It is proposed to use phototransductiondefective mutants of Drosophila and to identify the molecules which are involved in fly phototransduction and to study their modes of interactions. Electrophysiological, biochemical and genetical tools will be applied to study phototransduction at four levels: (a) the photopigment, (b) the quanine nucleotide binding protein (G- protein), (c) the phosphoinositide cycle, and (d) the light-activated membrane channels. It is aimed to develop the photoreceptors of the fly as a model system for studies of the phosphoinositide cycle. The phosphoinositides transduction system is of great interest since a large number of hormones, neurotransmitters, growth factors and perhaps also oncogenes mediate their effect through modulation of this process. Furthermore, the involvement, in this cycle, of gene products which cause lightinduced retinal degeneration in mutants Drosophila (the rdgB mutant) should give us clues as to the mechanism of retinal degeneration diseases and may indicate possible therapeutic measures. (1) The role of the photopigment in excitation and adaptation will be studied by analyzing the molecular mechanism underlying the prolonged depolarizing afterpotential (PDA). Site-directed mutants of Drosophila lacking phosphorylation sites in the rhodopsin will be used to test the hypothesis that the PDA is due to inhibition of phosphorylation turnoff of the photopigment. (2) The transient receptor potential (trp) mutant together with wild type Drosophila and Musca flies will be used to study the light-dependent cycle of the G-protein by measuring the effects of pharmacological agents in intact cells, by resolution and reconstitution of light-dependent GTPase, by measuring lightactivated binding of GTP(gamma)S, and by analysis of protein phosphorylation. (3) The no receptor potential A (norp A) and the retinal degeneration B (rdgB) mutants of Drosophila and white eyed Musca will be used to study the lightdependent phosphoinositide cycle. The cycle will be investigated electrophsiologically, by measuring chemically- induced excitation in intact cells and degeneration induced in mutant photoreceptors and biochemically, by measuring lightactivated phospholipase C in membranes and phosphorylation of polyphosphoinositides. (4) Whole-cell patch clamp will be used to measure light or chemically induced currents. Singel channel patch clamp will be applied to search for the identity of the second messenger(s) molecule(s) that open(s), or modulate(s) the opening, of the light sensitive channels in the fly.
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