Inactivation of single cardiac Na+ channels in three different gating modes

In small cell-attached patches containing one and only one Na+ channel, inactivation was studied in three different gating modes, namely, the fast-inactivating F mode and the more slowly inactivating S mode and P mode with similar inactivation kinetics. In each of these modes, ensemble-averaged currents could be fitted with a Hodgkin-Huxley-type model with a single exponential for inactivation (tauh). tauh declined from 1.0 ms at -60 mV to 0.1 ms at 0 mV in the F mode, from 4.6 ms at -40 mV to 1.1 ms at 0 mV in the S mode, and from 4.5 ms at -40 mV to 0.8 ms at +20 mV in the P mode, respectively. The probability of non-empty traces (net), the mean number of openings per non-empty trace (op/tr), and the mean open probability per trace (popen) were evaluated at 4-ms test pulses. net inclined from 30% at -60 mV to 63% at 0 mV in the F mode, from 4% at -90 mV to 90% at 0 mV in the S mode, and from 2% at -60 mV to 79% at +20 mV in the P mode. op/tr declined from 1.4 at -60 mV to 1.1 at 0 mV in the F mode, from 4.0 at -60 mV to 1.2 at 0 mV in the S mode, and from 2.9 at -40 mV to 1.6 at +20 mV in the P mode. popen was bell-shaped with a maximum of 5% at -30 mV in the F mode, 48% at -50 mV in the S mode, and 16% at 0 mV in the P mode. It is concluded that 1) a switch between F and S modes may reflect a functional change of inactivation, 2) a switch between S and P modes may reflect a functional change of activation, 3) tauh is mainly determined by the latency until the first channel opening in the F mode and by the number of reopenings in the S and P modes, 4) at least in the S and P modes, inactivation is independent of pore opening, and 5) in the S mode, mainly open channels inactivate, and in the P mode, mainly closed channels inactivate.     

Calcium current and inactivation in identified neurons in Hermissenda crassicornis

1. N-type (omega-conotoxin sensitive) calcium currents (ICa) were recorded in identified neurons in Hermissenda crassicornis using low-resistance patch electrodes (0.7 +/- 0.3 M omega; n = 101) under conditions that eliminated inward Na+ currents (choline ions substitution) and suppressed outward K+ currents (Cs+, tetraethylammonium, and 4-AP). Step depolarization from a holding potential of -60 mV to potentials above -30 mV elicited ICa, which peaked approximately 20 mV and declined with increasing depolarizations. 2. Evidence for a low-threshold current was present. Step depolarization from a more hyperpolarizing potentials (e.g., -90 mV) revealed a small shoulder (< 100 pA) at -60 to -40 mV that was sensitive to Co2+ and Ni2+. However, under the conditions examined here (holding potential of -60 mV), the high-voltage-activated current predominated. 3. Barium (Ba2+) and strontium (Sr2+) permeate the Ca2+ channel with similar activation kinetics (ease of permeation; Ba2+ > Ca2+ > Sr2+). Steady-state activation of permeability versus membrane potentials for Ca2+, Ba2+, and Sr2+ as charge carriers could be fitted with the Boltzmann equation, with half-activation voltage and slope factor of 2.9 and 7.7 mV for ICa, -13.1 mV and 7.8 for Ba2+ current (IBa) and -2.3 mV and 7.8 for Sr2+ current (ISr). The time course of activation was monotonic with time constant (tau) for ICa ranging from 2 to 8 ms. 4. The inactivation profile was complex. At negative step potentials (e.g., -20 mV), inactivation of the current was slow. Depolarization steps to relatively positive voltages (e.g., 10 mV) showed more rapid inactivation than those at more positive potentials (e.g., 40 mV). When extracellular Ca2+ was raised from 5 to 10 mM, a biphasic decay (tau fast of 25 +/- 4 ms; and tau slow of 473 +/- 64 ms; mean +/- SD, n = 9) was seen. Such an observation suggested a current-mediated inactivation. 5. With a pulse duration of approximately 350 ms, ISr showed inactivation whereas Ba2+ virtually removed the decay. However, IBa turned off with more prolonged depolarization. 6. A twin-pulse protocol was used to assess the voltage dependence of inactivation: an incomplete U-shaped inactivation curve was observed for ICa, IBa, and ISr. Channels available for inactivation were increased in the presence of Ca2+ ions. 7. Inactivation was further studied with the Ca2+ chelators, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and bis(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). With 10 mM of BAPTA, in the pipette, inactivation was reduced but not removed.(ABSTRACT TRUNCATED AT 250 WORDS)     

