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J. Wei, J. B. Hurley; On the Influence of Calcium and Recoverin on Rhodopsin Phosphorylation in Mice. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4641.
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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.
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).
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.
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.
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