Peak INa Research Articles

Terfenadine blocks time-dependent Ca2+, Na+, and K+ channels in guinea pig ventricular myocytes.

Terfenadine, which blocks delayed rectifier K+ channels (Ik), is structurally related to diphenylalkylamine L-type Ca2+ channel (ICa) blockers and has been reported to render Purkinje fibers inexcitable. We used standard whole-cell patch clamp techniques in isolated guinea pig ventricular myocytes to investigate the direct effect of terfenadine on ICa after discovering that the upstrokes of early afterdepolarizations in guinea pig myocytes were inhibited by the drug at concentrations > or = 10(-6)M. Some data analyzing the effect of terfenadine on time-dependent Na+ channels (INa) and IK also were obtained. All experiments were controlled for time of intracellular dialysis. Terfenadine (3 x 10(-6)M) reduced peak ICa (measured in either K+-containing or Cs+-substituted intracellular solutions from holding potentials of -40 mV) after 10 min exposure [peak at 0 mV in K+-deficient dialysis solution -4.2 +/- 2.3 pA/pF (mean +/- SD, n = 5) versus -13.02 +/- 4.33 pA/pF in control solution (n = 5), p < 0.01], and ICa was almost completely blocked after 15 min drug exposure. Ten minutes of exposure to terfenadine (3 x 10-6M) also caused near-complete blockade of peak INa when INa was measured at -40 mV after 300 ms conditioning pulses from a holding potential of -40 to potentials between -60 and -90 mV. The effect was much less pronounced when INa was measured from a holding potential of -90 mV. After exposure to terfenadine 3 x 10 (-6)M, IK density, measured as peak tail current at -40 mV after 300-ms depolarizations, was also reduced but not eliminated at membrane potentials between -20 and +60 mV. In contrast, exposure to terfenadine caused no significant change in the current-voltage relationship after 300-ms steps from -90 to +60 mV. Terfenadine had no effect on time constants of decay of IK or ICa. These results suggest that terfenadine blocks several time- and voltage-dependent channels, possibly by binding to a common protein structure, not related to ion selectivity, that is primarily associated with time-dependent activation of channel conductance.

Interaction of Na+ and Mg2+ ions in acetylcholine receptor channels of frog skeletal muscle changes in character with an increase in agonist concentration.

Measurements of agonist-induced single-channel currents of nicotinic acetylcholine receptor channels (nAChR) have shown that addition of divalent metal cations (Ca2+, Mg2+, Ba2+) in millimolar amounts causes a decrease of 40% or more in currents carried by monovalent cations (Na+, Cs+). Conventional voltage-clamp measurements were made at frog (Rana pipiens) sartorius endplates to determine if this occlusive interaction between mono- and divalent cations is preserved in macroscopic currents produced by higher concentrations of nAChR agonists, as would be expected on the basis of simple summation of unitary currents. Single ion nAChR currents of Na+ (INa+ ) and Mg2+(IMg2+ ) and the composite current when both cations were present (INa+, Mg2+ ) were measured in high-K+, low ionic strength bathing media during local superfusion with various concentrations of carbamylcholine. It was found that, at lower test carbamylcholine levels of 27 and 54 microM, peak values of INa+, Mg2+ were 40% and 53% of the respective INa+ , in agreement with the effects observed from single-channel measurements. At all higher carbamylcholine levels, however, peak INa+, Mg2+ exceeded INa+ and increased instead with the sum of INa+ and IMg2+ as if there was an independent movement of these ions through the channels rather than the occlusive interaction found at lower carbamylcholine levels. This suggests that with exposure to increasing agonist concentrations above those commonly used in single-channel measurements there are changes in the open state of nAChR affecting cation selectivity possibly in the narrow pore region of the channels.

Effect of intracellular and extracellular acidosis on sodium current in ventricular myocytes

Conduction slowing is an essential element in the generation of ischemic ventricular arrhythmias and is determined in part by the inward Na+ current (INa). Because intracellular acidosis is an early consequence of ischemia, we hypothesized that lowering intracellular pH (pHi) would reduce or kinetically modulate INa and thus affect cardiac conduction. To test this hypothesis, the whole cell patch-clamp method was used to measure INa in neonatal rat ventricular myocytes exposed to varying extracellular pH (pHo 6.4-7.4), while perfusing the cells with acidic solutions (pHi 6.2-7.2). With simultaneous acidification of pHo and pHi there was a progressive increase in time to peak current, a 31% decrease in peak INa (298 +/- 18 to 206 +/- 16 pA/pF), and a complex slowing of inactivation kinetics. At the most extreme levels of acidification, there was a 5-mV hyperpolarizing shift in steady-state inactivation and a 6-mV depolarizing shift in activation. Independent changes of pHo and pHi indicate that the reduction of peak INa is a function of pHo. However, steady-state inactivation is modulated by pHi. The time course of activation and inactivation appears to depend on both pHo and pHi. We conclude that both intracellular and extracellular acidosis are significant but distinct modulators of INa amplitude and kinetics in cardiac myocytes.

