Mechanisms of Cone Pigment Deactivation

描述（由申请人提供）：杆状和锥状光感受器都吸收光，触发放大级联，通过关闭选择性离子通道产生膜超极化。光引发事件的辐射是g蛋白偶联的次级活性反应，包括通过GRK1磷酸化关闭视紫红质或视锥蛋白受体，随后结合杆状或视锥蛋白。锥状光感受器在形态、光敏性、恢复速度、热稳定性、外节脱落的时间和细胞凋亡程序性死亡的抗性方面不同于杆状细胞。这些锥体基因及其基因产物的动态相互作用和功能的表征可能为年龄相关性黄斑变性和其他视网膜杆状和锥体变性的诊断、治疗或预防提供基础，从而为目前无法治疗的失明形式保留视力。为了解决锥体光传导途径固有的不同方面并实现我们的目标，实验旨在探索锥体阻滞蛋白（CAR），其靶向G蛋白偶联受体（s和M视蛋白色素）以及锥体突触中其他潜在的相关伙伴的功能。我们的工作假设，部分基于我们正在进行的生化和电生理研究，支持CAR通过结合光激活、GRK1磷酸化的S和M视蛋白来调节锥体光传导的作用。我们提出，当这种编码CAR的x染色体基因通过小鼠基因敲除（KO）技术被基因删除时，有缺陷的受体关闭将导致锥体光反应的延迟恢复。为了验证这一假设，具体的目的和实验设计包括：1)表征新生成的Car - KO的形态、生化和电生理视网膜表型。与野生型相比，进一步的实验将在具有两种遗传背景的Grk1/Car双KO小鼠（具有正常棒状形态但没有棒状反应的转导蛋白α -/-和具有纯锥体视网膜的Nrl -/-）中探索这些参数；2)研究Grk1 S和M视蛋白磷酸化和CAR结合对视锥视维甲酸循环通路的影响；3)鉴定CAR及其选择性剪接异构体的其他潜在相互作用的锥体突触伙伴。研究高敏性视力发生和终止的光导级联和分子触发因素对维持终身视力至关重要。

DESCRIPTION (provided by applicant): Both rod and cone photoreceptors absorb light, triggering an amplification cascade, which produces membrane hyperpolarization through closure of selective ion channels. Radiating from the light-initiated event is a G-protein-coupled response of secondary activities, including rhodopsin or cone opsin receptor shut-off through a GRK1 phosphorylation and subsequent binding of either rod or cone arrestin. Cone photoreceptors are distinct from rods in morphology, light sensitivity, recovery rate, thermal stability, timing of outer segment shedding and resistance to programmed cell death by apoptosis. Characterization of the dynamic interactions and functions of these cone gene and their gene products may provide a basis for diagnosis, treatment or prevention of age related macular degeneration and other retinal rod and cone degenerations, thus preserving vision for currently untreatable forms of blindness. To address the distinct aspects inherent to the cone photo-transduction pathway and to accomplish our goals, experiments are designed to explore the function(s) of cone arrestin (CAR), its targeted G protein-coupled receptors (S and M opsin pigments) and other potential relevant partners in the cone synapse. Our working hypothesis, based in part on our ongoing biochemical and electrophysiological studies, support a role for CAR in regulating cone photo-transduction through binding to light-activated, GRK1 phosphorylated S and M opsins. We propose that when this X-chromosomal gene encoding CAR is genetically deleted with mouse knockout (KO) technology, a defective receptor shut-off will lead to a delayed recovery of cone photoresponses. To test this hypothesis, the specific aims and experimental design include 1) characterization of the morphological, biochemical and electrophysiological retinal phenotypes of the newly generated Car KO. Further experiments will explore these parameters in Grk1/Car double KO mice on two genetic backgrounds (transducin alpha -/- with normal rod morphology but no rod response and Nrl -/- with pure cone retina) compared to wildtype; 2) examine the effects of Grk1 S and M opsin phosphorylation and CAR binding on the cone visual retinoid cycle pathway; and 3) identification of other potential interacting cone synaptic partners for CAR and its alternatively spliced isoforms. Studies of the photo-transduction cascade and the molecular triggers for initiation and termination of high acuity vision are vital for sustaining lifelong vision.