The properties and function of Ca(2+)-activated K+ (KCa) and voltage-dependent K+ (IK) currents of rabbit coronary myocytes were studied under whole cell voltage-clamp conditions (22 degrees C). Inhibition of KCa by tetraethylammonium chloride (1-10 mM) or charybdotoxin (50-100 nM) suppressed noisy outward rectifying current elicited by 5-s voltage steps or ramp at potentials > 0 mV, reduced the hump of the biphasic ramp current-voltage relation, and shifted by less than +5 mV the potential at which no net steady-state current is recorded (Enet; index of resting membrane potential). Inhibition of steady-state inward Ca2+ currents [ICa(L)] by nifedipine (1 microM) displaced Enet by -11 mV. Analysis of steady-state voltage dependence of IK supported the existence of a "window" current between -50 and 0 mV. 4-Aminopyridine (2 mM) blocked a noninactivating component of IK evoked between -30 and -40 mV, abolished the hump current during ramps, and shifted Enet by more than +15 mV; hump current persisted during 2-min ramp depolarizations and peaked near the maximum overlap of the steady-state activation and inactivation curves of IK (about -22 mV). A threefold rise in extracellular Ca2+ concentration (1.8-5.4 mM) enhanced time-dependent outward K+ current (6.7-fold at +40 mV) and shifted Enet by -30 mV. It is concluded that, under steady-state conditions, IK and ICa(L) play a major role in regulating resting membrane potential at a physiological level of intracellular Ca2+ concentration, with a minor contribution from KCa. However, elevation of intracellular Ca2+ concentration enhances KCa and hyperpolarizes the myocyte to limit Ca2+ entry through ICa(L).
Macroscopic currents were recorded in freshly dissociated smooth muscle cells from the rabbit portal vein using the tight seal whole cell recording mode (22 degrees C). In some experiments, the indo 1 fluorescence technique was used to simultaneously monitor the changes in the concentration of free intracellular Ca2+ ([Ca2+]i; indo 1 ratio, 400/500 nm). In cells exposed to tetraethylammonium chloride (TEA) to inhibit K+ channels and 1-10 microM nifedipine or nicardipine to inhibit L-type Ca2+ channels, cell dialysis with 30 mM Na+ increased [Ca2+]i and induced membrane current consistent with the activation of Ca(2+)-activated Cl- channels [ICl(Ca)]. From holding potential (HP) of -60 mV, high intracellular Na+ concentration ([Na+]i)-mediated current was instantaneous in response to 0.5- to 10-s voltage clamp pulses from -80 to +20 mV; steps ranging from +20 to +80 mV evoked slow time-dependent outward current (I(t); superimposed on the instantaneous current) and voltage-dependent Ca2+ transient; on return to HP, slow inward tail current appeared that reflected deactivation of I(t). Both current components 1) exhibited outward rectifying properties, 2) reversed near the predicted equilibrium potential for Cl-, 3) were stimulated by elevation of extracellular Ca2+ concentration, 4) were abolished when the cells were dialyzed with 5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 5) were inhibited by extracellular application of niflumic acid (50 microM). Complete replacement of extracellular Na+ concentration with tetramethylammonium increased both the instantaneous and time-dependent components of ICl(Ca), resting [Ca2+]i at -60 mV and Ca2+ transient at +40 mV. Cell dialysis with Na(+)-free pipette solution prevented these effects. Our results are consistent with an indirect mechanism of stimulation of ICl(Ca), which involves intracellular Ca2+ accumulation via reverse-mode electrogenic Na+/Ca2+ exchange activity.
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