Irreversible inhibition of sodium current and batrachotoxin binding by a photoaffinity-derivatized local anesthetic.

We have synthesized a model local anesthetic (LA), N-(2-di-N-butyl-aminoethyl)-4-azidobenzamide (DNB-AB), containing the photoactivatable aryl azido moiety, which is known to form a covalent bond to adjacent molecules when exposed to UV light (Fleet, G.W., J.R. Knowles, and R.R. Porter. 1972. Biochemical Journal. 128:499-508. Ji, T.H. 1979. Biochimica et Biophysica Acta. 559:39-69). We studied the effects of DNB-AB on the sodium current (INa) under whole-cell voltage clamp in clonal mammalian GH3 cells and on 3[H]-BTX-B binding to sheep brain synaptoneurosomes. In the absence of UV illumination, DNB-AB behaved similarly to known LAs, producing both reversible block of peak INa (IC50 = 26 microM, 20 degrees C) and reversible inhibition of 3[H]-BTX-B (50 nM in the presence of 0.12 microgram/liter Leiurus quinquestriatus scorpion venom) binding (IC50 = 3.3 microM, 37 degrees C), implying a noncovalent association between DNB-AB and its receptor(s). After exposure to UV light, both block of INa and inhibition of 3[H]-BTX-B binding were only partially reversible (INa = 42% of control; 3[H]-BTX-B binding = 23% of control) showing evidence of a light-dependent, covalent association between DNB-AB and its receptor(s). In the absence of drug, UV light had less effect on INa (post exposure INa = 96% of control) or on 3[H]-BTX-B binding (post exposure binding = 70% of control). The irreversible block of INa was partially protected by coincubation of DNB-AB with 1 mM bupivacaine (IC50 = 45 microM, for INa inhibition at 20 degrees C, Wang, G.K., and S.Y. Wang. 1992. Journal of General Physiology. 100:1003-1020), (post exposure INa = 73% of control). The irreversible inhibition of 3[H]-BTX-B binding also was partially protected by coincubation with bupivacaine (500 microM, 37 degrees C) (post exposure binding = 51% of control), suggesting that the site of irreversible inhibition of both INa and 3[H]-BTX-B binding is shared with the clinical LA bupivacaine.

Fast sodium currents induced by serum in human uterine leiomyosarcoma cells.

We previously demonstrated that fast Na+ channels are expressed in uterine smooth muscle cells during pregnancy [Y. Ohya, and N. Sperelakis. Am. J. Physiol. 257 (Cell Physiol. 26): C408-C412, 1989; Y. Inoue, and N. Sperelakis. Am. J. Physiol. 260 (Cell Physiol. 29): C658-C663, 1991]. In the present study, we investigated the possible expression of fast Na+ channels and resultant fast Na+ current [INa(f)] using whole cell voltage clamp in cultured uterine leiomyosarcoma cells (SK-UT-1B), which are derived from human myometrium. The expression of INa(f) was dependent on the concentration of fetal bovine serum (FBS) in the culture medium. The mean current densities of peak INa(f) in cells incubated with 0, 1, 5, and 10% FBS were (in pA/pF) 1.3 +/- 0.5, 1.6 +/- 0.9, 9.8 +/- 2.4, and 9.8 +/- 1.7, respectively. Two types of INa(f) were identified based on different time courses of current decay. Both types of current were equally tetrodotoxin sensitive (half-maximal inhibitory concentration approximately 50 nM) and had similar electrical properties. These characteristics are consistent with those in the pregnant myometrial cells, yet the current density in the sarcoma cells is much higher. These findings suggest that one or more factors in serum induce INa(f) in the uterine sarcoma cells, and this cell line could be a good model for the study of the fast Na+ channels in uterine smooth muscle.

Different voltage dependence of transient and persistent Na+ currents is compatible with modal-gating hypothesis for sodium channels.

