The long-term objectives of the research are to understand the cascades of reactions that mediate and modulate excitation in photoreceptors. Light is absorbed by rhodopsin, and the excitatory cascade ultimately induces a change in current that flows across the plasma membrane. The proposed research will examine several putative steps in that cascade. The action of a phospholipase on phosphatidylinositides has been proposed to be one step in the cascade. The research involves correlated electrophysiological and biochemical studies of: (1) the light-modulated metabolism of phosphoinositides and the water soluble hydrolysis products, the inositol polyphosphates; (2) the identification of the active inositol polyphosphate isomers; (3) the participation of calcium ions in the phosphoinositide metabolism; (4) the ability of exogenous compounds, such as phospholipase C and pp60src (the src gene product) to mimic or modulate excitation, and (5) the participation of GTP-binding proteins in the excitatory cascade. The biochemical studies involve the use of: HPLC to separate and identify the water soluble inositol polyphosphates, including the isomers;m TLC (and possible HPLC) to separate and identify phosphoinositides; and liquid scintillation counting to quantify the various fractions. Assays will be performed on single Limulus ventral eyes, and possibly on single photoreceptor cells. Intracellular recording of membrane voltage and voltage-clamp current will be used to monitor excitation electrophysiologically. The intracellular calcium ion concentration will be monitored by the aequorin luminesence technique.
