for the activation of the sodium conductance lay on a bell-shaped curve with a maximum under 180 microsec at -40 mV (at 18 degrees C). Values of the time constant for the inactivation of the sodium conductance were also fitted using a bell-shaped curve with a maximum under 7 msec at -70 mV. The effects of membrane potential on the fraction of Na channels available for activation studied using double pulse protocols suggest that hyperpolarizing potentials more negative than -100 mV lock a fraction of the Na channels in a closed conformation.     

Effects of N-alcohols on potassium conductance in squid giant axons

The effect of bath application of several short chain N-alcohols on voltage-dependent potassium conductance has been studied in intact giant axons of Loligo forbesi under voltage-clamp conditions. All tested alcohols (methanol, ethanol, propanol, butanol, heptanol and octanol) were found to depress potassium conductance only at concentrations much larger than those necessary to reduce sodium conductance. The efficacy of the different molecules was correlated with the carbon-chain length. In all cases the effects were found to be at least partly reversible. Low concentrations of propanol (100 mM) or heptanol (1 mM) were found to increase potassium conductance whereas higher concentrations had the usual depressing effect. The two alcohols were found to induce a slow inactivation of the potassium conductance. A detailed analysis of the time course of the turning-on of the potassium current for various pulse potentials in the presence of TTX revealed that, for membrane potential values more positive than -20 mV, the time constant of activation was reduced in the presence of propanol or heptanol. The delay which separates the change in potential and the turning-on of the potassium current, which was systematically analysed for different pulse and prepulse potential values, was increased by the two alcohols, the curve relating this delay to prepulse potential being shifted towards larger (positive) delays. This high degree of complexity in the effects on potassium conductance suggests that the alcohol molecules modify several more or less independent mechanisms associated with the turning-on of the potassium current.     

Gating of Na channels. Inactivation modifiers discriminate among models

Macroscopic Na currents were recorded from N18 neuroblastoma cells by the whole-cell voltage-clamp technique. Inactivation of the Na currents was removed by intracellular application of proteolytic enzymes, trypsin, alpha-chymotrypsin, papain, or ficin, or bath application of N-bromoacetamide. Unlike what has been reported in squid giant axons and frog skeletal muscle fibers, these treatments often increased Na currents at all test pulse potentials. In addition, removal of inactivation gating shifted the midpoint of the peak Na conductance-voltage curve in the negative direction by 26 mV on average and greatly prolonged the rising phase of Na currents for small depolarizations. Polypeptide toxins from Leiurus quinquestriatus scorpion and Goniopora coral, which slow inactivation in adult nerve and muscle cells, also increase the peak Na conductance and shift the peak conductance curve in the negative direction by 7-10 mV in neuroblastoma cells. Control experiments argue against ascribing the shifts to series resistance artifacts or to spontaneous changes of the voltage dependence of Na channel kinetics. The negative shift of the peak conductance curve, the increase of peak Na currents, and the prolongation of the rise at small depolarization after removal of inactivation are consistent with gating kinetic models for neuroblastoma cell Na channels, where inactivation follows nearly irreversible activation with a relatively high, voltage-independent rate constant and Na channels open only once in a depolarization. As the same kind of experiment does not give apparent shifting of activation and prolongation of the rising phase of Na currents in adult axon and muscle membranes, the Na channels of these other membranes probably open more than once in a depolarization.     

Modulation of K channels in dialyzed squid axons. ATP-mediated phosphorylation

In squid axons, internally applied ATP potentiates the magnitude of the potassium conductance and slows down its activation kinetics. This effect was characterized using internally dialyzed axons under voltage-clamp conditions. Both amplitude potentiation and kinetic slow-down effects are very selective towards ATP, other nucleotides like GTP and ITP are ineffective in millimolar concentrations. The current potentiation Km for ATP is near 10 microM with no further effects for concentrations greater than 100 microM. ATP effect is most likely produced via a phosphorylative reaction because Mg ion is an obligatory requirement and nonhydrolyzable ATP analogues are without effect. In the presence of ATP, the K current presents more delay, resembling a Cole-Moore effect due to local hyperpolarization of the channel. ATP effect induces a 10-20 mV shift in both activation and inactivation parameters towards more depolarized potentials. As a consequence of this shift, conductance-voltage curves with and without ATP cross at approximately -40 mV. This result is consistent with the hyperpolarization observed with ATP depletion, which is reversed by ATP addition. At potentials around the resting value, addition of ATP removes almost completely K current slow inactivation. It is suggested that a change in the amount of the slow inactivation is responsible for the differences in current amplitude with and without ATP, possibly as a consequence of the additional negative charge carried by the phosphate group. However, a modification of the local potential is not enough to explain completely the differences under the two conditions.     

Block and inactivation of sodium channels in nerve by amino acid derivatives. I. Dependence on voltage and sodium concentration.

The side chain of arginine, n-propylguanidinium (nPG), reversibly decreases peak sodium conductance and increases the speed of sodium current decay, when perfused internally. Effects are voltage dependent and are more pronounced at high depolarizations. Results are also dependent on the sodium concentration gradient. Both the decline in peak conductance and the speeding of inactivation are greater if the sodium concentration gradient is reversed from the normal. The decrease in peak sodium current is too large to be due solely to the faster decay kinetics. The difference is not due to a change in slow inactivation of the channel. Sodium current inactivation has also been studied with a double pulse procedure. Results show that at - 70 mV, nPG leaves sodium channels rapidly (less than 500 microseconds) in normal sodium gradient, but more slowly (greater than 1 ms) in reversed sodium gradient. Several structural analogs of nPG have been tested. Shortening the alkyl chain weakens effects significantly. Arginine itself, which contains extra charged groups, is also less effective. n-Propylammonium is active but with an apparent affinity only one-fifth that of arginine. We conclude that nPG acts within the sodium channel, and has at least two modes of action.     

蝎毒耐热蛋白对大鼠海马神经元钠通道的抑制作用

应用全细胞膜片钳技术观察蝎毒耐热蛋白（scorpion venom heat resistant protein,SVHRP）对急性分离大鼠海马神经元电压依赖性钠通道的影响。结果表明,急性分离大鼠海马神经元产生的河豚毒素（tetrodotoxin,TTX）敏感的电压依赖性钠电流被SVHRP浓度依赖性地抑制,半数抑制浓度为（0．0034±0．0004）μg／mL,Hill常数为0．4361±0．0318;SVHRP可使钠通道稳态激活曲线向电压的正方向移动,正常TTX敏感的钠通道的半数激活电压（V1/2）为（-34．38±0．62）mV（n=16）,给予0．1μg／mL的SVHRP后V1/2为（-23．96±0．41）mV（n=8,P〈0．05）,斜坡因子（κ）由正常的4．52±0．52变为3．73±0．08（n=8,P〈0．05）。SVHRP亦能改变电压依赖性钠通道的稳态失活曲线,使其向电位的负方向移动,SVHRP处理前钠通道半数失活电压（V1/2）为（-32．60±1．52）mV,κ为6．73±0．51（n=16）;0．1μg／mL的SVHRP处理后V1/2变为（-50．69±2．55）mV（n=8,P〈0．01）,κ为5．49±0．72（n=8,P〈0．05）。结果提示,SVHRP能抑制电压依赖性钠电流,改变钠通道的动力学特性,抑制其激活,促进其失活,从而影响神经元的兴奋性,这可能是其抗癫痫的机制之一。     

The sodium current underlying action potentials in guinea pig hippocampal CA1 neurons

Neurons were acutely dissociated from the CA1 region of hippocampal slices from guinea pigs. Whole-cell recording techniques were used to record and control membrane potential. When the electrode contained KF, the average resting potential was about -40 mV and action potentials in cells at -80 mV (current-clamped) had an amplitude greater than 100 mV. Cells were voltage-clamped at 22-24 degrees C with electrodes containing CsF. Inward currents generated with depolarizing voltage pulses reversed close to the sodium equilibrium potential and could be completely blocked with tetrodotoxin (1 microM). The amplitude of these sodium currents was maximal at about -20 mV and the amplitude of the tail currents was linear with potential, which indicates that the channels were ohmic. The sodium conductance increased with depolarization in a range from -60 to 0 mV with an average half-maximum at about -40 mV. The decay of the currents was not exponential at potentials more positive than -20 mV. The time to peak and half-decay time of the currents varied with potential and temperature. Half of the channels were inactivated at a potential of -75 mV and inactivation was essentially complete at -40 to -30 mV. Recovery from inactivation was not exponential and the rate varied with potential. At lower temperatures, the amplitude of sodium currents decreased, their time course became longer, and half-maximal inactivation shifted to more negative potentials. In a small fraction of cells studied, sodium currents were much more rapid but the voltage dependence of activation and inactivation was very similar.     