A single non-L-, non-N-type Ca2+ channel in rat insulin-secreting RINm5F cells

Single high-voltage-activated (HVA) Ca2+ channel activity was recorded in rat insulinoma RINm5F cells using cell-attached and outside-out configurations. Single-channel recordings revealed three distinct Ca2+ channel subtypes: one sensitive to dihydropyridines (DHPs)-(L-type), another sensitive to omega -conotoxin (CTx)-GVIA (N-type) and a third type insensitive to DHPs and omega -CTx-GVIA (non-L-, non-N-type). The L-type channel was recorded in most patches between -30 and +30 mV. The channel had pharmacological and biophysical features similar to the L-type channels described in other insulin-secreting cells (mean conductance 21 pS in control conditions and 24 pS in the presence of 5 microM Bay K 8644). The non-L-, non-N-type channel was recorded in cells chronically treated with omega -CTx-GVIA in the presence of nifedipine to avoid the contribution of N- and L-type channels. Channel activity was hardly detectable below -10 mV and was recruited by negative holding potentials (< -90 mV). The channel open probability increased steeply from -10 to + 40 mV. Different unitary current sublevels could be detected and the current voltage relationship was calculated from the higher amplitude level with a slope conductance of 21 pS. Channel activity lasted throughout depolarizations of 300-800ms with little sign of inactivation. Above 0 mV the channel showed a persistent flickering kinetics with brief openings (tau o 0.6 ms) and long bursts (tau burst 60 ms) interrupted by short interburst intervals. The third HVA Ca2+ channel subtype, the N-type, had biophysical properties similar to the non-L-, non-N-type and was best identified in outside-out patches by its sensitivity to omega -CTx-GVIA. The channel was detectable only above -10 mV from a -90 mV holding potential, exhibited a fast flickering behaviour, persisted during prolonged depolarizations and had a slope conductance of about 19 pS. The present data provide direct evidence for a slowly inactivating non-L-, non-N-type channel in insulin-secreting RINm5F cells that activates at more positive voltages than the L-type channel and indicate the possibility of identifying unequivocally single HVA Ca2+ channels in cell-attached and excised membrane patches under controlled pharmacological conditions.     

Conformational state-dependent effects of halothane on cardiac Na+ current

BACKGROUND: The Na+ channel is voltage gated and characterized by three distinct states: closed, open, and inactivated. To identify the effects of halothane on the cardiac Na+ current (I(Na)) at various membrane potentials, the effects of 1.2 mM halothane at different holding potentials (V(H)) on I(Na) were examined in single, enzymatically isolated guinea pig ventricular myocytes. METHODS: The I(Na) was recorded using the whole-cell configuration of the patch-clamp technique. Currents were generated from resting V(H)s of -110, -80, or -65 mV. State-dependent block was characterized by monitoring frequency dependence, tonic block, and removal of inactivation by veratridine. RESULTS: Halothane produced significant (P < 0.05) V(H)-dependent depressions of peak I(Na) (mean +/- SEM): 24.4 +/- 4.1% (V(H) = -110 mV), 42.1 +/- 3.4% (V(H) = -80 mV), and 75.2 +/- 1.5% (V(H) = -65 mV). Recovery from inactivation was significantly increased when cells were held at -80 mV (control, tau = 6.0 +/- 0.3 ms; halothane, tau = 7.1 +/- 0.4 ms), but not at -110 mV. When using a V(H) of -80 mV, halothane exhibited a use-dependent block, with block of I(Na) increasing from 8.6 +/- 1.4% to 30.7 +/- 3.5% at test pulse rates of 2 and 11 Hz, respectively. Use-dependent inhibition was not apparent at V(H) of -110 mV. When inactivation of I(Na) was removed by exposure to 100 microM veratridine, no significant difference was observed in the depressant effect of halothane at both V(H)s: 26.6 +/- 4.5% (V(H) = -80 mV) and 26.4 +/- 5.6% (V(H) = -110 mV). CONCLUSIONS: The present findings indicate that the depressant action of halothane on cardiac I(Na) depends on the conformational state of the channel. As more channels are in the inactivated state, the more potent is the effect of halothane. Removal of channel inactivation by veratridine abolished the dependence of the halothane effect on V(H), but depression of the current was still evident. These results indicate a complex interaction between halothane and the various conformational states of the Na+ channel.     