1. These experiments tested the hypothesis that the differing voltage dependence of the transient (INa) and persistent (INaP) Na+ currents in neocortical neurons results from the state of inactivation of one type of Na+ channel rather than from the existence of different types of Na+ channels. This question was examined in acutely isolated pyramidal neurons from the sensorimotor cortex of rats by using papain to remove inactivation from INa and comparing the resulting activation curve with that of INaP. 2. In control cells, INaP activated at more negative potentials than INa. Inclusion of papain in the recording pipette removed inactivation from INa and caused the INa activation curve to be shifted leftward to the position of the curve for INaP measured in control cells. Papain greatly increased both INa amplitude and the time to reach peak INa during smaller depolarizations, whereas the difference between control and test currents was reduced during large depolarizations. 3. We conclude that differences in the voltage dependence of INa and INaP activation does not provide sufficient evidence that these currents flow through separate sets of Na+ channels. Instead, our results are consistent with the idea that INaP largely arises from a fraction of the transient Na+ channels that intermittently lose their inactivation.

Modification of cardiac sodium current by intracellular application of cAMP

We examined the effects of intracellular perfusion of cyclic adenosine monophosphate (cAMP) on the sodium current (INa) of guinea-pig ventricular myocytes, using the whole-cell clamp technique. INa was elicited by depolarizing voltage steps (-20 mV) from a variety of holding potentials (-120 to -50 mV), under conditions of 60 mM extracellular Na+ concentration ([Na+]o) and at the temperature of 24-26 degrees C. Intracellular perfusion of cAMP decreased the INa elicited from the holding potentials less negative than -90 mV. In the presence of 1 mM cAMP, for example, the peak INa elicited from -80 mV decreased from 6.0 +/- 2.0 nA to 4.0 +/- 2.2 nA (mean +/- SD, P < 0.02, n = 7) within 3-6 min. In the presence of extracellular 3-isobutyl-1-methylxanthine (IBMX, 20 microM), much lower concentrations of cAMP (0.2 mM) yielded a comparable effect. On the other hand, intracellular perfusion of cAMP increased the INa elicited from very negative holding potentials (< -100 mV). For instance, the application of cAMP (1 mM) increased the INa elicited by step depolarizations from -120 mV (to -20 mV), from 9.9 +/- 2.1 nA to 11.0 +/- 3.1 nA (P < 0.05, n = 5). The former effect was attributed to a marked shift of the steady-state inactivation curve of INa to the negative direction; the voltage of half-inactivation shifted from -77.9 +/- 1.0 to -83.5 +/- 1.4 mV, or by -5.6 mV. The latter effect may be explained by increases in maximum available conductance of INa.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of sodium channel inactivation by alpha-chymotrypsin in canine cardiac Purkinje cells.

Studies of tetrodotoxin-sensitive sodium current (INa) after modification of inactivation by intracellular enzymes in mammalian cells have demonstrated a marked increase in peak INa at test potentials near current threshold causing a large, negative shift of the peak INa conductance-voltage relationship by approximately -20 mV. These findings support a kinetic model in which the unmodified Na channel has rapid and voltage-independent inactivation from the open state. However, the kinetics of cardiac Na channels differ from those of mammalian neuronal Na channels. In particular, inactivation of cardiac Na channels has been proposed to be more voltage dependent than that of tetrodotoxin-sensitive Na channels. To help understand the role of inactivation in cardiac Na channel kinetic behavior, we studied Na currents before and after modification of inactivation by the proteolytic enzyme, alpha-chymotrypsin. Whole cell INa was measured in single canine cardiac Purkinje cells that were voltage clamped and internally perfused with a large-bore suction pipette. The decay of INa in response to step depolarizations was dramatically slowed after perfusion with intracellular alpha-chymotrypsin consistent with modification of inactivation. In contrast to mammalian tetrodotoxin-sensitive Na current, Boltzmann distribution fits to peak INa conductance-voltage (GNa-V) relationships after alpha-chymotrypsin showed no change in either the potential at half maximum conductance (V 1/2), after correction for the spontaneous background shift of INa kinetics, or in the voltage-dependence of conductance (i.e., slope factor of GNa-V relationships). Maximal peak INa conductance increased by 18%. INa tail-current relaxations at potentials < or = -110 mV, after correction for spontaneous shifts in Na channel kinetics, were also similar before and after modification by alpha-chymotrypsin. alpha-chymotrypsin modified inactivation of cardiac INa with little or no change in activation, and cardiac Na channel inactivation was slow near threshold and played little role in determining V1/2 for peak INa conductance-voltage relationships.

