Molecular Mechanisms of Retinal cGMP-Activated Channels

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DESCRIPTION (provided by applicant): Cyclic nucleotide-gated (CNG) ion channels play a fundamental role in signal transduction in the retina. In photoreceptor outer segments, they signal the fall in intracellular cGMP concentration that results from receptor activation by absorption of light. At synapses between cone and horizontal cells, they regulate synaptic transmission and mediate presynaptic feedback by nitric oxide. The overall goal of our research is to elucidate the molecular mechanisms underlying the activity of CNG channels that are important for visual signaling and retinal disease. CNG channels are composed of four homologous subunits, each containing a single cyclic nucleotide-binding site. Ligand binding to these sites is coupled to conformational changes that lead to opening of the channel pore. Native cone photoreceptor CNG channels contain two different subunit types, CNGA3 and CNGB3; the assembly of these divergent subunits creates heteromeric CNG channels with properties optimized for their role in phototransduction. In this proposal, we will discover the structural elements and mechanisms regulating the assembly and trafficking of cone CNG channel subunits. Cone CNG channels are especially sensitive to calcium-feedback regulation, and this feature is critical to the extraordinary range of light adaptation in cones. Here, we will examine the structural determinants and interactions that are important for calcium-dependent regulation of cone CNG channels. In addition, we will elucidate the molecular pathology of mutations in cone CNG channel genes that have been linked previously to complete and incomplete achromatopsia and to progressive cone dystrophy. For these experiments, channels will be studied using electrophysiological recording of exogenously expressed cDNA clones in Xenopus oocytes, fluorescent microscopy to localize channels fused to green fluorescent protein, and biochemical protein interaction assays. These experiments will enhance our understanding of the molecular basis for the unique properties of cyclic nucleotide-gated ion channels essential to signal transduction in the retina, and of the molecular mechanisms that lead to color blindness and retinal degeneration.