THE CGMP-GATED CHANNEL OF THE ROD PHOTORECEPTOR CELL CHARACTERIZATION AND ORIENTATION OF THE AMINO TERMINUS

The molecular properties and orientation of the cGMP-gated cation channel of bovine rod outer segment membranes were studied using biochemical and immunochemical methods. Western blots labeled with anti-channel monoclonal antibodies indicate that the channel has a subunit M(r) of 63,000 in bovine rod outer segment membranes prepared in the presence and absence of protease inhibitors and in rod outer segments from other mammalian retinas. The channel has an apparent M(r) of 78,000 in both COS-1 cells and Xenopus oocytes expressing the cloned cDNA. NH-2-terminal sequence analysis indicates that the lower M(r) of the channel in rod outer segments is caused by the absence of the first 92 amino acids predicted by cDNA sequence analysis. Immunofluoreseent and immunogold labeling has confirmed that the 63,000 form of the channel is present in rod outer segments. These results indicate that photoreceptor cell-specific co-translational or post-translational cleavage of the NH-2-terminal segment of the channel occurs prior or during the incorporation of the channel into the rod outer segment plasma membrane. Immunogold labeling studies using site-directed antibodies also indicate that the NH-2-terminal segment of the rod outer segment channel is exposed on the cytoplasmic side of the plasma membrane.