Responsiveness of cardiac Na+ channels to a site-directed antiserum against the cytosolic linker between domains III and IV and their sensitivity to other modifying agents

Elementary Na+ currents were recorded in inside-out patches from neonatal rat heart cardiocytes to analyze the influence of a site-directed polyclonal anti-serum against the linker region between the domains III and IV (amino acids 1489-1507 of the cardiac Na+ channel protein) on Na+ channel gating and to test whether this part of the alpha-subunit may be considered as a target for modifying agents such as the (-)-enantiomer of DPI 201-106. Anti-SLP 1 serum (directed against amino acids 1490-1507) evoked, usually within 10-15 min after cytosolic administration, modified Na+ channel activity. Antiserum-modified Na+ channels retain a single open state but leave, at -60 mV for example, their conducting configuration consistently with an about threefold lower rate than normal Na+ channels. Another outstanding property of noninactivating Na+ channels, enhanced burst activity, may be quite individually pronounced, a surprising result which is difficult to interpret in terms of structure-function relations. Removal of inactivation led to an increase of reconstructed peak INa (indicating a rise in NPo) and changed INa decay to obey second-order kinetics, i.e., open probability declined slowly but progressively during membrane depolarization. The underlying deactivation process is voltage dependent and responds to a positive voltage shift with a deceleration but may operate even at the same membrane potential with different rates. Iodate-modified Na+ channels exhibit very similar properties including a conserved conductance. They are likewise controlled by an efficient, voltage-dependent deactivation process.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of TTX-sensitive and TTX-resistant sodium currents in small cells from adult rat dorsal root ganglia.

1. The whole-cell patch-clamp technique was used to investigate the characteristics of two types of sodium current (INa) recorded at room temperature from small diameter (13-25 microns) dorsal root ganglion (DRG) cells, isolated from adult rats and maintained overnight in culture. 2. Sodium currents were isolated pharmacologically. Internal Cs+ and external tetraethylammonium (TEA) ions were used to suppress potassium currents. A combination of internal EGTA, internal F-, a low (10 microM) concentration of external Ca2+ and a relatively high (5 mM) concentration of internal and external Mg2+ was used to block calcium channels. The remaining voltage-dependent currents reversed direction at the calculated sodium equilibrium potential. Both the reversal potential and magnitude of the currents exhibited the expected dependence on the external sodium concentration. 3. INa subtypes were characterized initially in terms of their sensitivity to tetrodotoxin (TTX). TTX-sensitive (TTXs) currents were at least 97% suppressed by 0.1 microM TTX. TTX-resistant (TTXr) INa were recorded in the presence of 0.3 microM TTX and appeared to be reduced in amplitude by less than 50% in 75 microM TTX (n = 1). 4. As in earlier studies, the peak of the current-voltage relationship, the mid-point of the normalized conductance curve and the potential (Vh) at which the steady-state inactivation parameter (h infinity) was 0.5 were found to be significantly more depolarized for the TTXr INa (by ca 10, 14 and 37 mV respectively). There was little difference in the slope at the mid-point of the normalized conductance curves (the mean slope factors were 5.1 mV for the TTXs INa and 4.9 mV for the TTXr current) but the h infinity curves for TTXr currents were significantly steeper than those for TTXs currents (mean slope factors of 3.8 and 11.5 mV respectively). Both the time to peak and the decay time constant of the peak current recorded from a holding potential of -67 mV were more than a factor of three slower for the TTXr INa than for the TTXs current. 5. However, in direct contrast to the difference in activation and decay kinetics, 'slow' TTXr INa recovered from inactivation at -67mV, or reprimed, more than a factor of ten faster than 'fast' TTXs INa. 6. The differences apparent in both the repriming kinetics of TTXs and TTXr INa at -67 mV and the kinetics of the decay phase of the peak INa are shown to be explicable largely in terms of the voltage dependence of their respective inactivation systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Modification of cardiac Na+ channels by anthopleurin-A: effects on gating and kinetics.

