On the Influence of Calcium and Recoverin on Rhodopsin Phosphorylation in Mice

Abstract

Purpose:: To seek experimental evidence that Ca²⁺ and recoverin influence rhodopsin phosphorylation under physiological conditions in mouse retinas. Previously, we showed that elevated free Ca²⁺ inhibits visual pigment phosphorylation in zebrafish cones¹. However, we were not able to evaluate in those experiments whether that effect of Ca²⁺ on cone visual pigment phosphorylation was mediated by recoverin. In this study we compared rhodopsin phosphorylation in normal and phototransducin-deficient mice. We also evaluated whether any effects of Ca²⁺ on rhodopsin phosphorylation under these conditions could be attributed to recoverin.

Methods:: We used a previously established mass-spectrometry-based method² to quantify the extent of rhodopsin phosphorylation in mouse retinas. Mice were illuminated in constant bright light for times ranging from 1 to 30 minutes before being quickly anaesthetized and euthanized. To measure the phosphorylation of light-activated rhodopsin in rods at low intracellular free Ca²⁺ concentrations we used wild-type mice. To measure phosphorylation at high intracellular free Ca²⁺ we used mice in which the rod transducin gene (GNAT1) was inactivated. (GNAT1-/- mice were produced by Janis Lem and colleagues). In GNAT1-/- retinas free intracellular Ca²⁺ concentrations do not fall as they do in normal retinas when they are illuminated³. To evaluate whether any effects of Ca²⁺ could be attributed to recoverin we used GNAT1-/-;Rv-/- double knockout mice. (Rv-/- mice were produced by Jeannie Chen and colleagues).

Results:: Elevated Ca²⁺ inhibits rhodopsin phosphorylation in mouse eyes by 20-30%. The 20-30% inhibition of rhodopsin phosphorylation also occurs in the absence of recoverin, i.e. in retinas of GNAT1-/-;Rv-/- mice.

Conclusions:: The effect of Ca²⁺ on rhodopsin phosphorylation in mice is less than the inhibition by Ca²⁺ of cone visual pigment phosphorylation in zebrafish retinas¹. The small inhibitory effect of Ca²⁺ on mouse rhodopsin phosphoryaltion occurs even in the absence of recoverin. Our findings do not provide physiological confirmation of in vitro studies in which recoverin inhibits rhodopsin phosphorylation in bright light. ¹Kennedy, M.J. et al., (2004). Neuron 41:915; ²Kennedy, M.J. et al., (2001). Neuron 31:87; ³Woodruff M.L. et al., (2002 ) J. Physiol. 542:843.

Keywords: phosphorylation • calcium • photoreceptors