Mammalian Phototransduction: Role of cGMP and Calcium

描述（由申请人提供）：视网膜光感受器中的视觉转导通过Ca2+和cGMP两种秒信使的动态相互作用进行操作。光刺激cGMP的水解，导致cGMP的下降导致cGMP门控通道的关闭和Ca2+的下降。参与调节cGMP和Ca2+的两个关键酶系统是cGMP特异性磷酸二酯酶（PDE）和鸟苷酸环化酶（GC）。PDE是一种多亚基酶（PDEalphabetagamma2delta2），与外周膜相关，而GC是一种单亚基完整膜蛋白。光感受器GC的Ca2+敏感性是由Ca2+结合蛋白GCAPs（鸟苷酸环化酶激活蛋白）介导的。gcap对GCs的调控，其复杂性因人类视网膜中至少存在三个gcap和两个GCs而增强，尚不完全清楚。本申请旨在调查关于PDE和GC/GCAPs的监管过程中未解决的问题。特异性目标1解决了PDE研究中尚未解决的问题，特别是在生物合成过程中对新生多肽正确折叠所必需的伴侣的鉴定，以及通过体外和体内表达阐明Pdelta（假定的第四个亚基）的作用。在第二项研究中，我们提出了在转基因小鼠视网膜中表达突变gcap时导致显性锥体营养不良的机制。其他问题涉及GCAP2和GC2在光转导中的作用。gcap1,2双敲除揭示了与激活因子缺失一致的暗适应延迟表型。我们发现（见初步结果/进展报告）转基因GCAP1挽救了GCAP1/2双敲除表型，这表明GCAP1可能单独调节杆和锥光传导。阐明视网膜中的特定作用，特别是GCAP2和GC2，是specific Aim 3的一部分。综上所述，本研究将增强我们对cGMP和ca2 +这两个重要的光转导次级信使的调节成分的结构和功能的认识。

DESCRIPTION (provided by applicant): Visual transduction in retinal photoreceptors operates through a dynamic interplay of two second messengers, Ca2+ and cGMP. Light stimulates the hydrolysis of cGMP and the resulting fall in [cGMP] leads to closure of cGMP-gated channels and a drop in Ca2+. The two key enzyme systems involved in regulation of cGMP and Ca2+are a cGMP-specific phosphodiesterase (PDE) and guanylate cyclase (GC). PDE is a multisubunit enzyme (PDEalphabetagamma2delta2) and peripherally membrane associated, while GC is a single subunit integral membrane protein. The Ca2+ sensitivity of photoreceptor GC is mediated by Ca2+-binding proteins termed GCAPs (guanylate cyclase-activating proteins). The regulation of GCs by GCAPs, the complexity of which is enhanced by the presence of at least three GCAPs and two GCs in human retina, is incompletely understood. This application aims to investigate outstanding and unsolved questions in the regulatory processes regarding PDE and GC/GCAPs. Specific Aim 1 addresses unsolved problems in PDE research, particularly the identification of chaperones essential for the correct folding of nascent polypeptides during biosynthesis, and elucidation of the role of Pdelta, a putative fourth subunit, by in vitro and in-vivo expression. In Aim 2, we propose to address mechanisms leading to dominant cone dystrophy when mutant GCAPs are expressed in the retina of transgenic mice. Additional questions concern the roles of GCAP2 and GC2 in phototransduction. GCAP1,2 double knockouts revealed a phenotype of delay in dark adaptation consistent with loss of activators. We showed (see preliminary results/progress report) that transgenic GCAP1 rescues the GCAP1/2 double knockout phenotype, suggesting that GCAP1 alone may regulate both rod and cone phototransduction. Elucidation of specific roles, particularly for GCAP2 and GC2, in the retina is part of Specific Aim 3. Taken together, this research will enhance our knowledge of the structure and function of components involved in the regulation of cGMP and Ca 2+, two important secondary messengers of phototransduction.