We used the whole cell patch clamp technique to investigate the characteristics of modification of cardiac Na+ channel gating by the sea anemone polypeptide toxin anthopleurin-A (AP-A). Guinea pig ventricular myocytes were isolated enzymatically using a retrograde perfusion apparatus. Holding potential was -140 mV and test potentials ranged from -100 to +40 mV (pulse duration 100 or 1000 ms). AP-A (50-100 nM) markedly slowed the rate of decay of Na+ current (INa) and increased peak INa conductance (gNa) by 38 +/- 5.5% (mean +/- SEM, P < 0.001, n = 12) with little change in slope factor (n = 12) or voltage midpoint of the gNa/V relationship after correction for spontaneous shifts. The voltage dependence of steady-state INa availability (h infinity) demonstrated an increase in slope factor from 5.9 +/- 0.8 mV in control to 8.0 +/- 0.7 mV after modification by AP-A (P < 0.01, n = 14) whereas any shift in the voltage midpoint of this relationship could be accounted for by a spontaneous time-dependent shift. AP-A-modified INa showed a use-dependent decrease in peak current amplitude (interpulse interval 500 ms) when pulse duration was 100 ms (-15 +/- 2%, P < 0.01, n = 17) but showed no decline when pulse duration was 100 ms (-3 +/- 1%). This use-dependent effect was probably the result of a decrease in the recovery from inactivation caused by AP-A which had a small effect on the fast time constant of recovery (from 4.1 +/- 0.3 ms in control to 6.0 +/- 1.1 ms after AP-A, P < 0.05) but increased the slow time constant from 66.2 +/- 6.5 ms in control to 188.9 +/- 36.4 ms (P < 0.002, n = 19) after exposure to AP-A. Increasing external divalent cation concentration (either Ca2+ or Mg2+) to 10 mM abolished the effects of AP-A on the rate of INa decay. These results demonstrate that modification of cardiac Na+ channels by AP-A markedly slowed INa inactivation and altered the voltage dependence of activation; these alterations in gating characteristics, in turn, caused an increase in gNa presumably by increasing the number of channels open at peak INa. AP-A slows the rate of recovery of INa from inactivation which is probably the basis for a use-dependent decrease in peak amplitude. Finally, AP-A binding is sensitive to external divalent cation concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation, inactivation and recovery in the sodium channels of the squid giant axon dialysed with different solutions

Comparisons were made between families of ion currents recorded in voltage-clamped squid axons dialysed with 20 mM NaF and 330 mM CsF or TMAF, and bathed in a solution in which four fifths of the Na was replaced by Tris. The permeability coefficient PNa,fast for the fast-inactivating current in the initial open state was calculated as a function of test potential from the size of the initial peak of INa. The permeability coefficient PNa,non for the non-inactivating open state was calculated from the steady-state INa that persisted until the end of the test pulse. Dialysis with TMA had no direct effect on the QV curve for gating charge. The reversal potential for INa,non was always lower than that for INa,fast, the mean difference being about -9 mV when dialysing with Cs, but only about -1 mV with TMA. Except close to threshold, PNa,fast was roughly halved by dialysis with TMA as compared with Cs, but PNa,non was substantially increased. The time constant tau h inactivation of the sodium system was slightly increased during dialysis with TMA in place of Cs, and there were small shifts in the steady-state inactivation curve, but the rate of recovery from inactivation was not measurably altered. The flattening off of the tau h curve at increasingly positive test potentials corresponded to a steady reduction of the apparent inactivation charge until a value of about 0.2e was reached for pulses to 100 mV. The instantaneous I-V relationship in the steady state was also investigated. The results have a useful bearing on the effects of dialysis with TMA, on the differences between the initial and steady open states of the sodium channel, and on the relative voltage-dependences of the transitions in each direction between the resting and inactivated states.

Extracellular divalent and trivalent cation effects on sodium current kinetics in single canine cardiac Purkinje cells.

