Changes in the currents across sodium channels as affected by blood serum on cultured neuroblastoma cells

Adding of 5% bovine serum to internally perfused voltage-clamped serum deprived neuroblastoma cells rapidly stimulates transient sodium current. This stimulating effect is mainly due to the increase in the peak sodium conductance by almost 24 per cent, on the average. Besides that a modifying effect was observed resulting in the 6 mV shift of the sodium peak conductance curve towards more negative potentials and in the 5 mV shift of steady inactivation curve towards more positive ones. The sign of the latter shift was changed to the opposite under the action of serum thermally pretreated at 100 degrees C. This procedure led also to more than two fold lowering of the stimulating effect. Experiments with serum deprivation demonstrate different degrees of reversibility of the serum effects, the most reversible being the inactivation curve shift. EGF, insulin, dexamethasone, transferrin, ATP, serotonin and their combinations in physiological concentrations failed to give the typical whole serum effects. The serum is supposed to contain at least two active components of unknown nature, one of which being thermoresistant.     

Removal of sodium inactivation and block of sodium channels by chloramine-T in crayfish and squid giant axons.

Modification of sodium channels by chloramine-T was examined in voltage clamped internally perfused crayfish and squid giant axons using the double sucrose gap and axial wire technique, respectively. Freshly prepared chloramine-T solution exerted two major actions on sodium channels: (a) an irreversible removal of the fast Na inactivation, and (b) a reversible block of the Na current. Both effects were observed when chloramine-T was applied internally or externally (5-10 mM) to axons. The first effect was studied in crayfish axons. We found that the removal of the fast Na inactivation did not depend on the states of the channel since the channel could be modified by chloramine-T at holding potential (from -80 to -100 mV) or at depolarized potential of -30 mV. After removal of fast Na inactivation, the slow inactivation mechanism was still present, and more channels could undergo slow inactivation. This result indicates that in crayfish axons the transition through the fast inactivated state is not a prerequisite for the slow inactivation to occur. During chloramine-T treatment, a distinct blocking phase occurred, which recovered upon washing out the drug. This second effect of chloramine-T was studied in detail in squid axons. After 24 h, chloramine-T solution lost its ability to remove fast inactivation but retained its blocking action. After removal of the fast Na inactivation, both fresh and aged chloramine-T solutions blocked the Na currents with a similar potency and in a voltage-dependent manner, being more pronounced at lower depolarizing potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiology of phagocytic membranes: III. Evidence for a calcium-dependent potassium permeability change during slow hyperpolarizations of activated macrophages

The roles of potassium and calcium in the slow hyperpolarizations of membranes of activated macrophages are investigated using standard intracellular electrical recording techniques.The amplitude of spontaneous slow hyperpolarizations decreases as a logarithmic function of the external potassium concentration in the culture medium. Similar dependence on the potassium gradient is observed when different levels of membrane potentials are imposed by constant current injection. The reversal potential for electrically evoked slow hyperpolarizations is ?90 mV. A 10-fold increase in external potassium concentration causes a 60 mV shift of the reversal potential towards zero.Divalent cation ionophores (A23187 and X537A) can induce slow hyperpolarization responses in quiescent cells or permanent hyperpolarization in spontaneously active cells. The amplitude of the ionophore-induced hyperpolarizations is reduced by an increase in external potassium concentration in a manner consistent with data on slow hyperpolarization responses in the absence of ionophore.The calcium antagonist, verapamil, depresses the slow hyperpolarization responses at the concentration of 10^?5 M.It is suggested that the development of the hyperpolarizing response is due to a calcium-dependent potassium channel. The data support the assumption that spontaneous and artificially elicited slow hyperpolarization responses share a common calcium-dependent mechanism.     

