We propose to study visual transduction as """"""""a nerve signal in a test tube"""""""" by utilizing purified suspensions of frog outer segments still attached to their mitochondria rich inner segments (OS-IS). Our development of such a peparation permits us to simultaneously assay and compare ion fluxes and biochemical changes accompanying excitation and adaptation in a chemically defined preparation, experiments of a sort that are not yet possible in other nerve membrane preparations. We plan to: 1. Characterize the processes of light adaptation, recovery from large rhodopsin bleaches in the dark, and quantum bump membrane noise, determining whether they correlate with simultaneous light-induced changes in the concentration or binding state of nucleotides or other small molecules, or with covalent protein modifications such as phosphorylation. 2. Begin a serarch for new putative intracellular messengers by systematically surveying light-induced changes in the concentration or binding of small organic molecules and proteins. 3. Develop an in vitro assay for the light-sensitive conductance that can be used to define the necessary components of the transduction mechanism and also to screen putative conductance regulators. The experimental approaches being used in this work and the mechanisms being elucidated are relevant to understanding events intervening between input and output in many other cellular systems. Such understanding is especially relevant to defining the perturbations underlying diseased states of the retina, and permits a more rational approach to appropriate thereapy.
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