Phosphorylation of Photolyzed Rhodopsin is Calcium-Insensitive in Retina Permeabilized by α -toxin

Light triggers the phototransduction cascade by activating the visual pigment rhodopsin (Rho → Rho*). Phosphorylation of Rho*by rhodopsin kinase (RK) is necessary for the fast recovery of sensitivity after intense illumination. Ca2+ions, acting through Ca2+-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells. In this report, we describe an in vitro assay system using isolated retinas that is well suited for a variety of biochemical assays, including assessing Ca2+effects on Rho*phosphorylation. Pieces of bovine retina with intact rod outer segments were treated with pore-forming staphylococcal α -toxin, including an α -toxin mutant that forms pores whose permeability is modulated by Zn2+. The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules