CYCLIC-GMP CONTACT POINTS WITHIN THE 63-KDA SUBUNIT AND A 240-KDA ASSOCIATED PROTEIN OF RETINAL ROD CGMP-ACTIVATED CHANNELS

Ion channels from retinal rods and a variety of other cells are directly gated by cyclic nucleotides. The rod channel is known to contain a 63-kDa subunit, and there is molecular genetic evidence for the existence, in human retina, of a second subunit with a deduced molecular mass of about 100 kDa. When purified from bovine rods, the channel consists of the 63-kDa subunit and a 240-kDa associated protein that has been shown recently to contain a version of the cloned second subunit as part of a larger complex. We had previously shown that a photoaffinity analog of cGMP, 8-(p-azidophenacylthio)-[P-32]cGMP, specifically labels both the 63- and 240-kDa proteins. Here the analog was used to identify cGMP-binding regions and amino acid contact points within these proteins. The specific labeling of the 63-kDa subunit was localized to a 66 amino acid fragment (Tyr-515-Met-58O) that is contained entirely within a 110 amino acid region proposed to be the cGMP-binding site on the basis of homology with other cyclic nucleotide-binding proteins. Within this fragment, amino acid residues Val-524, Val-525, and Ala-526 were found to contain label. These residues are part of a larger hydrophobic cluster that appears to line the binding pocket. The results also indicate that the 240-kDa protein contains a similar cGMP-binding site. Sequencing of a specifically labeled 8-kDa fragment through 16 amino acid residues indicated that the fragment was derived from the portion of the 240-kDa complex that contains the second subunit. Alignment of the amino acid sequences of the bovine 63-kDa subunit and the human second subunit based on homology indicates that the specifically labeled peptide from the 240-kDa protein is the corresponding fragment to that labeled from the 63-kDa subunit.