The voltage-dependent conductances of rat neocortical layer I neurons

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na+), calcium (Ca2+), and potassium (K+) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of gamma-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca2+-binding protein calretinin in a subset of cells, indicated the presence of Cajal-Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na+ current activated at about -45 mV, was half-maximal steady-state inactivated at -66.6 mV, and recovery from inactivation followed a two-exponential process (tau1 = 8.4 ms and tau2 = 858.8 ms). Na+ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (Ifast) activated approximately -40 mV, declined rapidly with a voltage-insensitive time constant (tau approximately 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mM). Ifast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 microM) and a second by high concentrations (6 mM). After elimination of Ifast by a conditioning prepulse (50 ms to -50 mV), a slow K+ current (I(KV)) could be studied in isolation. I(KV) was only moderately affected by 4-AP (6 mM), while TEA (20 mM) blocked most (> 80%) of the current. I(KV) activated at about -40 mV, declined monoexponentially in a voltage-dependent manner (tau approximately 850 ms at -30 mV), and revealed an incomplete steady-state inactivation. In addition to Ifast and I(KV), indications of a Ca2+-dependent outward current component were found. When Na+ currents, Ifast, and I(KV) were blocked by tetrodotoxin (TTX, 1 microM), 4-AP (6 mM) and TEA (20 mM) an inward current carried by Ca2+ was found. Ca2+ currents activated at depolarized potentials at about -30 mV, were completely blocked by 50 microM cadmium (Cd2+), were sensitive to verapamil (approximately 40% block by 10 microM), and were not affected by nickel (50 microM). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (approximately 2 ms, measured at half maximal amplitude) of relative small amplitude (approximately 83 mV, measured from the action potential threshold).     

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.     

Isolation and characterization of a persistent potassium current in neostriatal neurons

1. Depolarization-activated, calcium-independent potassium (K+) currents were studied with the use of whole cell voltage-clamp recording from neostriatal neurons acutely isolated from adult (> or = 4 wk old) rats. The whole cell K+ current was composed of transient and persistent components. The aims of the experiments were to isolate the persistent component and then to characterize its voltage dependence and kinetics. 2. Application of 10 mM 4-aminopyridine (4-AP) completely blocked the transient currents while reducing the persistent current by approximately 40% [50% inhibitory concentration (IC50), of blockable current = 125 microM]. The persistent K+ current also was reduced by tetraethylammonium (TEA). Two components to the TEA block were present, having IC50s of 125 microM (23% of the blockable current) and 5.9 mM (77% of the blockable current). Collectively, these results suggested that the persistent components of the total K+ current was pharmacologically heterogeneous. The properties of the 4-AP-resistant, persistent K+ current (IKrp) were subsequently studied. 3. The kinetics of activation and deactivation of IKrp were voltage dependent. Examination of the entire activation/deactivation time constant profile showed that it was bell shaped, with time constants being moderately rapid (tau approximately 50 ms) at membrane potentials corresponding to the resting potential of neostriatal cells (approximately -80 mV), becoming considerably longer (tau approximately 100 ms) at potentials near the cells' spike thresholds (approximately -45 mV), and decreasing to a minimum (tau approximately 5 ms) at potentials associated with the peak of the cells' action potentials (approximately +20 mV). The inactivation kinetics of IKrp also were voltage dependent. The time constants of inactivation varied between 1 and 8 s at potentials between -10 and +35 mV. 4. Unlike persistent K+ currents in many other cell types, IKrp activated at relatively hyperpolarized membrane potentials (approximately -70 mV). The Boltzmann function describing activation had a half-activation voltage of -13 mV and a slope factor of 12 mV. In addition, the Boltzmann function describing the voltage dependence of inactivation of IKrp had a relatively depolarized half-inactivation voltage of -55 and a large slope factor of 19 mV, indicating that this current was available over a broad range of membrane potentials (between -100 and -10 mV). 5. Neostriatal neurons recorded in vivo exhibit subthreshold shifts in membrane potential of variable duration (tens of ms to s) from a hyperpolarized resting state to a depolarized state that is limited in amplitude just below spike threshold. The voltage dependence of activation and inactivation of IKrp indicates that it will be available on depolarization from the hyperpolarized state. However, the slow activation rate of this current suggests that it will contribute little either to limiting the amplitude of the initial depolarization associated with entry into the depolarized state or to depolarizing episodes of short duration (e.g., < 50 ms). However, IKrp should limit the amplitude of membrane depolarizations associated with prolonged excursions into the depolarized state.     

