Mammalian Phototransduction: Role of cGMP and Calcium

光感受器鸟苷环化酶(GC1和GC2)产生cGMP，cGMP是 光转导。被称为鸟苷环化酶激活蛋白的钙结合蛋白介导的阳离子 (GCAP)，规范他们的活动。影响GCAP1基因(GUCA1A)钙结合的突变有 常染色体显性遗传性视锥细胞营养不良症(AdCD)和常染色体显性遗传性视锥细胞视锥细胞视杆细胞营养不良 (AdCORD)。生化缺陷表现为在黑暗中对Gc1的主要、持续的刺激[钙]。在……里面 具体目标1，我们将基于GCAP1突变来描述adCD和adCORD的动物模型，以及 开发一种RNA干扰策略，同时击倒突变的和正常的GCAP1基因。一个 部分或完全敲除将延迟或治愈锥体营养不良，因为GCAP缺失的小鼠只有一个 表型轻微(暗适应延迟)，无视网膜变性。这些实验只是一个前奏 最终的人类基因疗法。在特定目标2中，我们建议通过以下方式生成GCAP1基因敲除小鼠 GCAP2在GCAPS基因敲除背景下的转基因表达 GCAP1的缺失。进一步，我们将探索GCAP2(G157R)突变的生化后果 首先在体外，最后在转基因实验中，与显性RP相关联。在具体目标3中，我们建议 仅在GCAPs基因敲除背景上生成仅表达Gc1或GC2的小鼠以分析 在无钙刺激的情况下，各酶对暗电流的贡献。我们进一步建议 使用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。目的是为了 描述表达GC2而不表达GC1的锥体的生理特性，以及GC2在 GCAP1的存在(在体外对GC2刺激很差)。在分析GC1和GC2的表型时 在小鼠的基因敲除中，我们发现这两个GC作为完整的膜蛋白在 膜相关蛋白(PDE、GRK1、转导蛋白)到外节的运输。因此，我们 建议使用表达绿色荧光蛋白的构建物来鉴定转基因非洲爪哇中的靶向序列 缺少靶向序列VXPX的视紫红质C末端片段和C末端GC片段 包含建议的目标主题(S)。拟议的研究将有助于我们了解 光感受器中的cGMP/钙反馈系统以及导致视网膜疾病的机制 GCap突变。

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.