Holding potential affects the apparent voltage-sensitivity of sodium channel activation in crayfish giant axons.

Sodium channel activations, measured as the fraction of channels open to peak conductance for different test potentials (F[V]), shows two statistically different slopes from holding potential more positive than -90 mV. A high valence of 4-6e is indicated a test potentials within 35 mV of the apparent threshold potential (circa -65 mV at -85 mV holding potential). However, for test potentials positive to -30 mV, the F(V) curve shows a 2e valence. The F(V) curve for crayfish axon sodium channels at these "depolarized" holding potentials thus closely resembles classic data obtained from other preparations at holding potentials between -80 and -60 mV. In contrast, at holding potentials more negative than -100 mV, the high slope essentially disappears and the F(V) curve follows a single Boltzmann distribution with a valence of approximately 2e at all potentials. Neither the slope of this simple distribution nor its midpoint (-20 mV) was significantly affected by removal of fast inactivation with pronase. The change in F(V) slope, when holding potential is increased from -85 to -120 mV, does not appear to be caused by the contribution of a second channel type. The simple voltage dependence of sodium current found at Vh -120 mV be used by to discriminate between models of sodium channel activation, and rules out models with three particles of equal valence.     

Effects of SO2 derivatives on sodium currents in acutely isolated rat hippocampal lead-exposed neurons

In this study, the effects of acute SO_2 derivatives and chronic lead exposure together on sodium cur-rents (INa) were investigated in acutely isolated rat hippocampal neurons by using the whole-cell patch clamp techniques. We found that chronic lead exposure hardly reduced the amplitudes of INa. In the normal condition, sodium current started to appear at around ?70 mV, and reached the peak current at around ?40 mV. After chronic lead exposure, the data changed to ?70 and ?30 mV. After adding SO2 derivatives, the data changed to ?80 and ?40 mV, respectively. SO_2 derivatives caused a significant in-crease of INa in hippocampal chronic-lead exposed neurons. Chronic lead exposure induced a right shift of the activation curve and a left shift of the inactivation curve of sodium channels. SO_2 derivatives caused negative shifts of the activation and inactivation curves of INa in hippocampal chronic-lead ex-posed neurons. Lead exposure put off the time reaching the peak of INa activation. SO_2 derivatives in-creased the time constants of inactivation after lead exposure. The interaction of lead and SO_2 deriva-tives with voltage-dependent sodium channels may lead to changes in electrical activity and contribute to worsening the neurotoxicological damage.     

Modification of inactivation in cardiac sodium channels: ionic current studies with Anthopleurin-A toxin

The site 3 toxin, Anthopleurin-A (Ap-A), was used to modify inactivation of sodium channels in voltage-clamped single canine cardiac Purkinje cells at approximately 12 degrees C. Although Ap-A toxin markedly prolonged decay of sodium current (INa) in response to step depolarizations, there was only a minor hyperpolarizing shift by 2.5 +/- 1.7 mV (n = 13) of the half-point of the peak conductance- voltage relationship with a slight steepening of the relationship from - 8.2 +/- 0.8 mV to -7.2 +/- 0.8 mV (n = 13). Increases in Gmax were dependent on the choice of cation used as a Na substitute intracellularly and ranged between 26 +/- 15% (Cs, n = 5) to 77 +/- 19% (TMA, n = 8). Associated with Ap-A toxin modification time to peak INa occurred later, but analysis of the time course INa at multiple potentials showed that the largest effects were on inactivation with only a small effect on activation. Consistent with little change in Na channel activation by Ap-A toxin, INa tail current relaxations at very negative potentials, where the dominant process of current relaxation is deactivation, were similar in control and after toxin modification. The time course of the development of inactivation after Ap-A toxin modification was dramatically prolonged at positive potentials where Na channels open. However, it was not prolonged after Ap-A toxin at negative potentials, where channels predominately inactivate directly from closed states. Steady state voltage-dependent availability (h infinity or steady state inactivation), which predominately reflects the voltage dependence of closed-closed transitions equilibrating with closed-inactivated transitions was shifted in the depolarizing direction by only 1.9 +/- 0.8 mV (n = 8) after toxin modification. The slope factor changed from 7.2 +/- 0.8 to 9.9 +/- 0.9 mV (n = 8), consistent with a prolongation of inactivation from the open state of Ap-A toxin modified channels at more depolarized potentials. We conclude that Ap-A selectively modifies Na channel inactivation from the open state with little effect on channel activation or on inactivation from closed state(s).     