Voltage- and use-dependent inhibition of Na+ channels in rat sensory neurones by 4030W92, a new antihyperalgesic agent

1. Whole cell patch clamp techniques were used to study the effects of 4030W92 (2,4-diamino-5-(2,3-dichlorophenyl)-6-fluoromethylpyrimidine), a new antihyperalgesic agent, on rat dorsal root ganglion (DRG) neurones. 2. In small diameter, presumably nociceptive DRG neurones under voltage-clamp, 4030W92 (1-100 microM) produced a concentration-related inhibition of slow tetrodotoxin-resistant Na+ currents (TTXR). From a holding potential (Vh) of -90 mV, currents evoked by test pulses to 0 mV were inhibited by 4030W92 with a mean IC50 value of approximately 103 microM. 3. The inhibitory effect of 4030W92 on TTX(R) was both voltage- and use-dependent. Currents evoked from a Vh of -60 mV were inhibited by 4030W92 with a mean IC50 value of 22 microM, which was 5 fold less than the value obtained at -90 mV. Repeated activation of TTX(R) by a train of depolarizing pulses (5 Hz, 20 ms duration) enhanced the inhibitory effects of 4030W92. These data could be explained by a preferential interaction of the drug with inactivation states of the channel. In support of this hypothesis 4030W92 (30 microM) produced a significant hyperpolarizing shift of 10 mV in the slow inactivation curve for TTX(R) and markedly slowed the recovery from channel inactivation. 4. Fast TTX-sensitive Na+ currents (TTXs) were also inhibited by 4030W92 in a voltage-dependent manner. The IC50 values obtained from Vhs of -90 mV and -70 mV were 37 microM and 5 microM, respectively. 4030W92 (30 microM) produced a 13 mV hyperpolarizing shift in the steady-state inactivation curve of TTXs. 5. High threshold voltage-gated Ca2+ currents were only weakly inhibited by 4030W92. The reduction in peak Ca2+ current amplitude produced by 100 microM 4030W92 was 20+/-6% (n=6). Low threshold T-type Ca2+ currents were inhibited by 17+/-8% and 43+/-3% by concentrations of 4030W92 of 30 microM and 100 microM, respectively (n=6). 6. Under current clamp, some cells exhibited broad TTX-resistant action potentials whilst others showed fast TTX-sensitive action potentials in response to a depolarizing current injection. In most cells a long duration (800 ms) supramaximal current injection evoked a train of action potentials. 4030W92 (10-30 microM) had little effect on the first spike in the train but produced a concentration-related inhibition of the later spikes. The number of spikes per train was significantly reduced from 9.7+/-1.5 to 4.2+/-1.0 and 2.6+/-1.1 in the presence of 10 microM and 30 microM 4030W92, respectively (n=5). 7. Thus, 4030W92 is a potent voltage- and use-dependent inhibitor of Na+ channels in sensory neurones. This profile can be explained by a preferential action of the drug on a slow inactivation state of the channel that results in a delayed recovery to the resting state. This state-dependent modulation by 4030W92 of Na+ channels that are important in sensory neurone function may underlie or contribute to the antihyperalgesic profile of this compound observed in vivo.     

Pharmacological modulation of human cardiac Na+ channels

Pharmacological modulation of human sodium current was examined in Xenopus oocytes expressing human heart Na+ channels. Na+ currents activated near -50 mV with maximum current amplitudes observed at -20 mV. Steady-state inactivation was characterized by a V1/2 value of -57 +/- 0.5 mV and a slope factor (k) of 7.3 +/- 0.3 mV. Sodium currents were blocked by tetrodotoxin with an IC50 value of 1.8 microM. These properties are consistent with those of Na+ channels expressed in mammalian myocardial cells. We have investigated the effects of several pharmacological agents which, with the exception of lidocaine, have not been characterized against cRNA-derived Na+ channels expressed in Xenopus oocytes. Lidocaine, quinidine and flecainide blocked resting Na+ channels with IC50 values of 521 microM, 198 microM, and 41 microM, respectively. Use-dependent block was also observed for all three agents, but concentrations necessary to induce block were higher than expected for quinidine and flecainide. This may reflect differences arising due to expression in the Xenopus oocyte system or could be a true difference in the interaction between human cardiac Na+ channels and these drugs compared to other mammalian Na+ channels. Importantly, however, this result would not have been predicted based upon previous studies of mammalian cardiac Na+ channels. The effects of DPI 201-106, RWJ 24517, and BDF 9148 were also tested and all three agents slowed and/or removed Na+ current inactivation, reduced peak current amplitudes, and induced use-dependent block. These data suggest that the alpha-subunit is the site of interaction between cardiac Na+ channels and Class I antiarrhythmic drugs as well as inactivation modifiers such as DPI 201-106.     

Fast inactivation in Shaker K+ channels. Properties of ionic and gating currents

Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as "charge immobilization" (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567-590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at -90 mV return potential changed from a single fast component to at least two components, the slower requiring approximately 200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current (Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at -120 and -90 mV. In contrast, at higher potentials (-70 and -50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of "parallel" inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH2-terminus region.     

Voltage-operated potassium currents in the somatic membrane of rat dorsal root ganglion neurons: ontogenetic aspects

Whole-cell transmembrane potassium currents were studied in somatic membrane of freshly isolated rat dorsal root ganglion neurons. We defined three types of potassium currents, which were separated on the basis of their different potential dependence of activation and sensitivity to external tetraethylammonium and 4-aminopyridine. The potential dependence of kinetic and steady-state properties of a fast inactivating potassium current, a slow inactivating potassium current and a non-inactivating delayed rectifier current were described by the Hodgkin-Huxley equations. A transient fast inactivating potassium current was activated at the most negative membrane potentials and was not reduced in the presence of 10 mM tetraethylammonium in the external solution. 4-Aminopyridine (2 mM) caused an 80% inhibition of this current. The activation of the fast inactivating potassium current was properly described by fitting a single exponent raised to the fourth power. The time constant of activation changed from 4 to 1 ms in the voltage range between -30 and +40 mV. The time constant of inactivation decreased from 35 to 15 ms over the same range of potentials. Parameters for the fit of a Boltzmann equation to mean values for steady-state activation were V1/2=-20mV, k=11.8mV, and for steady-state inactivation V1/2= -85 mV, k=-9.8 mV. A transient slow inactivating potassium current had an activation threshold between -40 and -30 mV. At 2 mM 4-aminopyridine, the depression of the slow potassium current was 55%. The extracellular application of 10 mM tetraethylammonium was less effective and evoked a 40% reduction. The activation of the slow inactivating potassium current was also described by a single exponential function raised to the fourth power. The time constant of activation decreased from 12 ms at a membrane potential of -10 mV to 4 ms at the potential of 60 mV. The inactivation of slow inactivating potassium current was described by two exponents. The time constant for the fast exponent ranged from 300 ms at -20 mV to 160 ms at +60 mV. The slower exponent was also potential dependent and its time constant ranged from approximately 2600 to 1600 ms over the same potentials. Parameters for the Boltzmann equation fittings to mean values were V1/2= -12.8 mV, k=13.4 mV and V1/2= -54.6 mV, k= -12 mV for steady-state activation and inactivation, respectively. A non-inactivating delayed rectifier potassium current was activated at the most positive membrane potentials. This non-inactivating current did not change in the presence of 4-aminopyridine. Extracellular tetraethylammonium (10 mM) caused a 70% reduction of this current. The activation of the non-inactivating potassium current was described by one exponent raised to the fourth power. The time constant for activation ranged from 85 ms at -5 mV to 30 ms at 45 mV. No time-dependent inactivation was observed during 15-s testing potentials in the voltage range between 10 and +60 mV. The activation behavior was characterized by V1/2=15.3 mV, k=12.5 mV. The densities of these potassium currents were studied for three groups of animals: one, five to six and 14-15 days of postnatal development. Fifty cells were examined in each age group. All three types of potassium currents were found in each investigated neuron. The mean densities of slow and fast inactivating potassium currents increased during ontogenetic development. The densities of non-inactivating delayed rectifier potassium current decreased in the first week of ontogenetic development and did not change thereafter.     

