Inhibition of rhodopsin kinase by recoverin. Further evidence for a negative feedback system in phototransduction

Recoverin is a 23-kDa Ca²⁺-binding protein found predominantly in vertebrate photoreceptor cells. Recent electrophysiological and biochemical studies suggest that recoverin may regulate the photoresponse by inhibiting rhodopsin phosphorylation. We find in both cell homogenates and reconstituted systems that the inhibition of rhodopsin phosphorylation by recoverin occurs over a significantly higher free Ca²⁺ range than previously reported. Half- maximal inhibition occurs at 1.5-3 μM free Ca²⁺ and is cooperative with a Hill coefficient of ~2. Measurements of transducin activation demonstrate that this inhibition prolongs the lifetime of catalytically active rhodopsin. Ca²⁺-recoverin directly inhibits rhodopsin kinase activity, and Ca²⁺- dependent binding of recoverin to rod outer segment membranes is not required for its action. Extrapolation of the in vitro data to in vivo conditions based on simple mass action calculations places the Ca²⁺-recoverin regulation within the physiological free Ca²⁺ range in intact rod outer segment. The data are consistent with a model in which the fall in free Ca²⁺ that accompanies red excitation exerts negative feedback by relieving inhibition of rhodopsin phosphorylation.