Electrophysiology of phagocytic membranes. III. Evidence for a calcium-dependent potassium permeability change during slow hyperpolarizations of activated macrophages

The roles of potassium and calcium in the slow hyperpolarizations of membranes of activated macrophages are investigated using standard intracellular electrical recording techniques. The amplitude of spontaneous slow hyperpolarizations decreases as a logarithmic function of the external potassium concentration in the culture medium. Similar dependence on the potassium gradient is observed when different levels of membrane potentials are imposed by constant current injection. The reversal potential for electrically evoked slow hyperpolarizations is -90 mV. A 10-fold increase in external potassium concentration causes a 60 mV shift of the reversal potential towards zero. Divalent cation ionophores (A23187 and X537A) can induce slow hyperpolarization responses in quiescent cells or permanent hyperpolarization in spontaneously active cells. The amplitude of the ionophore-induced hyperpolarizations is reduced by an increase in external potassium concentration in a manner consistent with data on slow hyperpolarization responses in the absence of ionophore. The calcium antagonist, verapamil, depresses the slow hyperpolarization responses at the concentration of 10(-5) M. It is suggested that the development of the hyperpolarizing response is due to a calcium-dependent potassium channel. The data support the assumption that spontaneous and artificially elicited slow hyperpolarization responses share a common calcium-dependent mechanism.     

IK inactivation in squid axons is shifted along the voltage axis by changes in the intracellular pH.

The inactivation curve of the delayed rectifier in internally perfused squid giant axons is shifted along the voltage axis by changes in the pH of the internal perfusate. The amplitude of the shift is 9.5 mV per pH unit (6 less than or equal to pHi less than or equal to 10). No saturation of the effect was observed at either end of the pH range. This result suggests that the inactivation gating mechanism has several titratable groups accessible to protons from the intracellular side of the membrane.     

BTX modification of Na channels in squid axons. I. State dependence of BTX action

The state dependence of Na channel modification by batrachotoxin (BTX) was investigated in voltage-clamped and internally perfused squid giant axons before (control axons) and after the pharmacological removal of the fast inactivation by pronase, chloramine-T, or NBA (pretreated axons). In control axons, in the presence of 2-5 microM BTX, a repetitive depolarization to open the channels was required to achieve a complete BTX modification, characterized by the suppression of the fast inactivation and a simultaneous 50-mV shift of the activation voltage dependence in the hyperpolarizing direction, whereas a single long-lasting (10 min) depolarization to +50 mV could promote the modification of only a small fraction of the channels, the noninactivating ones. In pretreated axons, such a single sustained depolarization as well as the repetitive depolarization could induce a complete modification, as evidenced by a similar shift of the activation voltage dependence. Therefore, the fast inactivated channels were not modified by BTX. We compared the rate of BTX modification of the open and slow inactivated channels in control and pretreated axons using different protocols: (a) During a repetitive depolarization with either 4- or 100-ms conditioning pulses to +80 mV, all the channels were modified in the open state in control axons as well as in pretreated axons, with a similar time constant of approximately 1.2 s. (b) In pronase-treated axons, when all the channels were in the slow inactivated state before BTX application, BTX could modify all the channels, but at a very slow rate, with a time constant of approximately 9.5 min. We conclude that at the macroscopic level BTX modification can occur through two different pathways: (a) via the open state, and (b) via the slow inactivated state of the channels that lack the fast inactivation, spontaneously or pharmacologically, but at a rate approximately 500-fold slower than through the main open channel pathway.     

Effect of acidosis on transient outward potassium current in isolated rat ventricular myocytes

The effect of acidosis on the transient outward K(+) current (I(to)) of rat ventricular myocytes has been investigated using the perforated patch-clamp technique. When the holding potential was -80 mV, depolarizing pulses to potentials positive to -20 mV activated I(to) in subepicardial cells but activated little I(to) in subendocardial cells. Exposure to an acid solution (pH 6.5) had no significant effect on I(to) activated from this holding potential in either subepicardial or subendocardial cells. When the holding potential was -40 mV, acidosis significantly increased I(to) at potentials positive to -20 mV in subepicardial cells but had little effect on I(to) in subendocardial cells. The increase in I(to) in subepicardial cells was inhibited by 10 mM 4-aminopyridine. In subepicardial cells, acidosis caused a +8.57-mV shift in the steady-state inactivation curve. It is concluded that in subepicardial rat ventricular myocytes acidosis increases the amplitude of I(to) as a consequence of a depolarizing shift in the voltage dependence of inactivation.     

