Isolation of calcium current and its sensitivity to monovalent cations in dialysed ventricular cells of guinea-pig

Abstract

The ion selectivity of the Ca2+ channels in single ventricular cells of guinea-pig was studied using a 'giga-ohm seal' patch electrode for voltage clamp and internal dialysis. To isolate the Ca2+ channel current, currents through the Na+ channel and K+ channels were minimized by replacing external Na+ with Tris+ and removing K+ from both sides of the membrane. With 5 mM-ATP and 5 mM-EGTA in the pipette solution, the Ca2+ current was well maintained for more than 30 min in K+- and/or Na+-free external solution. Substitution of Cs+ for intracellular K+ eliminated the region of negative slope conductance in the steady-state current-voltage curve and shifted the zero-current potential or resting potential from -80 to -31 mV. After Cs+ substitution, a large inward current still flowed via inwardly rectifying K+ channels, but was abolished by removing external K+, which resulted in reduction of the resting membrane slope conductance to 1% of the control value. A decaying outward current attributable to the inwardly rectifying K+ channel was observed on depolarization in 5.4 mM-external K+ solution with Cs+-rich internal solution after blocking Ca2+ current. The induction of that current caused an apparent decrease of Ca2+ channel current when the K+-rich internal solution was switched to the Cs+-rich one at an external K+ concentration of 5.4 mM. When inwardly rectifying K+ current was suppressed by exposure to K+-free external solution, replacement of intracellular K+ with Cs+ caused no significant change in the Ca2+ current. With Cs+-rich solution in the electrode, the decaying outward current was responsible for an apparent depression of the Ca2+ current observed when extracellular K+ was increased. When the K+ current was abolished by 0.2 mM-extracellular Ba2+, changes in external K+ concentration did not affect the Ca2+ current, excluding the possibility of a direct inhibitory action of K+ on the Ca2+ channel. A time- and voltage-dependent outward current attributed to Cs+ was observed at potentials above +30 mV in Na+-, K+-free external solution with Cs+-rich internal solution. This current persisted in the presence of 20 mM-intracellular TEA Cl and 5 mM-extracellular 4-aminopyridine. Inorganic Ca2+ channel blockers, such as Co2+ or Cd2+, not only suppressed the inward Ca2+ current but also caused some reduction in outward current. Thus the blocker-sensitive peak current reversed at around +75 mV.(ABSTRACT TRUNCATED AT 400 WORDS)