The long-term objectives of this application are to elucidate the mechanisms that control light-dependent ion channels in photoreceptors, and the modulation of the photoresponse by ambient illumination. The proposed experiments take advantage of the unique double retina of organisms in which both evolutionary lines of visual receptors are represented: (1) rhabdomeric, which depolarize to light and utilize the phosphoinositide cascade for transduction, and (ii) ciliary, which hyperpolarize to light and, like vertebrate rods, rely on a cGMP-based cascade. Dissociated cells of both classes are exceptionally suitable for recording light-dependent whole-cell and single-channel currents, intracellular dialysis, and fluorescence measurements of cytosolic calcium. In rhabdomeric photoreceptors a light-regulated PLC is critically involved in excitation, but the IP3/Ca branch of the cascade has proved insufficient to account for phototransduction, and the importance of other elements of the PLC pathway has been gaining recognition. One component that was recently identified as a key signaling molecule in other systems is PIP2 itself. Proposed studies will examine its role in visual transduction. The pivotal role of Ca in light adaptation of rhabdomeric photoreceptors is undisputed, but its mode of action remains poorly understood. Although Ca-regulated molecules such as PKC and calmodulin have been implicated, direct physiological evidence is lacking. We will further our studies linking PKC activation to desensitization, and systematically test the involvement of CaM in light adaptation, especially the acceleration of the response. It is also becoming clear that some additional modulatory pathways operate independently of Ca. Ciliary photoreceptors allow these processes to be studied in isolation, because neither influx nor internal mobilization of Ca are coupled to photoexcitation. We have uncovered two novel Ca-independent regulatory schemes: (i) cGMP, in addition to controlling light-sensitive channels, is also implicated in negative feedback regulation of the cascade. (ii) Photoresponse kinetics during light adaptation is modulated by photopigment state (which, owing to its bi-stability, can be readily manipulated); we will probe the underlying mechanisms and their possible dependency on cAMP. Finally, we will further investigate the gating of the light-dependent conductance of ciliary photoreceptors, testing a recent conjecture that links it to the inactivation process of voltage-dependent K channels, to which cyclic nucleotide gated channels are related.
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