Physiology of Photoreceptors

描述（由申请人提供）：本提案中的实验旨在解决光感受器生理学中重要的突出问题，并使用目前可用的最强大的方法来回答这些问题。本项目将与分子生物学家合作，利用具有必需光感受器蛋白靶向突变的动物，探索光感受器功能的基本特征，直接响应NEI视网膜疾病项目“分析光转导后光适应和恢复机制”的项目目标。这些实验将利用PI实验室最近开发的技术，这些技术可以快速灌注小鼠杆状细胞的小外部部分，并在分离细胞中漂白和再生色素。这个提议的主要目标之一是询问在光刺激后杆状体的反应是如何恢复的，以及是什么导致这种恢复随着背景照明强度的增加而加速，这样我们就能更好地检测到强光下的变化和运动。光感受器的恢复速率是由Ca2+调节的吗？如果是，是通过什么过程调节的？类似的方法将用于研究光感受器的光适应机制。最近的实验表明，适应性不能完全由通常提出的观酰基环化酶、视紫红质磷酸化和通道打开的调节机制来解释，但一种新发现的可能由磷酸二酯酶调节产生的成分也做出了重要贡献。哺乳动物的光适应是否受外节段Ca2+浓度控制？如果是这样，Ca2+是否调节磷酸二酯酶的衰变速率以及cGMP的合成速率？明亮的漂白光产生的脱敏是否也有磷酸二酯酶成分，漂白脱敏产生的机制是否与维持背景光下产生脱敏的机制基本相同？如果这些问题能够得到解答，它们将把光感受器转导推向一个新的方向，并刺激对视觉生物化学的进一步研究。综上所述，这一提议的实验将有助于更详细地了解全身g蛋白级联的生理学。

DESCRIPTION (provided by applicant): The experiments in this proposal are designed to address important outstanding questions in photoreceptor physiology, and to use the most powerful methods presently available to answer these questions. In collaboration with molecular biologists, this project will use animals with targeted mutations in essential photoreceptor proteins in order to probe basic features of photoreceptor function, in direct response to the program objective of the Retinal Disease Program of the NEI to "analyze the mechanisms underlying light adaptation and recovery following phototransduction". These experiments will take advantage of techniques recently developed in the PI's laboratory that permit the fast perfusion of the small outer segments of mouse rods, and the bleaching and regeneration of pigment in isolated cells. One of the principal goals of this proposal is to ask how rod responses recover after stimulation with light, and what causes this recovery to be accelerated as the intensity of background illumination is increased, so that we become better able to detect change and motion in bright light. Is the rate of photoreceptor recovery modulated by Ca2+, and if so, by what process? Similar approaches will be used to study mechanisms of photoreceptor light adaptation. Recent experiments have shown that adaptation cannot be completely explained by the usually proposed mechanisms of regulation of guanylyl cyclase, rhodopsin phosphorylation, and channel opening, but that a newly discovered component probably produced by modulation of phosphodiesterase also makes an important contribution. Is light adaptation in mammals controlled by outer segment Ca2+ concentration? If so, does Ca2+ regulate the rate of phosphodiesterase decay as well as the rate of cGMP synthesis? Does the desensitization produced by bright bleaching light also have a phosphodiesterase component, and is bleaching desensitization produced by a mechanism essentially identical to the one producing desensitization in maintained background light? If these questions can be answered, they will move photoreceptor transduction in a new direction and stimulate additional research into the biochemistry of vision. Taken together, the experiments of this proposal will contribute to a more detailed understanding of the physiology of G-protein cascades throughout the body. PUBLIC HEALTH RELEVANCE: The great majority of diseases of the retina are caused by disorder or degeneration of the photoreceptors, the cells in the eye that convert light into an electrical signal. This proposal seeks to understand basic mechanisms of photoreceptor function, particularly adaptation to maintained illumination and recovery after exposure to light, which are known to be implicated in genetically inherited retinal diseases including night blindness, bradyopsin, and Leber's amaurosis. This study will support an important program objective of the National Eye Institute of the NIH to "analyze the mechanisms underlying light adaptation and recovery following phototransduction", and this work will also contribute to a more detailed understanding of the physiology of signal transduction throughout the body.