Regulation of Cyclic GMP Synthesis in Photoreceptors

描述(申请人提供)：这项拟议的研究的目的是定义调节视觉光感受器中光传导和适应的新过程。长期的目标是确定调节视网膜膜鸟苷酸环化酶(RetGC)合成第二光信号转导信使环GMP(CGMP)的分子机制。这一建议是基于这样的发现：光感受器特异的钙感受器蛋白(GCAPs)调节RetGC以响应细胞内游离钙浓度的变化，对于正常的光反应和光感受器细胞的生存是必不可少的。最近的证据支持我们的假设，即两个EF-Hand结构域上的镁结合控制着GCAP与光中环化酶的对接。因此，该提案的第一个目的是通过定向突变来鉴定retGC和GCAPs中的对接位点，并在体外和体内测试它们的功能。脊椎动物中普遍存在两种GCAP，GCAP1和GCAP2。最近利用GCAP1和GCAP2基因敲除小鼠模型的研究发现，光刺激cGMP合成的激活是一个两步过程，其中GCAP1和GCAP2根据不同的钙敏感性依次刺激cGMP的产生。还有两种retGC同工酶，retGC1和retGC2，是杆状细胞恢复所必需的。因此，第二个特定的目的是建立GCAP1和GCAP2如何在小鼠视杆中调节两种不同的retGC同工酶。我们计划建立单个GCAP1或GCAP2对单个视网膜GC的参数和生化调节，并确定它们在视杆生理学和GCAP1突变导致的光感受器细胞死亡过程中的贡献。锥体细胞对光的反应比杆状细胞恢复得更快，适应光的速度也非常快，因此导致了一种假设，即圆锥体中cGMP的合成比杆状细胞中调节得更快。因此，这项建议的第三个具体目标是确定视锥细胞中视网膜GC的生化特性以及单个GCAP在调节视锥视网膜GC活性中的作用。拟议的实验与理解控制光感受器活性并导致人类患者遗传性视网膜疾病的机制有关。 公共卫生相关性：拟议的研究与公共健康相关，因为它的完成将更好地理解对正常视力和视觉障碍都很重要的过程。这项研究的主要目标是了解视网膜鸟苷酸环化酶和钙结合蛋白是如何控制视网膜的电反应的，以及为什么影响这些蛋白的突变会导致先天性失明。本研究的具体目的是阐明它们相互作用的分子机制及其在视网膜生理学和视网膜退行性变中的作用。

DESCRIPTION (provided by applicant): The purpose of the proposed study is to define new processes that regulate phototransduction and adaptation in visual photoreceptors. The long-term goal is to determine the molecular mechanisms that regulate synthesis of a second messenger of phototransduction, cyclic GMP (cGMP), by retinal membrane guanylyl cyclases (retGC). The proposal is based on the finding that photoreceptor-specific calcium sensor proteins, GCAPs, that regulate retGC in response to the change in intracellular free calcium concentrations are essential for normal photoresponses and survival of photoreceptor cells. Recent evidence supports our hypothesis that magnesium binding in two EF-hand domains controls GCAP docking with the cyclase in the light. Therefore, the first aim of the proposal is to identify, by using directed mutagenesis, the docking sites in retGC and GCAPs and to test their function in vitro and in vivo. There are two ubiquitous GCAPs in vertebrates, GCAP1 and GCAP2. Recent findings using GCAP1 and GCAP2 gene knockout mouse models have lead to a hypothesis that activation of cGMP synthesis in response to light is a two-step process, in which GCAP1 and GCAP2 stimulate cGMP production sequentially, according to their different calcium sensitivity. There are also two isozymes of retGC, retGC1 and retGC2, required for rod recovery. Therefore, the second specific aim is to establish how the two different isozymes of retGC are regulated by GCAP1 and GCAP2 in mouse rods. We plan to establish parameters and biochemical regulation of individual retGCs by individual GCAP1 or GCAP2 and determine their contribution to the rod physiology and to the progression of photoreceptor cell death caused by mutations in GCAP1. Cone cells respond to light and recover faster than rods and adapt to light very rapidly, thus leading to a hypothesis that cGMP synthesis is regulated faster in cones than in rods. Therefore, the third specific aim of this proposal is to determine the biochemical properties of retGC in cones and the role of individual GCAPs in regulation of cone retGC activity. The proposed experiments are relevant to the understanding of the mechanisms that control photoreceptor activity and cause inherited retinal diseases in human patients. PUBLIC HEALTH RELEVANCE: The proposed study has relevance to public health, because its completion will provide better understanding of the processes important for both normal vision and visual disorders. The main goal for this research is to understand how retinal guanylyl cyclases and calcium-binding proteins control electrical responses of the retina and why the mutations that affect these proteins lead to congenital blindness. The specific purpose of this study is to elucidate molecular mechanisms of their interactions and their role in retinal physiology and retinal degeneration.