Mechanisms of Cone Pigment Deactivation

描述（由申请人提供）：视杆和视锥光感受器都吸收光，触发放大级联，通过关闭选择性离子通道产生膜超极化。从光引发的事件的辐射是G蛋白偶联的次级活动的响应，包括视紫红质或视锥细胞视蛋白受体关闭通过GRK 1磷酸化和随后的结合杆或锥细胞抑制蛋白。视锥光感受器在形态学、光敏感性、恢复率、热稳定性、外节脱落的时间和对通过细胞凋亡的程序性细胞死亡的抗性方面不同于视杆。这些视锥基因及其基因产物的动态相互作用和功能的表征可能为诊断、治疗或预防年龄相关性黄斑变性和其他视网膜杆和视锥变性提供基础，从而为目前无法治疗的失明形式保留视力。为了解决视锥光转导途径固有的不同方面并实现我们的目标，设计实验以探索视锥抑制蛋白（CAR）、其靶向G蛋白偶联受体（S和M视蛋白色素）和视锥突触中的其他潜在相关伴侣的功能。我们的工作假设，部分基于我们正在进行的生物化学和电生理学研究，支持CAR通过结合光激活的GRK 1磷酸化S和M视蛋白来调节视锥细胞光转导的作用。我们提出，当编码CAR的X染色体基因用小鼠敲除（KO）技术遗传缺失时，有缺陷的受体关闭将导致视锥光响应的延迟恢复。为了检验这一假设，具体目的和实验设计包括1）表征新产生的Car KO的形态学、生物化学和电生理学视网膜表型。进一步的实验将在两种遗传背景的Grk 1/Car双KO小鼠中探索这些参数（具有正常视杆形态但无视杆反应的转导素α-/-和具有纯视锥视网膜的Nrl -/-）; 2）检查Grk 1 S和M视蛋白磷酸化和CAR结合对视锥视色素循环途径的影响;和3）鉴定CAR及其可变剪接同种型的其它潜在相互作用锥突触配偶体。研究光传导级联反应和引发和终止高敏度视力的分子触发因素对于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.