11] Functional study of rhodopsin phosphorylation in vivo

Abstract

Publisher Summary Phosphorylation of rhodopsin is a key reaction in the termination of the light response. It is the first step in rhodopsin inactivation, beginning 100 msec after the light stimulus. A subsequent step, the binding of arrestin to phosphorylated rhodopsin, is necessary to complete inactivation. Extensive work has been done to address basic aspects of rhodopsin Phosphorylation. Its importance in rhodopsin shutoff was first identified by in vitro reconstitution studies. The sites of phosphorylation on rhodopsin were localized at several serine and threonine residues at the cytoplasmic carboxyl terminus of rhodopsin, and the most favored sites in vitro were reported as Ser-338 and Ser-343 by three independent studies. The carboxyl terminus of rhodopsin was isolated from photoreceptor disk membranes and analyzed by chromatographic separation and mass spectrometry. This chapter describes how to generate transgenic mice expressing rhodopsin mutants at the phosphorylation sites and how to characterize the lines in terms of transgene expression and retinal morphology. It also describes the criteria used in the selection of mice for electrophysiological recordings so that light responses elicited by mutant rhodopsins can be unequivocally assigned.