MOLECULAR MECHANISMS OF RETINAL CGMP ACTIVATED CHANNELS

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Cyclic nucleotide-gated ion channels play a fundamental role in signal transduction in the retina in the retina. In photoreceptor outer segments, they signal the fall in intracellular cGMP concentration that results from absorption of light by rhodopsin. 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 CNG channels. 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 CNG channels are thought to contain two different subunit types, alpha and beta; the assembly of these divergent subunits creates heteromeric CNG channels with properties optimized for their role in phototransduction. In this proposal, we will ascertain the structural determinants responsible for the assembly of these channels, the precise arrangement of their subunits and the molecular features that modulate their cyclic nucleotide specificity. In addition, we will examine the molecular mechanisms underlying mutations in CNG channel genes that have been linked to rod monochromasy and retinitis pigmentosa. The channels will be studied using electrophysiological recording of exogenously expressed cDNA clones in Xenopus oocytes and in a mammalian cell line, fluorescent microscopy of transfected cells to localize channels fused to green fluorescent protein, and biochemical and genetic protein interaction assays. These experiments will channels essential to signal transduction in the retina, and of the molecular mechanisms that lead to retinal degeneration and color-blindness.