Single channel studies of the phosphorylation of K+ channels in the squid giant axon. II. Nonstationary conditions

The effects of phosphorylation on the properties of the 20-pS channel of the squid giant axon were studied using the cut-open axon technique. Phosphorylation of the channel was achieved by photoreleasing caged ATP (inside the patch pipette) in the presence of the catalytic subunit of the protein kinase A. An inverted K+ gradient (500 K+ external parallel 5 K+ internal) was used to study the activation process. Phosphorylation decreased the frequency of openings of the channel at most potentials by shifting the probability vs. voltage curve toward more positive potentials. The mean open times showed no voltage dependence and were not affected by phosphorylation. The distribution of first latencies, on the other hand, displayed a sharp voltage dependence. Phosphorylation increased the latency to the first opening at all potentials, shifting the median first latency vs. voltage curve toward more positive potentials. The slow inactivation process was studied in the presence of a physiological K+ gradient (10 K+ external parallel 310 K+ internal). Pulses to 40 mV from different holding potentials were analyzed. Phosphorylation increases the overall ensemble probability by decreasing the number of blank traces. A single channel inactivation curve was constructed by computing the relative appearance of blank traces at different holding potentials before and after photoreleasing caged ATP. As determined in dialyzed axons, the effect of phosphorylation consisted in a shift of the inactivation curve toward more positive potentials. The 20-pS channel has the same characteristics as the delayed rectifier current in activation kinetics, steady-state inactivation, and phosphorylation effects.     

Phosphorylation modulates potassium conductance and gating current of perfused giant axons of squid

The presence of internal Mg-ATP produced a number of changes in the K conductance of perfused giant axons of squid. For holding potentials between -40 and -50 mV, steady-state K conductance increased for depolarizations to potentials more positive than approximately -15 mV and decreased for smaller depolarizations. The voltage dependencies of both steady-state activation and inactivation also appears shifted toward more positive potentials. Gating kinetics were affected by internal ATP, with the activation time constant slowed and the characteristic delay in K conductance markedly enhanced. The rate of deactivation also was hastened during perfusion with ATP. Internal ATP affected potassium channel gating currents in similar ways. The voltage dependence of gating charge movement was shifted toward more positive potentials and the time constants of ON and OFF gating current also were slowed and hastened, respectively, in the presence of ATP. These effects of ATP on the K conductance occurred when no exogenous protein kinases were added to the internal solution and persisted even after removing ATP from the internal perfusate. Perfusion with a solution containing exogenous alkaline phosphatase reversed the effects of ATP. These results provide further evidence that the effects of ATP on the K conductance are a consequence of a phosphorylation reaction mediated by a kinase present and active in perfused axons. Phosphorylation appears to alter the K conductance of squid giant axons via a minimum of two mechanisms. First, the voltage dependence of gating parameters are shifted toward positive potentials. Second, there is an increase in the number of functional closed states and/or a decrease in the rates of transition between these states of the K channels.     

Gating of cardiac Na+ channels in excised membrane patches after modification by alpha-chymotrypsin.

Single cardiac Na+ channels were investigated after intracellular proteolysis to remove the fast inactivation process in an attempt to elucidate the mechanisms of channel gating and the role of slow inactivation. Na+ channels were studied in inside-out patches excised from guinea-pig ventricular myocytes both before and after very brief exposure (2-4 min) to the endopeptidase, alpha-chymotrypsin. Enzyme exposure times were chosen to maximize removal of fast inactivation and to minimize potential nonspecific damage to the channel. After proteolysis, the single channel current-voltage relationship was approximately linear with a slope conductance of 18 +/- 2.5 pS. Na+ channel reversal potentials measured before and after proteolysis by alpha-chymotrypsin were not changed. The unitary current amplitude was not altered after channel modification suggesting little or no effect on channel conductance. Channel open times were increased after removal of fast inactivation and were voltage-dependent, ranging between 0.7 (-70 mV) and 3.2 (-10 mV) ms. Open times increased with membrane potential reaching a maximum at -10 mV; at more positive membrane potentials, open times decreased again. Fast inactivation appeared to be completely removed by alpha-chymotrypsin and slow inactivation became more apparent suggesting that fast and slow inactivation normally compete, and that fast inactivation dominates in unmodified channels. This finding is not consistent with a slow inactivated state that can only be entered through the fast inactivated state, since removal of fast inactivation does not eliminate slow inactivation. The data indicate that cardiac Na+ channels can enter the slow inactivated state by a pathway that bypasses the fast inactivated state and that the likelihood of entering the slow inactivated state increases after removal of fast inactivation.     

