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

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Abstract
The ion selectivity of the Ca2+ channels in single ventricular cells [from the heart] of guinea-pigs was studied using a giga-ohm seal patch electrode for voltage clamp and interanl dialysis. To isolate the Ca2+ channel current, currents through the Na+ channel and K+ channels were minimized by replacing external Na+ with Tris+ [2-amino-2-hydroxymethyl-1,3-propanediol] and removing K+ from both sides of the membrane. With 5 mM-ATP and 5 mM-EGTA [ethyleneglycol-bis(.beta.-aminocthylether)-N,N''-tetracetic acid] in the pipette solution, the Ca2+ current was well maintained for > 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 electode, 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 > + 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 [tetraethylammonium chloride] 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. The blocker-sensitive outward current was not inactivated by a conditioning pre-pulse to + 15 mV, suggesting that the outward Cs+ current flows mainly not through Ca2+ channnels but through some voltage-dependent leakage conductance. The Ca2+ channel current was not significantly changed when external Na+ was replaced with Tris+ in the presence of 3.1 .mu.M-tetrodotoxin. This finding indicates that Na+ do not contribute significanly to Ca2+ channel current. Neither K+ nor Na+ contribute significant charge to the Ca+ current. Under physiological conditions, the Ca2+ channel is believed to be much more selective for Ca2+ than previously recognized.