Calcium Feedback Mechanisms in Visual Adaptation

描述（由申请人提供）：本提案的主要目标是破译视杆细胞和视锥细胞中光适应的分子机制。一段时间以来，人们已经知道钙协调了几种反馈机制，用于防止视网膜光感受器中的信号饱和。到目前为止，已经在体外确定了视杆细胞光传导级联中的三个Ca 2+依赖性步骤;即视紫红质激酶（RK）活性、鸟苷酸环化酶（GC）活性和cGMP门控（CNG）通道对cGMP的亲和力。这些活性的Ca 2+依赖性分别由Ca 2+结合蛋白：恢复素、鸟苷酸环化酶激活蛋白（GCAP）和钙钙调蛋白（Ca 2 +-CaM）赋予。这些酶促步骤的Ca 2+调节最终转化为细胞适应行为的方式尚未完全了解。由于细胞适应光的整体能力很可能反映了单个Ca 2+敏感的转导步骤的总和，因此对适应的分子基础的完整理解将依赖于实验设计，该实验设计允许对完整光感受器中的个体贡献进行特异性分离和定量评估。为了实现这一目标，我们已经专门中断Ca 2+反馈RK和GC有针对性的破坏recoverin和GCAP，分别在转基因小鼠，他们的贡献杆适应光的能力进行了评估，通过抽吸电极记录。现在，我们建议继续研究杆适应通过评估的贡献的Ca 2+反馈的CNG通道，并使用定量视网膜电图分析，以调查的手段，通过相同的Ca 2+反馈调节控制光适应锥光感受器。我们建立的具体目标是：1）测试Ca 2+调节CNG通道对cGMP的亲和力有助于杆适应光的能力的假设。2)测试恢复蛋白对第一次磷酸化事件几乎没有影响，但在光响应的恢复阶段延迟磷酸化的假设。3)测试GCAP调节视锥细胞感光器灵敏度调节的假设。4)测试恢复蛋白调节视锥细胞PDE适应的假设。这些研究将帮助我们了解光适应过程中视杆细胞和视锥细胞之间的一些根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.