Voltage-clamp of cut-end skeletal muscle fibre: a diffusion experiment

Membrane potential and current were studied in cut end fibres of frog skeletal muscle under current and voltage clamp conditions, by the double sucrose gap technique. Similar action potentials were recorded under current clamp conditions with either the microelectrode or the double sucrose gap techniques. Under voltage clamp conditions, the control of the membrane potential was maintained adequately. The early current was sensitive to both TTX and external Na concentration suggesting that the current was carried by Na ions. Sodium current (INa) was subsequently analysed using the Hodgkin-Huxley formulae. INa half-activation and inactivation occurred at -34 mV and -60 mV, respectively. Na-rich solution applied internally by diffusion through cut ends produced a reduction of INa associated with a shift of the sodium current reversal potential (VNa) towards more negative membrane potentials. This suggested that the sodium electromotive force was reduced by the increase in internal Na content of the fibre. Iodate applied externally changed neither the activation nor the inactivation time courses of INa, but reduced the peak current. Conversely, internally applied by diffusion from the cut end of skeletal muscle fibre, iodate slowed down the time course of INa inactivation and decreased the current peak. In conclusion, the double sucrose gap technique adapted to cut end frog skeletal muscle fibre allows a satisfactory analysis of INa.     

Effect of exogenous ATP on canine jejunal smooth muscle

Intracellular recordings were made from the circular smooth muscle cells of the canine jejunum to study the effect of exogenous ATP and to compare the ATP response to the nonadrenergic, noncholinergic (NANC) inhibitory junction potential (IJP) evoked by electrical field stimulation (EFS). Under NANC conditions, exogenous ATP evoked a transient hyperpolarization (6.5 +/- 0.6 mV) and EFS evoked a NANC IJP (17 +/- 0.4 mV). Omega-conotoxin GVIA (100 nM) and a low-Ca(2+), high-Mg(2+) solution abolished the NANC IJP but had no effect on the ATP-evoked hyperpolarization. The ATP-evoked hyperpolarization and the NANC IJP were abolished by apamin (1 microM) and N(G)-nitro-L-arginine (100 microM). Oxyhemoglobin (5 microM) partially (38.8 +/- 5.5%) reduced the amplitude of the NANC IJP but had no effect on the ATP-evoked hyperpolarization. Neither the NANC IJP nor the ATP-evoked hyperpolarization was affected by P2 receptor antagonists or agonists, including suramin, reactive blue 2, 1-(N, O-bis-[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine , pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid, alpha, beta-methylene ATP, 2-methylthioadenosine 5'-triphosphate tetrasodium salt, and adenosine 5'-O-2-thiodiphosphate. The data suggest that ATP evoked an apamin-sensitive hyperpolarization in circular smooth muscle cells of the canine jejunum via local production of NO in a postsynaptic target cell.     

Distribution and kinetics of membrane dielectric polarization. 1. Long- term inactivation of gating currents

Gating currents were measured by subtracting the linear component of the capacitative current recorded at very positive or very negative potentials. When the membrane is depolarized for a few minutes, repolarized to the usual holding potential (HP) of --70 mV for 1 ms, and then pulsed to 0 mV, the charge transferred in 2--4 ms is approximately 50% of that which was transferred during the same pulse holding at --70 mV. This charge decrease, called slow inactivation of the gating current, was found to be consistent with a shift of the charge vs. potential (Q-V) curve to more hyperpolarized potentials. When the HP is 0 mV, the total charge available to move is the same as the total charge available when the HP is --70 mV. The time constants of the fast component of the ON gating current are smaller at depolarized holding potentials than at --70 mV. When the HP is --70 mV and a prepulse of 50 ms duration is given to 0 mV, the Q-V curve is also shifted to more hyperpolarized potentials (charge immobilization), but the effect is not as pronounced as the one obtained by holding at 0 mV. When the HP is 0 mV, a prepulse to --70 mV for 50 ms partially shifts back the Q-V curve, indicating that fast inactivation of the gating charge may be recovered in the presence of slow inactivation. A physical model consisting of a gating particle that interacts with a fast inactivating particle, and a slow inactivating particle, reproduces most of the experimental results.     

