The mechanisms by which sulfhydryl (SH-) binding reagents modulate the light-sensitive conductance of retinal rods were investigated by current recording from single rods, by patch clamp recording from the plasma membrane of the rod outer segment (ROS), and by biochemical study of their effects on the light-induced hydrolysis of cyclic GMP. The electrophysiology, as well as measurements of the reagents' ability to traverse the ROS plasma membrane, was done on amphibian (Rona and Ambystoma) rods, and the biochemistry on bovine rods. The main SH-reagents used were N-ethyl-maleimide (NEM) and iodoacetamide (IAA). Both transiently increased rod current, but part of the large current could not be turned off by light. After a few minutes' exposure, NEM, but not IAA, caused a continuous decay of the rod's light sensitivity. In patch-clamp recordings from the ROS plasma membrane, the reagents increased conductivity both in the presence and absence of cGMP, consistent with the observation that the drug-induced current increase in intact rods involved both light-sensitive and light-insensitive components. In vitro, NEM was found to be a powerful inhibitor of cGMP hydrolysis, which can explain the gradual loss of light sensitivity in the rod and could initially contribute to the increased dark current via elevated cGMP levels. Thus, SH-reagents act both by modifying the light-sensitive channel and by inhibiting phototransduction inside the rod.
- cyclic GMP
- patch clamp