EFFECTS OF CYSTEINE MODIFICATION ON THE ACTIVITY OF THE CGMP-GATED CHANNEL FROM RETINAL RODS

The effect of sulfhydryl reagents on the activity of the cGMP-gated ch annel from bovine retinal rods was studied by measurements of 8-Br-cGM P-(cGMP)-induced calcium efflux from rod membrane vesicles and records of 8-Br-cGMP-dependent sodium currents through channels incorporated into planar lipid bilayers. N-ethylmaleimide and mersalyl (thiol block ers) as well as diamide (dithiol-disulfide conversion agent) have a du al effect on the channels activity: at low concentration, they increas e the apparent affinity for cyclic nucleotide (''activation'') at the same time inducing a loss of cooperativity for nucleotide binding; at higher concentration, N-ethylmaleimide and diamide produce a reduction of the amplitude and initial rate of the calcium release at saturatin g nucleotide concentration, while mersalyl is shown to reduce the acti vity of the channels in bilayer experiments (''inhibition''). Nitric o xide precursors have no effect. The results suggest that blocking at l east 1 of the 3 cytoplasmic cysteine residues situated close to the cC MP-binding site in each channel subunit by N-ethylmaleimide, mersalyl, or diamide (forming a dimer between 2 subunits) increases the affinit y for the nucleotide. Inhibition is produced by blocking at least one of the 2 other cytoplasmic sulfhydryl groups (N-ethylmaleimide, mersal yl, oxidized glutathione) or the 2 others (diamide, intrasubunit bridg e), and may concern a process of channel inactivation. The 3 cytoplasm ic sulfhydryl groups are accessible when the channels are in the open state, but not (or much less) accessible when the channels are in the closed state.