Isolation and characterization of the alpha-subunit of the rat rod photoreceptor cGMP-gated cation channel

Abstract

A combination of genomic and PCR clones has been used to derive the full-length coding sequence of the alpha-subunit of the rat rod photoreceptor cGMP-gated cation channel. The sequence encodes a protein of 683 amino acids with a predicted molecular weight of 79,221. The sequence shows extensive homology with other rod cGMP-gated channels and also with the rat olfactory cyclic nucleotide-gated cation channel. When the full-length sequence of the rat rod channel was expressed in Xenopus oocytes it gave a conductance that responded to cGMP with an EC(50) of 42 mu M. No response to 2 mM cAMP was detected. The conductance was decreased in the presence of increasing concentrations of calcium. Both monoclonal and polyclonal antibodies were generated against a C-terminal peptide of the rat rod channel. On immunoblots of adult rat retinal membranes the antibodies recognized a band of 71 kDa, suggesting that the rat channel may undergo proteolytic cleavage in the retina, as has previously been found for the bovine rod channel. Immunocytochemical labeling of adult rat retinal sections detected prominent labeling over the rod photoreceptor outer segments with both monoclonal and polyclonal antibodies.