1. The effects of the extracellular divalent cations barium, calcium, cadmium, cobalt, magnesium, manganese, nickel and zinc and the trivalent cation lanthanum on macroscopic sodium current (INa) were characterized in enzymatically isolated single canine cardiac Purkinje cells under voltage clamp at 9-14 degrees C. 2. All di(tri)valent cations produced depolarizing shifts in the conductance-voltage relationship. The order of efficacy, taken as the concentration required to produce a 5 mV shift in the mid-point of peak INa conductance, from least to most effective was (mM): Ca2+ (2.97) approximately Mg2+ (2.67) approximately Ba2+ (1.93) > CO2+ (1.02) approximately Mn2+ (0.88) > Ni2+ (0.54) > La3+ (0.095) approximately Cd2+ (0.083) approximately Zn2+ (0.076). 3. Addition of di(tri)valent cations also produced depolarizing shifts in voltage-dependent availability. The order of efficacy from the least to most effective was (mM): Cd2+ (7.70) approximately Mg2+ (6.86) approximately Ba2+ (4.50) > Ca2+ (2.47) approximately CO2+ (1.87) approximately Mn2+ (1.24) approximately Ni2+ (1.20) > Zn2+ (0.300) > La3+ (0.060). 4. The Gouy-Chapman-Stern equations were used to evaluate di(tri)valent cation efficacy in binding to surface charges. Surface charge density was estimated as 0.72 sites nm-2, and it was assumed that Mg2+, the divalent cation that produced the smallest shift, screened but did not bind to surface charges. Based on voltage-dependent availability, KD from lowest to highest affinity were (mM): Ba2+ (2500) > CO2+ (1670) approximately Mn2+ (1430) approximately Ca2+ = Cd2+ = Ni2+ (1200) > Zn2+ (250) > La3+ (30). 5. All di(tri)valent cations also produced a concentration-dependent acceleration of INa tail current relaxation. The addition of Ca2+ and La3+ produced acceleration of tail current relaxations that could be accounted for by the surface charge effects predicted from the shift in voltage-dependent availability. Cd2+, which produced almost no change in voltage-dependent availability, dramatically accelerated tail current relaxation. Zn2+, Ni2+, Mn2+ and CO2+ also produced greater acceleration of tail current relaxation than could be accounted for by surface charge effects. 6. Di(tri)valent cations delayed time to peak INa in a concentration-dependent manner. The time to peak INa-voltage relationship was well described by an exponential plus a constant, and di(tri)valent cations did not affect the slope factor or constant but shifted the relationship in the depolarizing direction. Similar to their effect on tail currents, addition of some di(tri)valent cations produced larger effects on time to peak INa than expected from the shift of voltage-dependent availability.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential response of DPI-modified cardiac Na+ channels to antiarrhythmic drugs: no flicker blockade by lidocaine.

Elementary Na+ currents were recorded in cell-attached patches from short-time cultured neonatal cardiocytes in order to test the hypothesis whether the open state of DPI-modified, noninactivating cardiac Na+ channels is basically sensitive to blocking drug molecules such as antiarrhythmics. Lidocaine (300 mumol/liter) effectively reduced the open probability of cardiac Na+ channels and, at a stimulation rate of 1 Hz, depressed the reconstructed macroscopic peak INa to 40 +/- 3.5% of the predrug value. The same drug concentration failed to influence DPI-modified Na+ channels. Their open state proved almost insensitive to lidocaine. tau open decreased only slightly to 85 +/- 2%. Still more importantly, the number of transitions between the conducting and a nonconducting configuration did not increase. At -40 mV, lidocaine may interfere with the open state with an association rate constant of 1.3 x 10(5) mol-1 sec-1 which is about two orders of magnitude smaller than the rate constant obtained with propafenone or prajmalium. Moreover, propafenone (10-20 mumol/liter) or prajmalium (30 mumol/liter) led to a tremendous increase in the number of transitions between the open and a nonconducting configuration. Lidocaine also failed to evoke a fast flicker blockade with reaction kinetics in the microsecond range. It is concluded that DPI-modified cardiac Na+ channels discriminate between lidocaine and other antiarrhythmic drugs. As a tentative explanation, this might be indicative for multiple binding sites for those drugs in cardiac Na+ channels.

Inhibitory effects of palmitoylcarnitine and lysophosphatidylcholine on the sodium current of cardiac ventricular cells.

We investigated the effects of ischemia-related amphipathic compounds, palmitoylcarnitine (PamCar, 0.5-50 microM) and lysophosphatidylcholine (lysoPtdCho, 5-50 microM) on sodium current (INa) of guinea-pig ventricular myocytes. The cells were perfused with low-Na+ (60 mM) Tyrode's solution, and Ca2+ and K+ currents were blocked by external Co2+ (3 mM) and internal Cs+ (140 mM), respectively. INa was elicited by depolarizing voltage steps from a holding potential of -100 mV at a temperature of 33 degrees C. PamCar (5 microM) decreased the peak INa (attained at -20 mV or -30 mV) from 6.1 +/- 2.1 nA to 3.9 +/- 1.4 nA (n = 11), or by 36.1% within 2 min, and shifted the curve of steady-state INa inactivation by 5.4 mV in the positive direction (from -76.3 +/- 4.6 mV, control to -70.9 +/- 4.0 mV, in PamCar, n = 4). Partial restoration of the amplitude and the shift of the steady-state inactivation curve of INa was attained after washout of PamCar. In contrast, lysoPtdCho at concentrations over 10 microM irreversibly depressed the INa within 0.5-3 min and the reduction of INa was followed by cell contracture or cell death (n = 9). The survival time, defined as a period from the start of lysoPtdCho application to the time of the last successful recording of the INa (before evolution of sudden changes in the holding current), depended on the concentrations of lysoPtdCho. Both PamCar and lysoPtdCho retarded the time course of activation and inactivation of INa.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of beta-adrenergic stimulation on the fast sodium current in the intact rat papillary muscle.