Intracellular pH and ionic channels in the Loligo vulgaris giant axon.

Squid giant axons were used to investigate the reversible effects of intracellular pH(pHi) on the kinetic properties of ionic channels. The pharmacologically separated K+ and Na+ currents were measured under: (a) internal perfusion, (b) enzymatic Pronase treatment, and (c) continuous estimate of periaxonal ion accumulation. Variation of internal pH from 4.8 to 11 resulted in: (a) a decrease of steady-state sodium inactivation at positive potentials similar to the effect of the proteolytic enzyme Pronase, (b) a shift of the h infinity (E) curve toward depolarizing voltages, and (c) a decrease of the time constant of inactivation for potentials below -20 mV (an increase above). A plot of the steady-state sodium conductance at E = +40 mV as a function of pHi suggests that two groups with pKa 10.4 and 5.6 affect respectively the inactivation gate and the rate constants for the transition from the inactivated to the second open state (h2) (Chandler and Meves, 1970b). The voltage shifts of the kinetic parameters predicted by the Gouy-Chapman-Stern theory are well satisfied at high pHi and less at low. Once corrected for voltage shifts, the forward rate constants for channel opening were found to be slowed with the acidity of the internal or external solution.     

Selective modification of sodium channel gating in lobster axons by 2, 4, 6-trinitrophenol: Evidence for two inactivation mechanisms

Trinitrophernol (TNP) selectively alters the sodium conductance system of lobster giant axons as measured in current clamp and voltage clamp experiments using the double sucrose gap technique. TNP has no measurable effect on potassium currents but reversibly prolongs the time-course of sodium currents during maintained depolarizations over the full voltage range of observable currents. Action potential durations are increased also. Tm of the Hodgkin-Huxley model is not markedly altered during activation of the sodium conductance but is prolonged during removal of activation by repolarization, as observed in sodium tail experiments. The sodium inactivation versus voltage curve is shifted in the hyperpolarizing direction as is the inactivation time constant curve, measured with conditioning voltage steps. This shift speeds the kinetics of inactivation over part of the same voltage range in which sodium currents are prolonged, a contradiction incompatible with the Hodgkin-Huxley model. These results are interpreted as support for a hypothesis of two inactivation processes, one proceeding directly from the resting state and the other coupled to the active state of sodium conductance.     

Ultraviolet-induced alterations of the sodium inactivation in myelinated nerve fibres.

Ultraviolet radiation irreversibly reduces the sodium permeability in nerve membranes and, in addition, induces a change of the potential dependence of the kinetic parameters of sodium inactivation in the node of Ranvier. This second ultraviolet effect shifts the kinetic parameters of sodium inactivation h infinity (V), alpha h (V), and beta h (V) to more negative potentials (no changes of the slopes of the curves). The amount of the displacement delta V along the potential axis is equal for the three parameters and depends on the ultraviolet dose. It is about delta V = --10 mV after an irradiation dose of 0.7 Ws/cm2 at 280 nm. Both ultraviolet-induced effects depend on membrane potential and on the wavelength of the applied radiation. But while the potential shift is enhanced at more negative holding potentials, the ultraviolet blocking is diminished and vice versa. Further, the ultraviolet-induced potential shift is greater at 260 nm than at 280 nm, whereas a maximum sensitivity of ultraviolet blocking is found at 280 nm. Therefore, the two radiation effects are the result of two separate photoreactions. For explanation of the radiation-induced potential shift it is assumed that ultraviolet radiation decreases the density of negative charges at the inner surface of the nodal membrane. From this hypothesis a value for the inner surface potential psii was derived. --19 mV less than or equal to psii less than or equal to --14 mV.     

