Cyclic nucleotide-gated (CNG) ion channels are opened by the direct binding of cyclic guanosine monophosphate (cGMP). The closure of CNG channels, following the hydrolysis of cGMP, produces the first electrical response to light in rods and cones. In addition, mutations in human rod CNG channels are associated with some forms of retinitis pigmentosa. The apparent affinity of CNG channels for cGMP is modulated by intracellular factors, including phosphatases, Ca2+ binding proteins, transition metal cations and diacylglycerol (DAG). The concentration and/or effectiveness of some of these modulatory factors may depend on the amount of light absorbed by the photoreceptor. Functional modulation of the CNG channels is significant in two ways: 1) it may be involved in some aspect of normal visual transduction, such as in light or dark adaptation; and 2) its pharmacologic or genetic manipulation may be useful in treating some forms of retinal degeneration in which either channel function or regulation of the cGMP concentration is abnormal. Preliminary evidence suggests that these channels are modulated by retinoids in a manner that could contribute to bleaching adaptation. The three specific aims for this project are to: 1) provide a thorough, quantitative description of rod CNG channel modulation by retinoids; 2) investigate the mechanism of action of the retinoids on CNG channels; and 3) initiate tests for functional significance of CNG channel modulation by retinoids. All three specific aims will involve patch clamp studies on CNG channels expressed in Xenopus oocytes, but specific aim 3 will also involve suction electrode and patch clamp recording on intact rods.
For specific aim 1, properties to be examined include: retinoid dose-response relations at high and low cGMP; all-trans-retinal effects on cyclic GMP dose-response relations, voltage dependence, gating kinetics, Na/Ca selectivity, and single-channel amplitude and kinetics.
Specific aim 2 will examine whether all-trans-retinal stabilizes the closed state(s) of the channel, and begin testing for putative interaction sites.
Specific aim 3 will examine retinoid modulation of cone channels, alpha/beta heteromultimeric rod channels and native rod channels, as well as testing whether buffering retinoids (with transduction blocked) increases the dark current in intact rods.
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