Action-potential-like depolarizations relieve opioid inhibition of N-type Ca2+ channels in NG108-15 cells

The ability of action-potential-like waveforms (APWs) to attenuate opioid-induced inhibition of N-type Ca2+ channels was investigated in the neuroblastoma x glioma cell line NG108-15 using whole-cell voltage clamp methods. In in vitro differentiated NG108-15 cells, the opioid agonist [d-ala2]-methionine-enkephalin (DAME) reversibly decreased omega-conotoxin-GVIA-sensitive Ba2+ currents (N-type currents). Agonist-mediated inhibition of N-type currents could be transiently relieved by strong unphysiological depolarizing prepulses to +80 mV (facilitation). Significant facilitation was also achieved by conditioning the cell with a train of 15 APWs, which roughly mimicked physiological action potentials (1- to 6-ms-long depolarizations to +30 mV from a holding potential of -40 mV). The APW-induced facilitation depended on both conditioning pulse frequency and duration. Summation of the disinhibition produced by each APW was possible because reinhibition following repolarization to -40 mV was a much slower process (tau=88 ms) than the onset of facilitation at +80 mV (tau=7 ms). These results provide evidence that N-type Ca2+ channel facilitation may be a physiologically relevant process, and suggest that neuronal firing may relieve agonist-induced inhibition of N-type currents to an extent depending on both the shape of action potentials and the frequency of firing.     

Differential expression of voltage-gated K+ and Ca2+ currents in bipolar cells in the zebrafish retinal slice

Whole-cell voltage-gated currents were recorded from bipolar cells in the zebrafish retinal slice. Two physiological populations of bipolar cells were identified. In the first, depolarizing voltage steps elicited a rapidly activating A-current that reached peak amplitude < or = 5 ms of step onset. IA was antagonized by external tetraethylammonium or 4-aminopyridine, and by intracellular caesium. The second population expressed a delayed rectifying potassium current (IK) that reached peak amplitude > or = 10 ms after step onset and did not inactivate. IK was antagonized by internal caesium and external tetraethylammonium. Bipolar cells expressing IK also expressed a time-dependent h-current at membrane potentials < -50 mV. Ih was sensitive to external caesium and barium, and was also reduced by Na+-free Ringer. In both groups, a calcium current (ICa) and a calcium-dependent potassium current (IK(Ca)) were identified. Depolarizing voltage steps > -50 mV activated ICa, which reached peak amplitude between -20 and -10 mV. ICa was eliminated in Ca+2-free Ringer and blocked by cadmium and cobalt, but not tetrodotoxin. In most cells, Ica was transient, activating rapidly at -50 mV. This current was antagonized by nickel. The remaining bipolar cells expressed a nifedipine-sensitive sustained current that activated between -40 and -30 mV, with both slower kinetics and smaller amplitude than transient ICa. IK(Ca) was elicited by membrane depolarizations > -20 mV. Bipolar cells in the zebrafish retinal slice preparation express an array of voltage-gated currents which contribute to non-linear I-V characteristics. The zebrafish retinal slice preparation is well-suited to patch clamp analyses of membrane mechanisms and provides a suitable model for studying genetic defects in visual system development.     

