The long-term objective of this project is a biophysical and molecular understanding of the processes of transduction in general, and in photoreceptor cells in particular. The details of the cascade of reactions that mediates excitation by linking the absorption of photons by photopigments to changes in conductance of the plasma membrane are not entirely understood. Moreover, the reactions that modulate the phototransduction cascade to produce """"""""adaptation are also incompletely understood.
Our specific aims are to investigate certain steps in the transduction cascade for excitation and adaptation in invertebrate photoreceptors. The candidacy of cGMP as the excitatory messenger that gates the light-induced channels in Limulus ventral photoreceptors will be investigated. (I) A light-induced increase in intracellular cGMP near the plasma membrane of these cells will be assayed by introducing exogenous channels that are gated by cGMP into the plasma membrane. mRNA that is specific for cGMP-gated channels from bovine rods or olfactory receptors will be injected intracellularly and allowed to express the channel proteins. Expressed channels will be identified by recording electrophysiological responses to intracellular injections of cGMP and identified by immunohistochemistry for the expressed proteins. (II) We will attempt to purify cGMP-gated channels from squid photoreceptors by affinity chromatography. To examine if cGMP participates in the transduction cascade in a way other than by gating channels, (III) activated bovine cGMP-specific phosphodiesterase will be injected intracellularly into Limulus photoreceptors to attempt to perturb the electrophysiological responses to light and (IV) the time course of light-induced changes in phosphodiesterase activity will be measured by a continuous fluorescence assay in broken cell preparations of squid photoreceptors.
