DESCRIPTION (provided by applicant): The long-term scientific goal of the research project is to understand the molecular mechanism underlying Drosophila phototransduction. Drosophila photoreceptor cells are photosensitive neurons with a phototransduction cascade similar to that of the Retinal Ganglion Cells (RGC), which contain the photopigment melanopsin, thus being a "Rosetta Stone" for RGC and other photosensitive neurons. Both Drosophila and presumably the light sensitive RGC are characterized by bi-stable rhodopsin that initiates phototranduction with Transient Receptor Potential (TRP) channels as the final target. The specific aims are: i) to elucidate the mechanisms that make the photoreceptor rhabdomere a signaling compartment as a strategy to optimize function. The unknown functional properties of the Ca2+ binding protein, calphotin, will be studied. To this end calphotin levels will be modulated in vivo by overexpression or by RNAi and the physiological consequences will be studied. Moreover, we will examine the putative role of calphotin as a dynamic barrier controlling the free diffusion of Ca2+ between the rhabdomere and the cell body. ii) To study the unknown mechanism underlying light dependent translocation of the TRP-like (TRPL) channel. We will use TRP-TRPL chimeras expressed in the living fly eye to study the effects of various domains of the immobile TRP on translocation of TRPL. Since TRP but not TRPL binds to the INAD scaffold protein, the chimeras will allow elucidating new roles for INAD in phototransduction and whether or not TRP and TRPL form heteromultimeres. iii) To elucidate the unknown mechanism of TRP and TRPL activation by light. Potentially, a phospholipase C (PLC) - mediated phototransduction can activate the channels by at least three putative mechanisms: by second messengers, by removal of inhibition and by change in membrane curvature. More specifically: (a) Activation by the second messengers Inositol 1,4,5 trisphosphate (InsP3), Diacylglycerol (DAG) and subsequently Polyunsaturated Fatty Acids (PUFA) due to hydrolysis of DAG. (b) Activation by a decrease in the concentration of PIP2, thereby removing its inhibitory effect on the channels. (c) Activation by conversion of PIP2 to DAG or PUFA causing membrane lipid packing modifications that changes the curvature of the plasma membrane and thereby activate the channels in a manner similar to activation of mechanosensitive channels. We will examine these possibilities in expression system of Drosophila cell-line which express TRPL and in the native photoreceptor cells by inducing PIP2 hydrolysis without activation of PLC under conditions that change, or do not change membrane curvature. The activation mechanism of the putative mechanosensitive and Ca2+ permeable TRP channel will be used to acquire insights on the involvement of pathological activation of mammalian TRP channels in retinal degeneration and glaucoma. PUBLIC HEALTH RELEVANCE: A powerful approach to identify potentially important proteins for mammalian retinal cells is to look for homologue proteins that have been identified in Drosophila by genetic screens, leading to isolation of mutants defective in retinal processes. Retinal degeneration or glaucoma, which leads by unknown ways to photoreceptor degeneration and apoptosis of retinal ganglion cells (RGC) respectively, may Involve Ca2+ permeable TRP channels activated by mechanical stretch and hypoxia like the Drosophila channels. Therefore, studies on Drosophila TRP and TRPL channels are likely to be relevant for understanding retinal degeneration and mechanisms underlying the damage to RGC by glaucoma.