Three kinetically distinct Ca2+-independent depolarization-activated K+ currents in callosal-projecting rat visual cortical neurons

Three kinetically distinct Ca2+-independent depolarization-activated K+ currents in callosal-projecting rat visual cortical neurons. J. Neurophysiol. 78: 2309-2320, 1997. Whole cell, Ca2+-independent, depolarization-activated K+ currents were characterized in identified callosal-projecting (CP) neurons isolated from postnatal day 7-16 rat primary visual cortex. CP neurons were identified in vitro after in vivo retrograde labeling with fluorescently tagged latex microbeads. During brief (160-ms) depolarizing voltage steps to potentials between -50 and +60 mV, outward K+ currents in these cells activate rapidly and inactivate to varying degrees. Three distinct K+ currents were separated based on differential sensitivity to 4-aminopyridine (4-AP); these are referred to here as IA, ID, and IK, because their properties are similar (but not identical) K+ currents termed IA, ID, and IK in other cells. The current sensitive to high (>/=100 mu M) concentrations of 4-AP (IA) activates and inactivates rapidly; the current blocked completely by low (相似文献   

Novel, ultraslow inactivating sodium current in human ventricular cardiomyocytes

BACKGROUND: Alterations in K+ channel expression and gating are thought to be the major cause of action potential remodeling in heart failure (HF). We previously reported the existence of a late Na+ current (INaL) in cardiomyocytes of dogs with chronic HF, which suggested the importance of the Na+ channel in this remodeling process. The present study examined whether this INaL exists in cardiomyocytes isolated from normal and failing human hearts. METHODS AND RESULTS: A whole-cell patch-clamp technique was used to measure ion currents in cardiomyocytes isolated from the left ventricle of explanted hearts from 10 patients with end-stage HF and from 3 normal hearts. We found INaL was activated at a membrane potential of -60 mV with maximum density (0.34+/-0.05 pA/pF) at -30 mV in cardiomyocytes of both normal and failing hearts. The steady-state availability was sigmoidal, with an averaged midpoint potential of -94+/-2 mV and a slope factor of 6.9+/-0.1 mV. The current was reversibly blocked by the Na+ channel blockers tetrodotoxin (IC50=1.5 micromol/L) and saxitoxin (IC50=98 nmol/L) in a dose-dependent manner. Both inactivation and reactivation of INaL had an ultraslow time course (tau approximately 0.6 seconds) and were independent of voltage. The amplitude of INaL was independent of the peak transient Na+ current. CONCLUSIONS: Cardiomyocytes isolated from normal and explanted failing human hearts express INaL characterized by an ultraslow voltage-independent inactivation and reactivation.     

Two types of TTX-resistant and one TTX-sensitive Na+ channel in rat dorsal root ganglion neurons and their blockade by halothane

The clinically employed general anaesthetic halothane was shown to exert action on the peripheral nervous system by suppressing spinal reflexes, but it is still unclear which mechanisms underlie this action. The present study addressed the question whether blockade of tetrodotoxin-sensitive (TTXs) and -resistant (TTXr) Na+-channels in rat dorsal root ganglia (DRG) neurons by halothane could explain its peripheral effects. Two types of TTXr Na+-currents, fast and slow, with distinct activation and inactivation kinetics were found in small (< 25 micrometer) and medium sized (25-40 micrometer) DRG neurons. These currents were blocked by halothane with IC50 values of 5.4 and 7.4 mmol/L, respectively. Additionally, in a concentration-dependent manner halothane accelerated the inactivation kinetics of both currents and shifted the inactivation curves to more hyperpolarized potentials. Neither the activation curves of both TTXr Na+-currents were influenced by halothane nor a voltage-dependent block at test potentials of the currents was seen. In contrast to that of fast current, the time-to-peak for slow current was changed in the presence of halothane. The TTXs Na+-current which prevailed in large neurons (> 40 micrometer) was blocked by halothane with an IC50 of 12.1 mmol/L. Its inactivation curve was also shifted to more hyperpolarized potentials and the inactivation kinetics accelerated with increasing halothane concentration. Similarly to TTXr Na+-currents, the activation curve of TTXs Na+-current and its time-to-peak were not influenced by halothane. It is suggested that two types of TTXr Na+-currents can explain the heterogeneity in kinetic data for TTXr Na+-currents. Furthermore, the incomplete blockade of Na+-currents might underlie the incomplete reduction of spinal reflexes at clinically used concentrations of halothane.     

