Molecular Mechanisms of Retinal cGMP-Activated Ion Channels

项目概要/摘要 关于编码环核苷酸的基因如何发生突变，在知识上存在着根本性的差距。 门控 (CNG) 离子通道可导致人类全色盲、视锥细胞营养不良和黄斑变性 勒芒。我们的长期目标是了解控制这些渠道活动的机制 以及与 CNG 通道突变相关的视网膜疾病的病理生理学。的核心目标 该应用旨在确定导致锥形 CNG 通道效应的细胞机制 对细胞活力的门控或运输突变，以及对通道控制至关重要的结构特征 磷酸肌醇。最近，我们对一些与疾病相关的突变进行了功能表征 锥 CNG 通道的 CNGA3 和 CNGB3 亚基，并发现对通道门的巨大影响 缺陷、调节和/或运输，但这些缺陷对细胞的影响尚未确定。 中心假设是锥体 CNG 通道的功能获得突变导致光感受器死亡 通过增强或不受控制的通道活性、细胞内钙 (Ca2) 稳态的紊乱和 随后的 Ca2 依赖性细胞凋亡。相反，运输缺陷预计会损害细胞活力 通过内质网（ER）应激。拟议研究的基本原理是开发一种欠 光感受器功能障碍和与异常 CNG 通道活性相关的损失的状态将提供 深入了解几种相关视锥细胞营养不良的可能治疗方法。在强有力的初步数据的指导下，我们 将通过追求两个具体目标来解决这些问题：（1）确定疾病与疾病之间的联系 锥体 CNG 通道的功能变化以及导致光感受器异常的细胞机制 功能和死亡； (2) 确定 CNGB3 能力的机制和相互作用 亚基赋予对磷酸肌醇通道控制的敏感性。这些研究将利用分子 以及人类 CNG 的细胞操作、生化方法和/或电生理学研究 通道在视锥光感受器衍生的 661W 细胞或非洲爪蟾卵母细胞中表达，并作为转基因 斑马鱼视锥细胞光感受器。拟议的研究具有创新性，因为信息丰富的体外研究将 扩展到体内突变 CNG 通道的转基因表达。总体而言，拟议的工作意义重大 很重要，因为它有望增强我们对导致视网膜变性的机制的理解 和失明，并深入了解预防光感受器损失的潜在方法。

PROJECT SUMMARY/ABSTRACT There is a fundamental gap in knowledge regarding how mutations in the genes encoding cyclic nucleotide- gated (CNG) ion channels can produce achromatopsia, cone dystrophy and macular degeneration in hu- mans. Our long-term objective is to understand the mechanisms controlling the activity of these channels and the pathophysiology of retinal diseases associated with CNG channel mutations. The core objectives of this application are to determine the cellular mechanisms responsible for the effect of cone CNG channel gating or trafficking mutations on cell viability, and the structural features critical for control of channels by phosphoinositides. Recently, we have functionally characterized several disease-associated mutations in the CNGA3 and CNGB3 subunits of cone CNG channels and discovered dramatic effects on channel gat- ing, regulation and/or trafficking, but the cellular consequences of these defects have not been determined. The central hypothesis is that gain-of-function mutations in cone CNG channels lead to photoreceptor death via enhanced or uncontrolled channel activity, disturbance of intracellular calcium (Ca2+) homeostasis and subsequent Ca2+-dependent apoptosis. Conversely, trafficking defects are expected to impair cell viability via endoplasmic reticulum (ER) stress. The rationale for the proposed research is that developing an under- standing of photoreceptor dysfunction and loss associated with abnormal CNG channel activity will provide insight into possible treatments for several related cone dystrophies. Guided by strong preliminary data, we will address these issues by pursuing two specific aims: (1) identify the connection between disease asso- ciated functional changes in cone CNG channels and the cellular mechanisms leading to photoreceptor dys- function and death; and (2) determine the mechanisms and interactions underlying the ability of CNGB3 subunits to confer sensitivity to channel control by phosphoinositides. These studies will utilize molecular and cellular manipulations, biochemical approaches and/or electrophysiological studies of human CNG channels expressed in cone photoreceptor derived 661W cells or Xenopus oocytes, and as transgenes in zebrafish cone photoreceptors. The proposed research is innovative in that informative in vitro studies will be extended to transgenic expression of mutant CNG channels in vivo. Overall, the proposed work is signif- icant because it is expected to enhance our understanding of the mechanisms that lead to retinal degenera- tion and blindness, and to provide insight into potential approaches for prevention of photoreceptor loss.