The objective of the proposal is to investigate the regulatory mechanisms that control light-sensitive channels in the plasma membrane of photoreceptors, and their dependence on changes in intracellular calcium. The proposed experiments will use dissociated cells from the double retina of two organisms that possess both rhabdomeric and ciliary photoreceptors. These two classes share salient features with the depolarizing photoreceptors commonly found in invertebrates (which utilize the IP3/Ca cascade for transduction), and with vertebrate rods and olfactory neurons (which rely on cyclic nucleotides), respectively. These cells lend themselves to voltage-clamp recording with intracellular dialysis and measurements of light-dependent single-channels. In rhabdomeric photoreceptors and several other sensory cells, calcium is intimately involved in the modulation of sensitivity (gain) but its mode of action is poorly understood. Ca-regulated protein kinases such as PKC have been postulated as a mediator, but direct evidence is lacking. As for possible sites of action, the gating process of light-dependent channels may be a target, but single-channel measurements are not available. Using intracellular perfusion, the effect of several activators and inhibitors of kinases and phosphatases will be tested on light adaptation. In addition, the changes in single-channel kinetics will be examined as a function of the adaptation state. The contribution of the DAG branch of the PLC cascade to the visual excitation process will also be assessed. Certain components of sensory adaptation are known to proceed independently of calcium, but these are difficult to tease out, owing to the widespread influence of this ion on the transduction process as well. Ciliary photoreceptors are unusually favorable to isolate these processes, because light.responsiveness and its modulation are preserved under conditions in which influx and internal mobilization of Ca do not occur. A particularly promising candidate that will be evaluated is a feedback mechanism that may involve cGMP. Owing to the photopigment bi-stability in these cells, such procedures can be carried out while independently manipulating rhodopsin state, using pigment photoconversion with monochromatic flashes. An aspect of Ca signalling whose importance has recently been recognized is the activation of a Ca influx mechanism which plays a central role in sustained responsiveness and in the homeostasis on internal Ca stores. In rhabdomeric cells a current that underlies such sustained Ca influx can be recorded. Experiments will be conducted to characterize its properties and physiological role in the maintenance and regulation of the visual response; emphasis will be placed on the possible role of cyclic nucleotides, which have been recently demonstrated to modulate Ca influx in other cells.
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