Influence of extracellular pH on the modulatory effect of zinc ions on Kv1.3 potassium channels.

In the present study we applied the whole-cell patch-clamp technique to study the influence of extracellular pH (pH(o)) on the modulatory effect exerted by zinc ions (Zn(2+)) on voltage-gated potassium channels Kv1.3 expressed in human lymphocytes. Obtained data provide evidence that lowering of pH(o) from the 7.35 to 6.4 slowed significantly the current activation rate, shifted the activation midpoint by about 16 mV towards positive membrane potentials and reduced the current amplitude to about 0.55 of the control value. In contrast, raising the pH(o) from 7.35 to 8.4 did not affect significantly the activation midpoint and current amplitude. Application of Zn(2+) in the concentration range from 100 microM to 1 mM at pH(o)=6.4 slowed additionally the activation rate, shifted the activation midpoint by about 20 mV towards positive membrane potentials and reduced the current amplitude in a concentration-dependent manner. The total effect exerted by Zn(2+) and protons at pH(o) = 6.4 was more significant than the effect exerted by Zn alone. Both the magnitude of the shift and the degree of current inhibition by Zn(2+) were independent on pH(o) in the range from 6.4 to 8.4. The data might suggest that the effects exerted by protons and zinc ions occur independently on each other and probably involve different mechanisms. Changing the holding potential from -90 mV to -60 mV at pH(o)=7.35 abolished the Zn(2+)-induced inhibition of the current amplitudes at concentrations below 300 microM. At pH(o)=6.4 the total inhibition caused by Zn(2+) and protons was also diminished, however, a significant reduction was observed at 100 microM concentration. In contrast, changing the holding potential did not change the Zn(2+)- and proton-induced shift of the activation midpoint. Altogether, obtained data suggest that extracellular protons exert the modulatory effects that are additive to the effects exerted by Zn(2+) on the channels. Possible physiological significance of these additive effects is discussed.     

Effect of sea anemone toxins on the sodium inactivation process in crayfish axons

The effect of sea anemone toxins from Parasicyonis actinostoloides and Anemonia sulcata on the Na conductance in crayfish giant axons was studied under voltage-clamp conditions. The toxin slowed the Na inactivation process without changing the kinetics of Na activation or K activation in an early stage of the toxin effect. An analysis of the Na current profile during the toxin treatment suggested an all-or-none modification of individual Na channels. Toxin-modified Na channels were partially inactivated with a slower time course than that of the normal inactivation. This slow inactivation in steady state decreased in its extent as the membrane was depolarized to above -45 mV, so that practically no inactivation occurred at the membrane potentials as high as +50 mV. In addition to inhibition of the normal Na inactivation, prolonged toxin treatment induced an anomalous closing in a certain population of Na channels, indicated by very slow components of the Na tail current. The observed kinetic natures of toxin-modified Na channels were interpreted based on a simple scheme which comprised interconversions between functional states of Na channels. The voltage dependence of Parasicyonis toxin action, in which depolarization caused a suppression in development of the toxin effect, was also investigated.     

Isoform-specific lidocaine block of sodium channels explained by differences in gating

