Mammalian Phototransduction: Role of cGMP and Calcium

描述（由申请人提供）：光感受器鸟苷酸环化酶（GC1和GC2）产生cGMP，这是光导的内部信使。阳离子由Ca2+结合蛋白介导，称为鸟苷酸环化酶激活蛋白（GCAPs），调节其活性。影响GCAP1基因（GUCA1A） Ca2+结合的突变与常染色体显性锥体营养不良（adCD）和常染色体显性锥体棒状营养不良（adCORD）有关。生化缺陷表现为显性的，持续刺激GC1在黑暗[Ca2+]。在具体的目标1中，我们将基于GCAP1突变来描述adCD和adCORD的动物模型，并开发一种RNA干扰策略来敲除突变和正常的GCAP1基因。部分或完全敲除GCAP将延迟或治愈锥体营养不良，因为GCAP缺失小鼠只有轻微的表型（黑暗适应延迟），没有视网膜变性。这些实验是最终人类基因治疗的前奏。在特定目标2中，我们提出在GCAP1敲除背景下通过转基因表达GCAP2来产生GCAP1敲除小鼠，以研究GCAP1缺失时的恢复动力学。此外，我们将首先在体外和最后在转基因实验中探索与显性RP相关的GCAP2（G157R）突变的生化后果。在特定目标3中，我们建议在GCAPs敲除背景下产生仅表达GC1或仅表达GC2的小鼠，以解剖在没有Ca2+刺激的情况下每种酶对暗电流的贡献。我们进一步提出利用小鼠锥体特异性启动子在小鼠锥体中产生表达GC2的转基因小鼠。目的是表征表达GC2而不表达GC1的锥体的生理特征，以及GCAP1存在时GC2的Ca2+敏感性（GCAP1在体外对GC2的刺激很差）。在分析小鼠GC1和GC2敲除的表型时，我们发现这两种GCs作为完整的膜蛋白，在膜相关蛋白（PDE, GRK1, Transducin）向外段的运输中起关键作用。因此，我们建议使用表达EGFP、缺乏靶向序列VXPX的视紫红质c端片段和含有所提出的靶向基序的c端GC片段的构建体来鉴定转基因非洲爪蟾的靶向序列。这项研究将有助于我们了解光感受器中的cGMP/Ca2+反馈系统，以及基于GCAP突变导致视网膜疾病的机制。

DESCRIPTION (provided by applicant): Photoreceptor guanylate cyclases (GC1 and GC2) produce cGMP, the internal messenger of phototransduction. Cations, mediated by Ca2+-binding proteins termed guanylate cyclase activating proteins (GCAPs), regulate their activities. Mutations affecting Ca2+-binding in the GCAP1 gene (GUCA1A) have been linked to autosomal dominant cone dystrophy (adCD) and autosomal dominant cone-rod dystrophy (adCORD). The biochemical defects present as a dominant, persistent stimulation of GC1 at dark [Ca2+]. In specific aim 1, we will characterize animal models of adCD and adCORD based on GCAP1 mutations, and develop an RNA interference strategy to knock down both the mutant and the normal GCAP1 genes. A partial or complete knockdown will delay onset or cure the cone dystrophy since GCAP null mice have only a minor phenotype (delay in dark adaptation) and no retinal degeneration. These experiments are a prelude to eventual human gene therapy. In specific aim 2, we propose to generate a GCAP1 knockout mouse by transgenic expression of GCAP2 on a GCAPs knockout background to investigate the kinetics of recovery in the absence of GCAP1. Further, we will explore the biochemical consequence of a GCAP2(G157R) mutation linked to dominant RP, first in-vitro and finally in a transgenic experiment. In specific aim 3, we propose to generate mice expressing GC1 only or GC2 only on a GCAPs knockout background to dissect the contribution of each enzyme to the dark current in the absence of Ca2+ stimulation. We further propose to generate transgenic mice expressing GC2 in mouse cones using a cone specific promoter. The purpose is to characterize the physiology of cones expressing GC2 but not GC1, and the Ca2+ sensitivity of GC2 in the presence of GCAP1 (which poorly stimulates GC2 in vitro). While analyzing the phenotypes of GC1 and GC2 knockouts in mouse, we discovered that both GCs, as integral membrane proteins, have key roles in transport of membrane-associated proteins (PDE, GRK1, Transducin) to the outer segments. We therefore propose to identify the targeting sequences in transgenic Xenopus using constructs expressing EGFP, a rhodopsin C-terminal segment lacking the targeting sequence VXPX, and C-terminal GC fragments containing the proposed targeting motif(s). The proposed research will contribute to our understanding of the cGMP/Ca2+ feed back system in photoreceptors, as well mechanisms leading to retina disease based on GCAP mutations.