Recovery of Phototransduction

描述(申请人提供)：由异三聚体G蛋白介导的信号转导在自然界中广泛存在，最好的代表是视网膜视杆中的光转导。然而，三聚体G蛋白如何在光感受器外削弱脊椎动物的视觉仍然知之甚少。同样，光转导的基本信息，如敏感度的潜在机制和视杆和视锥之间的动力学差异，也缺乏。视觉G蛋白通路的突变会导致衰弱的疾病，如先天性静止性夜盲、视力减慢和光感受器退化。这一应用建立在三个新的初步发现的基础上：1)视紫红质激酶可能作为GTP酶加速蛋白(GAP)，以确保在暗光条件下光传导的及时恢复。2)锥体光传导恢复受锥体转导关断的速率限制。3)不止一种类型的G？亚基介导去偏振双极细胞的光反应。我们建议使用一种综合的方法，结合小鼠遗传学、生物化学、基因组学、细胞生物学、成像、ERG和单细胞电生理学来研究上述发现的机制和生理相关性。AIM-1将研究视紫红质激酶介导的光传导恢复加速的机制和功能，以确定视紫红质激酶在视锥细胞中的较高水平是否有助于改善人类迟缓患者的症状。AIM-2将寻求对锥体光传导恢复的限速步骤进行定量验证，并探索第二个最慢的杆状光传导恢复步骤的一致性。将开发有用的小鼠品系和程序，以便对来自同一动物的杆状和圆锥体进行比较。AIM-3将检测G？3和G？13在视网膜中的作用，以了解它们的表达水平及其与G？亚基赋予不同类型的去极化双极细胞不同的光响应特性。通过完成这些目标，我们希望了解三聚体G蛋白如何用于光传导以外的视觉，以及视杆细胞和视锥细胞在对光的反应的敏感度和速度上可能有何不同。许多有用的小鼠品系将在这里制作、表征、保存和分布，以方便未来的发现。最后，了解正常的视觉机制对于有效预防、保护和恢复患有各种致残致盲疾病的患者的视力是必不可少的。

DESCRIPTION (provided by applicant): Signal transduction mediated by heterotrimeric G-proteins occurs throughout nature and is best represented by phototransduction in retinal rods. However, how trimeric G-proteins subserve vertebrate vision outside photoreceptors is still poorly understood. Likewise, basic information in phototransduction, such as the mechanisms underlying sensitivity and kinetic differences between rods and cones, is missing. Mutations in visual G-protein pathways cause debilitating diseases such as congenital stationary night blindness, bradyopsia, and photoreceptor degeneration. This application is built on three novel preliminary findings: 1) rhodopsin kinase may act as a GTPase accelerating protein (GAP) to ensure timely recovery of phototransduction under dim light condition. 2) cone phototransduction recovery is rate-limited by cone transducin turn-off. 3) more than one type of G¿? subunits mediate depolarizing bipolar cell light responses. We propose here to use an integrated approach combining mouse genetics, biochemistry, genomics, cell biology, imaging, ERG, and single cell electrophysiology to study the mechanisms and physiological relevance of the aforementioned findings. Aim-1 will investigate mechanism and function of rhodopsin kinase mediated acceleration of phototransduction recovery to determine whether a higher level of rhodopsin kinase in cone help to ameliorate symptoms of human bradyopsia patients. Aim-2 will seek quantitative validation of the rate- limiting step of cone phototransduction recovery and explore the identity of the second slowest step of rod phototransduction recovery. Useful mouse strains and procedures will be developed to allow comparisons between rods and cones derived from the same animal. Aim-3 will examine the role of G¿3 and G?13 in retina to see whether their expression levels and interactions with G? subunit confer different light response properties to different types of depolarizing bipolar cells. By completing these aims we expect to know how trimeric G-proteins are used in vision beyond phototransduction and how rod and cone may differ in their sensitivity and speed of their responses to light. Many useful mouse lines will be made, characterized, preserved, and distributed here to facilitate future discovery. Finally, knowing the normal visual mechanism is indispensable for efficient prevention, preservation, and restoration of vision in patients suffering from various debilitating blinding diseases.