COVALENT ACTIVATION OF RETINAL ROD CGMP-GATED CHANNELS REVEALS A FUNCTIONAL-HETEROGENEITY IN THE LIGAND-BINDING SITES

Ion channels gated by the binding of multiple ligands play a critical role in synaptic transmission and sensory transduction. It has been di fficult to resolve the contribution of individual binding events to ch annel gating because ligands are continuously binding and unbinding at each site. In examining the allosteric mechanism of retinal rod cGMP- gated channels, we have circumvented this problem by making use of a c GMP derivative, 8-p-azidophenacylthio-cGMP (APT-cGMP), that can be cov alently tethered to the binding sites in the presence of long-waveleng th UV light. In excised membrane patches, a population of channels was isolated that contained covalently-attached ligands at all but one si te. Activation of these channels by cGMP revealed a previously unknown heterogeneity in the ligand-binding sites. The dose-response relation s were much shallower than predicted by single-site activation models, but were well described by models in which there are two populations of sites, in roughly equal proportion, that bind cGMP with apparent af finities that differ by a factor of similar to 25. The two apparent af finities, incorporated into a four-site model of the channel, provided an accurate description of the patch's original dose-response relatio n. A comparison of results on native and expressed channels suggests t hat the heterogeneity in the native channel arises at least in part fr om the presence of two different cGMP-binding subunits.