Mammalian Phototransduction: Role of cGMP and Calcium

超出提供的空间。视网膜光感受器的视觉转导通过两个第二信使--钙和cGMP的动态相互作用发挥作用。光刺激cGMP的水解，导致[cGMP]下降，导致cGMP门控通道关闭，钙下降。参与cGMP和Ca~(2+)调控的两个关键酶系统是cGMP专一性的磷酸二酯酶(PDE)和鸟苷环化酶(GC)。PDE是一种多亚单位酶(PDEc_I3y282)，与外周膜结合，而GC是一种单亚基完整的膜蛋白。光感受器GC的钙敏感性是由钙结合蛋白GCAPs(鸟苷环化酶激活蛋白)介导的。人类视网膜中至少存在三个GCAP和两个GCAP，GCAP对GCAP的调控作用并不完全清楚，但GCAP的复杂性却增加了GCAP的复杂性。本申请旨在调查有关PDE和GC/GCAP的监管过程中悬而未决和悬而未决的问题。具体目的1解决PDE研究中尚未解决的问题，特别是鉴定在生物合成过程中新生多肽正确折叠所必需的伴侣，并通过体外和体内表达来阐明P&A可能的第四亚单位的作用。在目标2中，我们建议解决当突变的GCAP在转基因小鼠的视网膜中表达时导致显性视锥细胞营养不良的机制。其他问题涉及GCAP2和GC2在光转导中的作用。GCAP1，2双基因敲除显示了与激活剂丢失相一致的延迟暗适应的表型。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，表明GCAP1单独可能调节杆和锥体的光转导。阐明视网膜中的特定作用，特别是GCAP2和GC2，是特定目标3的一部分。综上所述，这项研究将加深我们对参与调控cGMP和钙的成分的结构和功能的了解，这两个重要的光转导第二信使。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. Visual transduction in retinal photoreceptors operates through a dynamic interplay of two second messengers, Ca 2¿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 Ca 2 . 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 (PDEc_I3y282)and peripherally membrane associated, while GC is a single subunit integral membrane protein. The Ca 2+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 P& 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 knock-outs 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. PERFORMANCE SITE ========================================Section End===========================================