The loose-patch-clamp technique was used on intact cardiac papillary muscle of the rat to examine whether the fast sodium inward current (INa+) is influenced by the beta-adrenergic stimulant isoproterenol (ISO) or by 8-bromo-3',5'-cyclic adenosine monophosphate (8-Br-cAMP), respectively. The amplitude of INa+ evoked by test pulses of 5 ms to a transmembrane potential of 0 mV and its time to peak were analyzed. The availability of INa+ was tested with conditioning pulses of 2.5 s to potentials between -130 mV and -50 mV. The potential of half-maximal availability was slightly shifted to more negative values by 1 microM ISO (2.0 mV, n.s.), as well as by 50 microM 8-Br-cAMP (4.0 mV; p less than 0.05). The peak amplitude of INa+ elicited from strongly negative potentials was increased by ISO (18%, n.s.), while 8-Br-cAMP exerted no directional effect. Depolarizing conditioning pulses (-60 mV) decreased INa+ to 13.3% of the maximal attainable current under control conditions, while ISO decreased INa+ to 9.1% of control (p less than 0.1). Corresponding values under the influence of 8-Br-cAMP were 11.4% and 8.3% (p less than 0.05). Moreover, in the presence of ISO there was a significant shortening of the time to peak of INa+ (0.56 ms to 0.50 ms at -80 mV conditioning potential, p less than 0.05) which could not be detected in the presence of 8-Br-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute effects of repetitive depolarization on sodium current in chick myocytes.

Acute effects of repetitive depolarization on the inward Na+ current (INa) of cultured embryonic chick atrial cells were studied using the whole cell patch-clamp technique. Stimulation rates of 1 Hz or greater produced a progressive decrement of peak INa. With depolarizations to 0 mV of 150-ms duration, applied at 2 Hz from a holding potential of -100 mV, the steady-state decrement was approximately 20%. The magnitude of this effect increased with stimulation frequency and with test potential depolarization and decreased with membrane hyperpolarization. Analysis of INa kinetics revealed that reactivation was sufficiently slow to preclude complete recovery from inactivation with interpulse intervals less than 1,000 ms. Moreover, reactivation accelerated markedly with membrane hyperpolarization, in parallel with the response to repetitive stimulation. The multiexponential time course of recovery of peak INa from repetitive depolarization was similar to that observed after single stimuli; however, there was a shift toward a greater proportion of current recovering with the slower of two time constants. It is concluded that incomplete recovery from inactivation is responsible for the decrement in INa observed with short interpulse intervals.

Characterization of sodium current in developing rat diencephalic neurons in serum-free culture.

1. Dissociated, synchronized (G1 phase of cell cycle), and birth-dated fetal rat diencephalic neurons were grown in a serum-free defined medium. The gigaseal whole-cell voltage-clamp technique was used to measure the inward Na+ currents (INa) from morphologically identified bipolar neurons. The earliest expressed somatic INa has been characterized and compared with that present at a later date. 2. The identity of the INa was established on the basis of its reversal potential and reversible blockade by tetrodotoxin (TTX). Close agreement between the measured reversal potentials (68.5 +/- 1.3 and 38.3 +/- 2.4 mV, mean +/- SE) and calculated Nernst equilibrium potentials (64.6 and 34.7 mV) at two different bath Na+ concentrations (120 and 35 mM, respectively) suggests that the channels are highly selective for Na+. 3. The peak INa density increased from 47.7 +/- 2.9 pA/pF in younger neurons (5-6 days in culture) to 93.9 +/- 6.4 pA/pF in older neurons (12-13 days in culture). The activation voltage and the voltage for peak current were also shifted by 10 mV in the hyperpolarizing direction, from -30 and +10 mV in younger neurons to -40 and 0 mV in older neurons, respectively. However, the reversal potential did not change (69.2 +/- 2.3 and 68.5 +/- 1.3 mV in younger and older neurons, respectively). 4. In older neurons the steady-state inactivation parameters (V1/2, the voltage at which inactivation was 50% of maximum, and kh, the voltage at which there is an e-fold change in inactivation) were significantly altered. V1/2 was shifted from -41.5 +/- 2.3 to -48.8 +/- 1.8 mV, and kh was increased from 6.2 +/- 0.5 to 8.9 +/- 0.4 mV. However, the time course of activation and the rates of inactivation and recovery from inactivation were unchanged. 5. In both groups, the INa decays were best described by a sum of two exponentials. The corresponding time constants were voltage dependent. Also, the amplitudes of the two components were differentially affected by membrane potential and niflumic acid. 6. The extrapolated amplitudes of both the fast and the slow components of INa were larger in older neurons, but the ratio of the amplitudes of the two components did not change with age. The voltage dependencies of the time constants of both components were altered. 7. We conclude that INa in fetal rat diencephalic neurons grown in a defined medium with only essential nutrients undergoes in vitro changes in current density and in some, but not all, kinetic parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual effect of the local anaesthetic penticainide on the Na+ current of guinea-pig ventricular myocytes.

