The objective of this research proposal is to investigate the underlying mechanism(s) that mediate and control stimulus-induced secretion. In a sensory photoreceptor cell the stimulus is a photon absorbed by a membrane-bound photopigment and the secretion is exocytosis of neurotransmitter molecules contained in vesicles. The ventral photoreceptors of Limulus polyphemus offers several experimental advantages that include the stimulus is repeatable and easily controlled in amplitude and timing, the secretion is controlled by a change in voltage across the membrane that can be easily measured, the cells are large enough to permit microinjection of water-soluble substances into the cytosol of intact cells, and biochemistry can be done on small clusters of photoreceptor cells. The goal of the proposal is to establish the nature of guanine nucleotide-binding regulatory proteins (N-proteins) and of phosphoproteins in Limulus photoreceptors and to elucidate the role of N-proteins and phosphorylation in visual excitation. Bacterial toxins that catalyze the ADP-ribosylation of mammalian and invertebrate N-proteins will be used to probe N-protein function via both microinjection in cells and whole-animal intoxication and will be used to radiolabel N-proteins. Radiolabeled N-proteins of Limulus will be compared structurally and immunologically to those of mammalian sources that have been well-defined with respect to function. Light-induced changes in phosphorylation of cells metabolically labeled with [32P]Pi will be further studied in order to define the kinetics of the process and its role in visual excitation. Purified protein kinases, phosphatases and phosphatase inhibitors will be used to probe the role of phosphorylation in visual excitation of intact cells (via microinjection) and the nature of Limulus phosphoproteins identified in parallel biochemical experiments. The status of N-proteins and phosphoproteins will be evaluated in Limulus under conditions of dark-adaptation, light-stimulation, light-adaptation and dark-readaptation. The overall strategy is to perform electrophysiological and biochemical measurements of N-proteins and phosphoproteins of Limulus ventral photoreceptors, in tandem, in order to delineate their roles in visual excitation.
| Brown, J E; Watkins, D C; Malbon, C C (1987) Light-induced changes in the content of inositol phosphates in squid (Loligo pealei) retina. Biochem J 247:293-7 |