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 influence of membrane lipid metabolites on lymphocyte potassium channel activity

In the present study, the whole-cell patch-clamp technique was applied to elucidate modulatory effects of high-density lipoproteins (HDL), sphingosine (SPH), sphingosine-1-phosphate (SPP), lysophosphatidic acid (LPA) and sphingosyl- phosphorylcholine (SPC) on the activity of Kv1.3 channels in human T lymphocytes (TL). Obtained data provide evidence that application of SPC at micromolar concentrations shifts the channel activation midpoint by about 20 mV towards positive membrane potentials. This effect occurs in a concentration-dependent manner and is saturated at SPC concentrations higher than 10 micro M. The shift of channel activation midpoint is accompanied by a pronounced slowing of the activation kinetics. The modulatory effect of SPC is clearly voltage-dependent, being most potent at -20 mV and least potent at +60 mV. The steady-state inactivation curve is also shifted by about 20 mV towards positive membrane potentials. The kinetics of channel inactivation and deactivation (closure) remain unaffected upon SPC treatment. In contrast, application of HDL (250 micro g/ml), SPH (50 and 100 micro M), SPP (10 micro M) and LPA (10 and 36 micro M) does not exert any modulatory effect on the channel activity. The effect of SPC on Kv1.3 channel gating resembles the effect exerted by extracellular zinc at the concentration of 10 micro M. It is concluded that the effect of SPC is specific and may be due to the presence of a choline residue in SPC molecules. The possible mechanism and the physiological significance of this modulatory effect on Kv1.3 channels are discussed.     

Action potential modulation of connexin40 gap junctional conductance

Connexin40 (Cx40) is abundantly expressed in the atrial myocardium, ventricular conduction system, and vascular endothelial and smooth muscle cells of the mammalian cardiovascular system. Rapid conduction through cardiac tissues depends on electrotonic transfer of the action potential between neighboring cells. To determine whether transjunctional voltages (Vj) elicited by an action potential can modulate conductance of Cx40 gap junctions, simulated myocardial action potentials were applied as voltage-clamp waveforms to Cx40 gap junctions expressed in mouse neuro2A (N2A) cells. Junctional currents resembled the action potential morphology but declined by >50% from peak to near-constant plateau values. Kinetics of Cx40 voltage gating were examined at peak voltages > or =100 mV, and decay time constants changed e-fold per 17.6 mV for Vj > +/-40 mV. Junctional conductance recovered during phase 3 repolarization and early diastole to initial values. These phasic changes in junctional conductance were due to rapid decay kinetics, increasing to tens of milliseconds at peak Vj of 130 mV, and the increase in the steady-state conductance curve as Vj returned toward 0 mV. Time-dependent conductance curves for Cx40 were modeled with one inactivation and two recovery Vj-dependent components. There was a temporal correlation between development of conduction delay or block and the inactivation phase of junctional conductance. Likewise, recovery of junctional conductance was coincident with recovery from refractoriness, suggesting that gap junctions may play a role in the genesis and propagation of cardiac arrhythmias.     

Comparison of heterologously expressed human cardiac and skeletal muscle sodium channels.

In this study we have expressed and characterized recombinant cardiac and skeletal muscle sodium channel alpha subunits in tsA-201 cells under identical experimental conditions. Unlike the Xenopus oocyte expression system, in tsA-201 cells (transformed human embryonic kidney) both channels seem to gate rapidly, as in native tissue. In general, hSkM1 gating seemed faster than hH1 both in terms of rate of inactivation and rate of recovery from inactivation as well as time to peak current. The midpoint of the steady-state inactivation curve was approximately 25 mV more negative for hH1 compared with hSkM1. In both isoforms, the steady-state channel availability relationships ("inactivation curves") shifted toward more negative membrane potentials with time. The cardiac isoform showed a minimal shift in the activation curve as a function of time after whole-cell dialysis, whereas hSkM1 showed a continued and marked negative shift in the activation voltage dependence of channel gating. This observation suggests that the mechanism underlying the shift in inactivation voltage dependence may be similar to the one that is causing the shift in the activation voltage dependence in hSkM1 but that this is uncoupled in the cardiac isoform. These results demonstrate the utility and limitations of measuring cardiac and skeletal muscle recombinant Na+ channels in tsA-201 cells. This baseline characterization will be useful for future investigations on channel mutants and pharmacology.     

