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的重要悬而未决的问题，并使用目前可用的最强大的方法来回答这些问题。与分子生物学家合作，该项目将使用基本光感受器蛋白质的定向突变动物，以探索光感受器功能的基本特征，以直接响应NEI视网膜疾病计划的计划目标，即分析光转导后潜在的光适应和恢复机制。这些实验将利用PI实验室最近开发的技术，允许快速灌流小鼠棒的小外段，并在分离的细胞中漂白和再生色素。这项提议的主要目标之一是询问光刺激后杆状反应如何恢复，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，从而使我们能够更好地检测强光下的变化和运动。光感受器的恢复速度是否受钙离子的调节？如果是，是通过什么过程调节的？类似的方法将被用来研究光感受器光适应的机制。最近的实验表明，这种适应不能完全用通常提出的鸟苷酸环化酶、视紫红质磷酸化和通道开放的调节机制来解释，但新发现的一种可能由磷酸二酯酶调节产生的成分也起到了重要作用。哺乳动物的光适应是否受外段钙离子浓度的控制？如果是这样的话，钙离子是否调节磷酸二酯酶的衰退速度和cGMP的合成速度？由明亮的漂白光产生的脱敏是否也有磷酸二酯酶成分，以及漂白脱敏产生的机制是否与在保持的背景光中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.