The outer segment region of the vertebrate photoreceptor is the only light- sensitive element in the entire visual system. The ultimate goal of this research is to understand how it works. The highly specialized purpose of the outer segment is to couple the absorption of light to the reduction of a standing cation current that steadily flows into the outer segment in darkness. Whole-cell voltage clamp is used to measure inward dark current through light-regulated ion channels in the plasma membrane of functionally intact isolated rod outer segments that have been mechanically detached from the rest of the receptor cell. Diffusional exchange between outer segment cytoplasm and whole-cell pipet filling solution is used to study the effects of specific changes in the biochemical composition of the intracellular compartment on the generation, maintenance and light- regulation of recorded dark current. The underlying processes are mediated by changes in two intracellular messengers, cGMP and Ca. The investigation will describe the reciprocal interactions between cGMP and Ca during the generation of the light response in darkness and adapting background light. More direct information about the molecular events that couple light to a change in outer segment dark current will be obtained by combining whole- cell voltage clamp with optical measurements: fluorescence, IR-light scattering, and light absorption. Optical methods will be used to assess the extent of cytoplasmic manipulations produced by internal dialysis and to study light-evoked changes in internal Ca, G protein state and intradiskal membrane potential. Hypotheses about the mechanism of phototransduction and its regulation in dark and light adaptation will be tested further using light-sensitive excised patches of outer segment plasma membrane. The presence of the transduction machinery in inside-out excised patches makes it accessible to bath applied reagents and the effects of specific molecular components will be studied by reconstitution. Basic research on the physiology, biochemistry and cell biology of the rod outer segment provides a more clear understanding of """"""""how it works"""""""" in health and in so doing expands the foundation for understanding """"""""how it does not work"""""""" in disease.
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