The overall goal of this research is to elucidate the molecular basis of visual excitation and adaptation in retinal rod cells. The focus will be on how calcium signals are read and processed in visual recovery and adaptation. The following enzymatic, spectroscopic, and electrophysiological studies of rod outer segment (ROS) proteins will be carried out: (1) The molecular mechanism of action of recoverin as a calcium sensor in vision will be defined by functional reconstitution of calcium-sensitive guanylate cyclase activity. Proteins in addition to recoverin and guanylate cyclase that are essential for conferring calcium- sensitive activation will be purified and characterized. In particular, the potential participation of the alpha subunit of transducin bearing GDP (T-alpha-GDP) will be assessed. (2) Does recoverin also control the activity of the cGMP phosphodiesterase (PDB), as does S-modulin in frog ROS? If so, the site of action of recoverin - whether it acts directly on photoexcited rhodopsin, rhodopsin kinase, transducin, or another component - will be delineated. (3) Retinal recoverin is acylated at its amino- terminus by myristic acid (14:0) or a related acyl group (14:1, 14:2, 12:0). Recombinant recoverins containing each of these acyl groups will be prepared. The effect of the attached acyl group on the calcium-induced insertion of recoverin into membranes will be measured. The geometry of membrane-inserted recoverin will be delineated by fluorescence energy transfer spectroscopy. (4) Proteins that bind to recoverin in a calcium- specific manner will be identified by affinity chromatography on recoverin-agarose, covalent crosslinking, and fluorescence emission anisotropy spectroscopy. (5) Site-specific mutants of recoverin will be produced to ascertain the functional significance of different regions. Of especial interest are the calcium-binding sites of EF hands 2 and 3, the hydrophobic crevice between EF hands 1 and 2, the linker between EF hands 3 and 4, and the highly charged C-terminal tail. (6) Electrophysiological studies of the effects of native and mutant recoverins and of T-alpha-GDP on membrane currents of truncated rods will be carried out to complement biochemical and spectroscopic studies. Studies of the mode of action of recoverin are likely to provide insight into a whole new class of retinal and brain calcium sensors. Interest in recoverin is further heightened by the finding that it is the antigen in cancer-associated retinopathy, an autoimmune retinal degenerative disease caused by a primary tumor in a distant tissue.
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