Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by alpha-toxin

Light triggers the phototransduction cascade by activating the visual pigme nt rhodopsin (Rho --> Rho*). Phosphorylation of Rho* by rhodopsin kinase (R K) is necessary for the fast recovery of sensitivity after intense illumina tion. Ca2+ ions, acting through Ca2+-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RR activity contributing to adaptation to bac kground illumination in retinal photoreceptor cells. In this report, we des cribe an in vitro assay system using isolated retinas that is well suited f or a variety of biochemical assays, including assessing Ca2+ effects on Rho * phosphorylation. Pieces of bovine retina with intact rod outer segments w ere treated with pore-forming staphylococcal alpha-toxin, including an alph a-toxin mutant that forms pores whose permeability is modulated by Zn2+. Th e pores formed through the plasma membranes of rod cells permit the diffusi on of small molecules <2 kDa but prevent the loss of proteins, including re coverin (25 kDa). The selective permeability of these pores was confirmed b y using the small intracellular tracer N-(2-aminoethyl) biotinamide hydroch loride. Application of [gamma(-32)P]ATP to alpha-toxin-treated, isolated re tina allowed us to monitor and quantify phosphorylation of Rho*. Under vari ous experimental conditions, including low and high [Ca2+](free), the same level of Rho* phosphorylation was measured. No differences were observed be tween low and high [Ca2+](free), conditions, even when rods were loaded wit h ATP and the pores were closed by Zn2+. These results suggest that under p hysiological conditions, Rho* phosphorylation is insensitive to regulation by Ca2+ and Ca2+-binding proteins, including recoverin.