The goal of this project is determine whether vertebrate opsin or rhodopsin can function as a cyclic GMP-activated channel Opsin will be purified, reconstituted into phospholipid vesicles, and single channel activity will be recorded from patches excised from the vesicles. The ion specificity and voltage sensitivity of the rhodopsin conductance will be characterize, as will the sensitivity of the rhodopsin channel activity to cyclic GMP and to other nucleotides. The effect of divalent cations upon the conductance properties of opsin and rhodopsin will be investigated. The effect of L-cis-Diltiazem upon channel activity will be studied. Reconstituted opsin will be regenerated with 9-cis-retinal or 11-cis-retinal and the effect of its bleaching state on single channel activity will be studied. Opsin species of different phosphorylation states will be isolated and reconstituted and the effect of extent of phosphorylation on the conductance properties of opsin will be examined. Rhodopsin has long been studied in terms of its receptor properties. Its contribution to photoreceptor physiology has, until now, been thought to be limited to receptor functions. The finding that the phototransducing pigment of the rod may also function as a channel is extremely important and may drastically change the way we view the phototransduction process as well as a host of other physiological and biochemical processes in the photoreceptors. The experiments described in this proposal are designed to determine whether or not this is the case and, if so, determine the significance of this new-found role for opsin/rhodopsin.