When depolarized from typical resting membrane potentials (V(rest) approximately -90 mV), cardiac sodium (Na) currents are more sensitive to local anesthetics than brain or skeletal muscle Na currents. When expressed in Xenopus oocytes, lidocaine block of hH1 (human cardiac) Na current greatly exceeded that of mu1 (rat skeletal muscle) at membrane potentials near V(rest), whereas hyperpolarization to -140 mV equalized block of the two isoforms. Because the isoform-specific tonic block roughly parallels the drug-free voltage dependence of channel availability, isoform differences in the voltage dependence of fast inactivation could underlie the differences in block. However, after a brief (50 ms) depolarizing pulse, recovery from lidocaine block is similar for the two isoforms despite marked kinetic differences in drug-free recovery, suggesting that differences in fast inactivation cannot entirely explain the isoform difference in lidocaine action. Given the strong coupling between fast inactivation and other gating processes linked to depolarization (activation, slow inactivation), we considered the possibility that isoform differences in lidocaine block are explained by differences in these other gating processes. In whole-cell recordings from HEK-293 cells, the voltage dependence of hH1 current activation was approximately 20 mV more negative than that of mu1. Because activation and closed-state inactivation are positively coupled, these differences in activation were sufficient to shift hH1 availability to more negative membrane potentials. A mutant channel with enhanced closed-state inactivation gating (mu1-R1441C) exhibited increased lidocaine sensitivity, emphasizing the importance of closed-state inactivation in lidocaine action. Moreover, when the depolarization was prolonged to 1 s, recovery from a "slow" inactivated state with intermediate kinetics (I(M)) was fourfold longer in hH1 than in mu1, and recovery from lidocaine block in hH1 was similarly delayed relative to mu1. We propose that gating processes coupled to fast inactivation (activation and slow inactivation) are the key determinants of isoform-specific local anesthetic action.     

Calcium channel currents in pars intermedia cells of the rat pituitary gland. Kinetic properties and washout during intracellular dialysis

Ca channel currents in primary cultured pars intermedia cells were studied using whole-cell recording with patch pipettes. Experiments were carried out at 18-21 degrees C in cells internally dialyzed with K-free, EGTA-containing solutions and in the presence of 10 mM Ca or 10 mM Ba in the external solution. Ca and Ba currents depended on the activity of two main populations of channels, SD and FD. With Ca as the charge carrier, these two populations differed in their closing time constants at -80 mV (SD, 1.8 ms; FD, 110 microseconds), apparent activation levels (SD, -40 mV; FD, -5 mV), half-maximal activation levels (SD, +5 to +10 mV; FD, +20 to +25 mV), half-times of activation at +20 mV (SD, 2.5-3.5 ms; FD, 1.0-1.3 ms), and time courses of inactivation (SD, fast; FD, slow). Functional FD channels were almost completely lost within 20-25 min of breaking into a cell, whereas SD channels retained most of their functional activity. In addition, the conductance-voltage curve for FD channels shifted approximately 15 mV toward more negative membrane potentials within 11-14 min under whole-cell recording. At that time, 60-70% of the FD channel maximum conductance was lost. However, the conductance-voltage curve for SD channels shifted less than 5 mV within 25 min. The addition of 3 mM MgATP and 40 microM GTP to the internal solution slowed down the loss of FD channels and prevented the shift in their activation curve. It was also found that the amplitude of the current carried by FD channels tends to increase as a function of the age of the culture, with no obvious changes in the kinetic properties of the channels or in SD channel activity.     

A delayed rectifier potassium current in Xenopus oocytes.

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

Modulation of nerve membrane sodium channels by chemicals

1. Modulations of sodium channel kinetics by grayanotoxins and pyrethroids have been studied using voltage clamped, internally perfused giant axons from crayfish and squid. 2. Grayanotoxin I and alpha-dihydrograyanotoxin II greatly depolarize the nerve membrane through an increase in resting sodium channel permeability to sodium ions. 3. Grayanotoxins modify a fraction of sodium channel population to give rise to a slow conductance increase with little or no inactivation, and the slow conductance-membrane potential curve is shifted toward hyperpolarization. This accounts for the depolarization. 4. The tail current associated with step repolarization during the slow current in grayanotoxins decays with a dual exponential time course. 5. (+)-trans tetramethrin and (+)-trans allethrin also modify a fraction of sodium channel population in generating a slow current, which attains a maximum slowly and decays very slowly during a maintained depolarizing step. The membrane is depolarized only slightly. 6. The tail current associated with step repolarization during the slow current in the pyrethroids is very large in initial amplitude and decays very slowly. 7. The rate at which the sodium channel arrives at the modified open state in the presence of pyrethroids is expressed by a dual exponential function, and the slow phase disappears following removal of the sodium inactivation mechanism by internal perfusion of pronase. 8. A kinetic model is proposed to account for the actions of both grayanotoxins and pyrethroids on sodium channels. Both chemicals interact with the channel at both open and closed states to yield a modified open state which results in a slow sodium current.     

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.