This project is focused on two problems: the reconstitution and functional analysis of sodium channels and the mechanism of visual transduction. Sodium channels from muscle plasma membranes are isolated and purified in a functionally intact state and reconstituted into vesicular and planar lipid bilayers. Reconstituted channels are studied by patch clamp and conventional recording techniques. Gating kinetics, ion permeation mechanism and specificities are investigated. Single channel gating kinetics are correlated with multi-channel data obtained from reconstituted bilayers as well as native membranes. Chemical modification of the channel and its lipid environment combined with pharmacological studies of the effects of neurotoxins serve as further tools for the elucidation of the channel function. The visual transduction studied are designed to test the possible role of protons is the light-induced Ca++ release. The effects of protons directly injected into isolated rod outer segments on dark current and light responses is measured and correlated with measurements of protons generated through light-induced cyclic GMP hydrolyis. Proton-induced Ca++ release is investigated in broken rod outer segments and disks. The ion specificities of the Ca++ release pathway are determined through Ca++ ion exchange experiments. The role of phosphatidylinositol in the visual transduction cycle is investigated. In particular it will be determined if the observed light-induced phosphatidylinositol turnover is linked to Ca++ release via the formation of phosphatidic acid.
| Sitaramayya, A; Casadevall, C; Bennett, N et al. (1988) Contribution of the guanosinetriphosphatase activity of G-protein to termination of light-activated guanosine cyclic 3',5'-phosphate hydrolysis in retinal rod outer segments. Biochemistry 27:4880-7 |
| Tanaka, J C; Furman, R E; Cobbs, W H et al. (1987) Incorporation of a retinal rod cGMP-dependent conductance into planar bilayers. Proc Natl Acad Sci U S A 84:724-8 |
| Sitaramayya, A (1986) Rhodopsin kinase prepared from bovine rod disk membranes quenches light activation of cGMP phosphodiesterase in a reconstituted system. Biochemistry 25:5460-8 |
| Sitaramayya, A; Harkness, J; Parkes, J H et al. (1986) Kinetic studies suggest that light-activated cyclic GMP phosphodiesterase is a complex with G-protein subunits. Biochemistry 25:651-6 |
| Furman, R E; Tanaka, J C; Mueller, P et al. (1986) Voltage-dependent activation in purified reconstituted sodium channels from rabbit T-tubular membranes. Proc Natl Acad Sci U S A 83:488-92 |
