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杆状和锥状光感受器细胞的光适应分子机制。一段时间以来，人们已经知道钙协调了几种反馈机制，这些机制有助于防止视网膜光感受器中的信号饱和。到目前为止，已经在体外鉴定了杆细胞光转导级联中的三个ca2 +依赖步骤；即紫红质激酶（RK）活性、鸟苷酸环化酶（GC）活性以及cGMP门控（CNG）通道对cGMP的亲和力。这些活性的Ca2+依赖性是由Ca2+结合蛋白赋予的：恢复蛋白、鸟苷酸环化酶激活蛋白（GCAPs）和钙调蛋白（Ca2+-CaM）。Ca2+调节这些酶促步骤最终转化为细胞适应行为的方式尚不完全清楚。由于细胞适应光的整体能力很可能反映了个体Ca2+敏感转导步骤的总和，因此对适应的分子基础的完整理解将依赖于实验设计，该实验设计允许对其在完整光感受器中的个体贡献进行特定的分离和定量评估。为了实现这一目标，我们在转基因小鼠中分别通过靶向破坏恢复和gcap来特异性地破坏Ca2+对RK和GC的反馈；它们对杆状体适应光的能力的贡献已经通过吸电极记录进行了评估。我们现在建议通过评估Ca2+反馈对CNG通道的贡献来继续研究杆适应，并使用定量视网膜电图分析来研究相同的Ca2+反馈调节控制锥状光感受器光适应的方法。我们确定的具体目标是：1)验证Ca2+调节CNG通道对cGMP的亲和力有助于杆状细胞适应光的能力的假设。2)验证恢复素对第一次磷酸化事件影响不大，但在光响应的恢复阶段延迟磷酸化的假设。3)验证GCAPs调节锥状光感受器灵敏度调节的假说。4)检验恢复调节锥体PDE适应的假说。这些研究将帮助我们理解在光适应过程中杆状和锥状感光细胞之间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.