Voltage-gated calcium and Ca2+-activated chloride channels and Ca2+ transients: voltage-clamp studies of perfused and intact cells of Chara

The voltage-clamp technique was used to study Ca²⁺ and Cl^– transient currents in the plasmalemma of tonoplast-free and intact Chara corallina cells. In tonoplast-free cells perfused medium with ethylene glycol bis(2-aminoethyl ether)tetraacetic acid] long-term inward and outward currents through Ca channels consisted of two components: with and without time-dependent inactivation. The voltage dependence of the Ca channel activation ratio was found to be sigmoid-shaped, with about –140-mV activation threshold, reaching a plateau at V>50 mV. As the voltage increased, the characteristic activation time decreased from approximately 10³ ms in the threshold region to approximately 10 ms in the positive region. The positive pulse-activated channels can then be completely deactivated, which is recorded by the Ca²⁺ tail currents, at below-threshold negative voltages with millisecond-range time constants. This tail current is used for fast and brief Ca²⁺ injection into tonoplast-free and intact cells, to activate the chloride channels by Ca²⁺ . When cells are perfused with EDTA-containing medium in the presence of excess Mg²⁺, this method of injection allows the free submembrane Ca²⁺ concentration, Ca²⁺]_c, to be raised rapidly to several tens of micromoles per liter. Then a chloride component is recorded in the inward tail current, with the amplitude proportional to . When Ca²⁺ is thus injected into an intact cell, it induces an inward current in the voltage-clamped plasmalemma, having activation–inactivation kinetics qualitatively resembling that in EDTA-perfused cells, but a considerably higher amplitude and duration (approximately 10 A m^–2 and _inact~0.5 s at –200 mV). Analysis of our data and theoretical considerations indicate that the Ca²⁺]_c rise during cell excitation is caused mainly by Ca²⁺ entry through plasmalemma Ca channels rather than by Ca²⁺ release from intracellular stores.