Mammalian Phototransduction: Role of cGMP and Calcium

光感受器鸟苷酸环化酶（GC 1和GC 2）产生cGMP，cGMP是光感受器的内部信使。 光传导阳离子，由称为鸟苷酸环化酶激活蛋白的钙结合蛋白介导 （GCAP），规范他们的活动。GCAP 1基因（GUCA 1A）中影响Ca 2+结合的突变， 常染色体显性视锥细胞营养不良（adCD）和常染色体显性视锥-视杆细胞营养不良 （adCORD）.生化缺陷目前作为一个占主导地位的，持续刺激GC 1在黑暗[Ca 2 +]。在 具体目标1，我们将基于GCAP 1突变表征adCD和adCORD的动物模型， 开发RNA干扰策略来敲除突变体和正常GCAP 1基因。一 部分或完全敲除将延迟视锥细胞营养不良的发作或治愈视锥细胞营养不良， 轻微表型（暗适应延迟）和无视网膜变性。这些实验是 最终的人类基因疗法在具体目标2中，我们提出通过以下方法产生GCAP 1敲除小鼠： 在GCAPs敲除背景下转基因表达GCAP 2，以研究GCAP 2的恢复动力学。 GCAP 1的缺失。此外，我们将探讨GCAP 2（G157 R）突变的生化后果， 与显性RP连锁，首先在体外，最后在转基因实验中。在具体目标3中，我们建议： 在GCAP敲除背景下产生仅表达GC 1或仅表达GC 2的小鼠， 在不存在Ca 2+刺激的情况下，每种酶对暗电流的贡献。我们进一步建议， 使用视锥细胞特异性启动子产生在小鼠视锥细胞中表达GC 2的转基因小鼠。目的是 表征表达GC 2但不表达GC 1的视锥细胞的生理学，以及GC 2在视锥细胞中的Ca 2+敏感性。 GCAP 1的存在（其在体外对GC 2的刺激很差）。在分析GC 1和GC 2的表型时， 在小鼠中敲除后，我们发现两种GC作为完整的膜蛋白， 将膜相关蛋白（PDE、GRK 1、转导蛋白）转运至外节。因此我们 建议使用表达EGFP的构建体鉴定转基因非洲爪蟾中的靶向序列， 缺乏靶向序列VXPX的视紫红质C末端片段和C末端GC片段 含有所提出的靶向基序。拟议的研究将有助于我们了解 cGMP/Ca 2+反馈系统在光感受器，以及机制导致视网膜疾病的基础上 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.