Voltage dependencies of the fast and slow gating modes of RIIA sodium channels

Rat brain IIA sodium channel alpha-subunits were expressed in Xenopus oocytes, and the sodium currents were measured by intracellular voltage clamping with large agarose-tipped electrodes and by excised membrane patch-clamp recording to separate and characterize the properties of the fast and slow channel gating modes. The currents showed biexponential inactivation properties with fast and slow phases that could be isolated as distinct gating modes through differences in their inactivation properties. At holding potentials more negative than -55 mV, fast mode currents inactivated within a few milliseconds of depolarization, and could be distinguished by their rapid recovery from inactivation. Single sodium channels in the fast mode opened early after depolarization and rarely showed re-openings. At holding potentials positive to -55 mV, fast mode currents were inactivated, revealing slow mode currents which had slower activation and inactivation kinetics and showed sustained single channel activity during depolarizing pulses. The steady-state voltage dependencies of fast and slow mode activation were very similar. In contrast, slow mode inactivation occurred at potentials 27 mV more positive than fast mode inactivation. The slow mode appears to be due to destabilization of a voltage-insensitive conformation of the channel. The fast gating process dominated at high current levels, perhaps due to alpha-subunit interactions.     

K+ channel blocking actions of flecainide compared with those of propafenone and quinidine in adult rat ventricular myocytes

The K+ channel blocking action of the class Ic antiarrhythmic agent flecainide was compared with that of propafenone and quinidine in isolated adult rat ventricular myocytes by using the whole-cell patch-clamp technique. In rat ventricular myocytes, depolarization activates both an inactivating (ITO) and a maintained (IK) outward K+ current. Flecainide, propafenone and quinidine all were potent inhibitors of ITO with IC50s of 3.7, 3.3 and 3.9 microM, respectively. Flecainide and quinidine were less potent inhibitors of IK than was propafenone with IC50s of 15 and 14 microM compared with an IC50 of 5 microM for propafenone. By contrast with their effects on outward currents, these agents produced little or no inhibition of the inward rectifier K+ current, even when present at 300 microM. All three agents produced a concentration-dependent increase in the rate of inactivation of ITO but they only produced minor hyperpolarizing shifts (approximately 3 mV) in the voltage dependence of steady-state inactivation. Although propafenone had little effect on the rate of ITO recovery from inactivation (tau CONTROL = 64 +/- 5 ms; tau PROPAFENONE = 84 +/- 9 ms), ITO recovery in the presence of flecainide and quinidine was biexponential; it exhibited an additional slow component (tau FAST = 67 +/- 5 ms and tau SLOW = 2580 +/- 1500 ms for flecainide; tau FAST = 55 +/- 5 ms and tau SLOW = 871 +/- 99 ms for quinidine). Consistent with these observations, flecainide and quinidine, but not propafenone, produced use-dependent block of ITO at a stimulation frequency of 1 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action potential and sodium current in the slowly and rapidly adapting stretch receptor neurons of the crayfish (Astacus astacus)

Action potentials (APs) and sodium current from the slowly and the rapidly adapting stretch receptor neurons in the crayfish (Astacus astacus) were recorded with a two microelectrode voltage- and current-clamp technique. In the rapidly adapting neuron the APs had a duration of 3.2 +/- 0.2 ms (means +/- SE) and an amplitude of 55.2 +/- 1.5 mV. In the slowly adapting receptor neuron APs had a duration of 4.1 +/- 0.2 ms and an amplitude 79.9 +/- 2.0 mV. APs in the rapidly adapting neuron had a larger amplitude if they were recorded from the axon. In the rapidly adapting neuron adaptation of the impulse response was prolonged by hyperpolarization or by exposure to scorpion venom. Also, sinusoidal current stimulation added to the current steps prevented impulse adaptation. Block of the potassium currents in the slowly adapting neuron resulted in a rapid adaptation of the impulse response. The maximum sodium current amplitude was 313 +/- 15 nA in slowly adapting neuron and 267 +/- 11 nA in the rapidly adapting neuron. The current-voltage relationship showed a hump most marked in the slowly adapting neuron and abolished when a depolarizing prepulse was given. In the rapidly adapting neuron the inactivation starts at a more negative potential (Eh = -45 mV) and is faster compared with the slowly adapting neuron (Eh = -41 mV). The crude scorpion venom of Leiurus quinquestriatus (ScVLq) shifted hinfinity curve toward more positive potentials and slowed down the rate of inactivation. The results indicate the possible presence of more than one Na+ channel population and that the relative density and the spatial distribution is different in the slowly and rapidly adapting neuron. The difference contributes to the adaptive properties of the two receptor neurons.