Sodium currents in the giant axon of the crabCarcinus maenas

Summary Measurements were made of the kinetics and steady-state properties of the sodium conductance changes in the giant axon of the crabCarcinus maenas. The conductance measurements were made in the presence of small concentrations of tetrodotoxin and as much electrical compensation as possible in order to minimize errors caused by the series resistance. After an initial delay of 10–150 sec, the conductance increase during depolarizing voltage clamp pulses followed the Hodgkin-Huxley kinetics. Values of the time constant for the activation of the sodium conductance lay on a bell-shaped curve with a maximum under 180 sec at –40 mV (at 18°C). Values of the time constant for the inactivation of the sodium conductance were also fitted using a bell-shaped curve with a maximum under 7 msec at –70 mV. The effects of membrane potential on the fraction of Na channels available for activation studied using double pulse protocols suggest that hyperpolarizing potentials more negative than –100 mV lock a fraction of the Na channels in a closed conformation.     

Enhancement of closed-state inactivation in long QT syndrome sodium channel mutation DeltaKPQ

DeltaKPQ, a three amino acid [lysine (K), proline (P), glutamine (Q)] deletion mutation of the human cardiac Na channel (hH1), which is one cause of long QT syndrome (LQT3), has impaired inactivation resulting in a late sodium current. To better understand inactivation in DeltaKPQ, we applied a site-3 toxin anthopleurin A, which has been shown to inhibit inactivation from the open state with little or no effect on inactivation from the closed state(s) in wild-type hH1. In contrast to the effect of site-3 toxins on wild-type hH1, inactivation from closed state(s) in toxin-modified DeltaKPQ demonstrated a large negative shift in the Na channel availability curve of nearly -14 mV. Recovery from inactivation showed that toxin-modified DeltaKPQ channels recovered slightly faster than those in control, whereas development of inactivation at potentials negative to -80 mV showed that inactivation developed much more rapidly in toxin-modified DeltaKPQ channels compared with control. An explanation for our results is that closed-state inactivation in toxin-modified DeltaKPQ is enhanced by the mutated inactivation lid being positioned "closer" to its receptor resulting in an increased rate of association between the inactivation lid and its receptor.     

Effects of the Enantiomers of BayK 8644 on the Charge Movement of L-type Ca Channels in Guinea-pig Ventricular Myocytes

The effects of the agonist enantiomer S(-)Bay K 8644 on gating charge of L-type Ca channels were studied in single ventricular myocytes. From a holding potential (Vh) of -40 mV, saturating (250 nm) S(-)Bay K shifted the half-distribution voltage of the activation charge (Q1) vs. V curve -7.5 +/- 0.8 mV, almost identical to the shift produced in the Ba conductance vs. V curve (-7.7 +/- 2 mV). The maximum Q1 was reduced by 1.7 +/- 0.2 nC/microF, whereas Q2 (charge moved in inactivated channels) was increased in a similar amount (1.4 +/- 0.4 nC/microF). The steady-state availability curves for Q1, Q2, and Ba current showed almost identical negative shifts of -14.8 +/- 1.7 mV, -18.6 +/- 5.8 mV, and -15.2 +/- 2.7 mV, respectively. The effects of the antagonist enantiomer R(+)BayK 8644 were also studied, the Q1 vs. V curve was not significantly shifted, but Q1max (Vh = -40 mV) was reduced and the Q1 availability curve shifted by -24.6 +/- 1.2 mV. We concluded that: a) the left shift in the Q1 vs. V activation curve produced by S(-)BayK is a purely agonistic effect; b) S(-)BayK induced a significantly larger negative shift in the availability curve than in the Q1 vs. V relation, consistent with a direct promotion of inactivation; c) as expected for a more potent antagonist, R(+)Bay K induced a significantly larger negative shift in the availability curve than did S(-)Bay K.