1. The effect of the local anaesthetic penticainide (2-alkyl-(4-dialkylamino)-2-pyridyl-butyramide) on macroscopic and single-channel sodium current (INa) of guinea-pig ventricular myocytes was studied with the patch-clamp technique in the cell-attached and inside-out mode. 2. Penticainide (3-60 microM) affected the INa from the outside as well as from the cytoplasmic side. 3. Peak INa was reduced by penticainide at concentrations of 6, 30 and 60 microM, and this decrease of peak INa was more pronounced when the holding potential was more negative. Despite a reduction of peak INa, the time integral of the Na+ current was not changed (60 microM) or was even enhanced (6 microM), and this enhancement became more pronounced at less negative potentials. 4. At a concentration of 3 microM, penticainide increased both the time integral of the current and peak INa. 5. The shape of the steady-state current-voltage relationship and the steady-state inactivation curve were not influenced by penticainide. 6. In pronase-modified inside-out patches penticainide reduced INa at the beginning of a depolarizing pulse to the same extent as at the end (400 ms), indicating a very fast blockade of the bursting Na+ channel. The most prominent effects on pronase-modified single-channel INa were an increase of sweeps without activity, and a fast, repeatedly occurring block (flickering) of the bursting Na+ channel. 7. The amplitude of the unitary current was not altered. 8. It is concluded that penticainide blocks the open Na+ channel, and in addition shows the macroscopic inactivation.

Na(+)‐activated K+ channels and voltage‐evoked ionic currents in brain stem and parasympathetic neurones of the chick.

1. Patch-clamp and computer-modelling techniques were used to study the activation of Na(+)-activated K+ channels (IK(Na] in dissociated neurones from the embryonic chick ciliary ganglion and the embryonic chick brain stem. 2. Numerical solutions of diffusion equations suggested that Na+ accumulation as a result of Na+ influx through voltage-sensitive Na+ channels (INa) is insufficient to allow for alteration in the gating of IK(Na) channels. 3. Whole-cell recordings using two independent micropipettes were made from chick ciliary-ganglion neurones. These showed that transient outward currents were present only when there were clear indications of incomplete voltage clamp. 4. Single-electrode whole-cell recordings from ciliary-ganglion neurones showed that transient tetrodotoxin (TTX)-sensitive outward currents were present, but only when partial TTX blockade produced significant alterations in the kinetics of INa. In cells that were properly voltage clamped, there was no effect of TTX on the kinetics of INa or on voltage-evoked outward currents. 5. Examination of the relationship between peak INa and the command potential showed that transient outward currents were only present in neurones that showed sharp deviations from the behaviour expected of a cell that is adequately voltage clamped. Transient outward currents were not present in cells that were adequately voltage clamped. 6. Application of TTX to isolated outside-out patches obtained from ciliary ganglion neurones eliminated voltage-evoked inward currents but had no effect on outward currents. 7. Isolated inside-out patches obtained from ciliary-ganglion neurones did not contain IK(Na) channels. These patches usually contained Ca(2+)-activated K+ channels (IK(Ca] with a unitary conductance of around 200 pS when [K+]o = 150 mM and [K+]i = 75 mM. 8. Two-electrode whole-cell recordings from cultured brain stem neurones showed that transient outward currents were present only when there were clear indications of incomplete voltage clamp. 9. Application of TTX caused blockade of inward but not outward currents in brain stem neurones voltage clamped with a single whole-cell pipette. TTX had no effect on the kinetics of INa. Application of TTX to outside-out patches isolated from the same cells blocked only the inward currents. 10. Isolated inside-out patches obtained from brain stem neurones contained IK(Na) channels that could be activated by exposure of the cytoplasmic face of the patch membrane to 75 mM-Na+. These channels had a predominant conductance state of around 100 pS when [K+]o = 150 mM and [K+]i = 75 mM. The IK(Na) channels were not activated by 1 mM-Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)