Effect of sulfhydryl reagents on the regulatory system of the L-type Ca channel in frog ventricular myocytes.

Abstract

The effects of sulfhydryl (SH) reagents on the L-type Ca current (ICa) were studied in frog ventricular myocytes using the whole-cell patch-clamp method. Methanethiosulfonate ethylammonium (MTSEA+) was found to enter the cell through the membrane and cause a remarkable increase in Ica from the intracellular side. Methanethiosulfonate ethyltrimethylammonium (MTSET+) and methanethiosulfonate ethylsulfonate (MTSES-) could not penetrate the membrane and were effective only when directly applied to the intracellular side. In addition, suppressive effects on ICa of these MTS reagents were indicated by the following observation. A progressive decay in the peak amplitude of ICa after establishing maximal ICa, stimulated by intracellular MTSET+, was prevented by adding extracellular dithiothreitol (DTT). The SH-oxidizing agents N-ethylmaleimide (NEM), chloramine-T (CL-T), 2,2'-dithiodipyridine (DTDP) and 2,2'-dithio-bis-5-nitropyridine (DTBNP) also exerted a stimulatory effect on Ica. The effect of SH reagents persisted even when cAMP production was inhibited with Rp-cAMP-S, or when G-protein was inhibited with 1 mM GDPbetaS, indicating that the effect is not due to cAMP production or G-protein stimulation. It is concluded that there are sites on the Ca channels that are subject to direct modification by SH reagents.