RESTING MEMBRANE-POTENTIAL AND POTASSIUM CURRENTS IN CULTURED PARASYMPATHETIC NEURONS FROM RAT INTRACARDIAC GANGLIA

1. Whole-cell K+ currents contributing to the resting membrane potential and repolarization of the action potential were studied in voltage-clamped parasympathetic neurones dissociated from neonatal rat intracardiac ganglia and maintained in tissue culture. 2. Rat intracardiac neurones had a mean resting membrane potential of -52 mV and mean input resistance of 850 MOMEGA. The current-voltage relationship recorded during slow voltage ramps indicated the presence of both leakage and voltage-dependent currents. The contribution of Na+, K+ and Cl- to the resting membrane potential was examined and relative ionic permeabilities P(Na)/P(K) = 0.12 and P(Cl)/P(K) < 0.001 were calculated using the Goldman-Hodgkin-Katz voltage equation. Bath application of the potassium channel blockers, tetraethylammonium ions (TEA; 1 mM) or Ba2+ (1 mM) depolarized the neurone by approximately 10 mV. Inhibition of the Na+-K+ pump by exposure to K+-free medium or by the addition of 0.1 mM ouabain to the bath solution depolarized the neurone by 3-5 mV. 3. In most neurones, depolarizing current pulses (0.5-1 s duration) elicited a single action potential of 85-100 mV, followed by an after-hyperpolarization of 200-500 ms. In 10-15% of the neurones, sustained current injection produced repetitive firing at maximal frequency of 5-8 Hz. 4. Tetrodotoxin (TTX; 300 nM) reduced, but failed to abolish, the action potential. The magnitude and duration of the TTX-insensitive action potential increased with the extracellular Ca2+ concentration, and was inhibited by bath application of 0.1 mM Cd2+. The repolarization rate of the TTX-insensitive action potential was reduced, and after-hyperpolarization was replaced by after-depolarization upon substitution of internal K+ by Cs+. The after-hyperpolarization of the action potential was reduced by bath application of Cd2+ (0.1 mM) and abolished by the addition of Cd2+ and TEA (10 mM). 5. Depolarization-activated outward K+ currents were isolated by adding 300 nM TTX and 0.1 mM Cd2+ to the external solution. The outward currents evoked by step depolarizations increased to a steady-state plateau which was maintained for > 5 s. The instantaneous current-voltage relationship, examined under varying external K+ concentrations, was linear, and the reversal (zero current) potential shifted in accordance with that predicted by the Nernst equation for a K+-selective electrode. The shift in reversal potential of the tail currents as a function of the extracellular K+ concentration gave a relative permeability, P(Na)/P(K) = 0.02 for the delayed outward K+ channel(s). 6. The outward K+ current was reduced by 35% when extracellular Ca2+ was replaced by Mg2+, or when Cd2+ (0.1 mM) was added to the external solution. These data suggest that the outward K+ current in rat parasympathetic cardiac neurones is comprised of at least two components: a voltage-dependent delayed rectifying K+ current (I(Kv)) and a Ca2+-activated K+ current (I(KCa)). The macroscopic K+ current showed no rapidly activating and inactivating component. 7. The activation kinetics of the outward K+ current were voltage dependent, with the rate of activation increasing at more depolarized potentials. The half-time to peak K+ current amplitude was reduced from 4.7 ms at 0 mV to 3.2 ms at +60 mV. The slow decline of the outward current amplitude during prolonged depolarization followed an exponential time course with a time constant of decay of approximately 10 s at +60 mV. This slow 'inactivation' of K+ currents was accelerated by increasing depolarization. 8. The conductance-voltage relationship of the Ca2+-insensitive K+ current, I(Kv), was fitted by a Boltzmann equation with half-maximal activation (V(h)) at +2.6 mV and a slope factor (k) of 18.6 mV. The steady-state inactivation curve for the I(Kv) was fitted by a Boltzmann equation with V(h) of -22.0 mV and k of 9.4 mV. 9. The delayed outward K+ current was abolished upon replacement of internal K+ with either Cs+ or arginine. Bath application of TEA blocked the total and Ca2+-insensitive K+ currents in a reversible and dose-dependent manner with K(i) values (concentrations producing 50% inhibition of maximum response) of 2.8 and 2.3 mM, respectively. 10. Bath application of charybdotoxin (CTX) inhibited the total outward K+ current by approximately 35% and the remaining current was not further reduced by the addition of 0.1 mM Cd2+ to the bath solution. CTX (100 nM) selectively inhibited I(KCa) in these neurones. 11. A maximally effective concentration of 4-aminopyridine (1 mM) inhibited the total outward K+ current by 30% and produced a 4-5-fold increase in the half-time of activation. Bath application of 2 mM Ba2+ reversibly abolished I(KCa). and inhibited I(Kv) by approximately 70%. 12. We conclude that I(Kv) and I(KCa) contribute to the repolarization of the action potential and the after-hyperpolarization observed in rat intracardiac neurones and hence influence vagally mediated neurotransmission in mammalian cardiac ganglia.