The broad goal of this proposal is to decipher the molecular mechanisms of light adaptation in both rod and cone photoreceptor cells. It has been known for some time that calcium orchestrates several feedback mechanisms that serve to prevent signal saturation in retinal photoreceptors. Thus far, three Ca 2+-dependent steps in the phototransduction cascade of rods have been identified in vitro; namely, rhodopsin kinase (RK) activity, guanylate cyclase (GC) activity, and affinity of cGMP-gated (CNG) channel for cGMP. The Ca2+ dependence of these activities is conferred by Ca2+-binding proteins: recoverin, guanylate cyclase activating proteins (GCAPs), and calcium calmodulin (Ca2+-CaM), respectively. The manner by which Ca2+ regulation of these enzymatic steps ultimately translates to cellular adaptation behavior is not fully understood. Since the overall ability of the cells to adapt to light very likely reflects the summation of individual Ca2+-sensitive transduction steps, a complete understanding of the molecular basis of adaptation will rely upon an experimental design that allows for specific isolation and quantitative assessment of their individual contributions in intact photoreceptors. Toward the attainment of this goal, we have specifically disrupted Ca2+ feedback to RK and GC by targeted disruption of recoverin and GCAPs, respectively, in transgenic mice; their contribution to the ability of rods to adapt to light has been evaluated by suction electrode recordings. We now propose to continue the study of rod adaptation by assessing the contribution of Ca2+ feedback to the CNG channel, and to use quantitative electroretinographic analysis in order to investigate the means by which the same Ca2+ feedback regulations control light adaptation in cone photoreceptors. Our established Specific Aims are to: 1) Test the hypothesis that Ca2+ modulation of the CNG channel's affinity for cGMP contributes to the ability of rods to adapt to light. 2) Test the hypothesis that recoverin has little effect on the first phosphorylation events but delays phosphorylation during the recovery phase of the light response. 3) Test the hypothesis that GCAPs regulate sensitivity adjustment in cone photoreceptors. 4) Test the hypothesis that recoverin regulates cone PDE adaptation. These studies will help us understand the molecular mechanisms behind some of the fundamental differences between rod and cone photoreceptor cells during light adaptation.
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