Calcium Feedback Mechanisms in Visual Adaptation

描述(申请人提供)：这项建议的主要目标是破译视杆细胞和视锥感光细胞中光适应的分子机制。众所周知，钙可以协调几种反馈机制，以防止视网膜光感受器中的信号饱和。到目前为止，在体外已经确定了视紫红质激酶(RK)活性、鸟苷环化酶(GC)活性和cGMP门控(CNG)通道对cGMP的亲和力等三个依赖于钙的步骤。这些活性的钙依赖关系分别由钙结合蛋白Recovery in、鸟苷环化酶激活蛋白(GCAPs)和钙调素(CaM)决定。这些酶步骤中的钙离子调节最终转化为细胞适应行为的方式还没有完全被理解。由于细胞对光的整体适应能力很可能反映了单个对钙离子敏感的转导步骤的总和，完全理解适应的分子基础将依赖于允许特定分离和定量评估它们在完整光感受器中的个体贡献的实验设计。为了实现这一目标，我们通过分别在转基因小鼠中有针对性地干扰Recovery in和GCAPs，特别干扰了钙离子对RK和GC的反馈；它们对杆适应光的能力的贡献已经通过吸入电极记录来评估。我们现在建议通过评估钙离子反馈对CNG通道的贡献来继续视杆适应的研究，并使用定量视网膜电图分析来研究同样的钙离子反馈调节控制视锥体光感受器的光适应的方法。我们已经确定的具体目标是：1)测试这一假设，即钙离子对CNG通道对cGMP的亲和力有助于视杆细胞适应光的能力。2)验证这样的假设，即恢复素对光反应的第一次磷酸化事件几乎没有影响，但在光反应的恢复阶段延迟了磷酸化。3)验证GCAPs调控视锥感光细胞敏感度调节的假设。4)检验恢复素调节视锥细胞PDE适应的假说。这些研究将有助于我们理解视杆细胞和视锥细胞在光适应过程中的一些基本差异背后的分子机制。

DESCRIPTION (provided by applicant): 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.