Calcium channel activation and inactivation inParamecium biochemically measured by cyclic GMP production

Abstract

The Ca-inward current ofParamecium is related to cGMP production by a Ca-dependent guanylate cyclase. Excitation with Ba2+ increases cGMP levels about ninefold to 45 pmol/ mg within 15 sec. Inhibition of cGMP hydrolysis reveals a large rate of synthesis of up to 25 pmol cGMP/mg·sec−1, or about 1.2 ·108 molecules/cell·sec−1. Because no other factors than the Ca-inward current were found to affect cGMP formation inParamecium, we used it as a quantitative measure of Ca2+ channel activity. After a transient stimulation of cGMP formation by 1mm Ba2+, an additional increase of Ba2+ to 5mm did not result in a renewed elevation of cGMP levels. The extent of desensitization towards a second stimulus was graded with the strength of the first stimulus. Termination of the first stimulus after various time intervals and restimulation after 3 min with 1mm Ba2+ revealed a time-dependent inactivation of the Ca2+ channel, which could be fitted by a single exponential. The inactivated form of the channel was stable for a few minutes at room temperature. The partial desensitization ofParamecium reduced the maximal response, but did not shift the dose-response curve for Ba2+. Veratridine, which activates the Ca2+ channel, was also used as a first stimulus. It effectively and transiently inactivated the channel resulting in a complete loss of both a behavioral response ofParamecium and cGMP elevation towards a second stimulus. The time course of reactivation of channel excitability was studied at different temperatures. Half times of recovery were 51 and 7.5 min at 12 and 25°C, respectively. Reactivation curves can be described by a single exponential, indicating a first order reaction. The activation energy was 100 kJ/mol.