Molecular Mechanisms of Retinal cGMP-Activated Ion Channels

描述（由申请人提供）：关于编码环核苷酸门控（CNG）离子通道的基因突变如何在人类中产生色盲、锥体营养不良和黄斑变性的知识存在根本空白。我们的长期目标是了解控制这些通道活性的机制以及与CNG通道突变相关的视网膜疾病的病理生理学。本应用程序的核心目标是确定锥体CNG通道门控或运输突变对细胞活力的影响的细胞机制，以及磷酸肌苷控制通道的关键结构特征。最近，我们在锥形CNG通道的CNGA3和CNGB3亚基中发现了几种与疾病相关的突变的功能特征，并发现了对通道门控、调节和/或运输的巨大影响，但这些缺陷的细胞后果尚未确定。核心假设是锥体CNG通道的功能获得突变通过通道活性增强或不受控制、细胞内钙（Ca2+）稳态紊乱和随后的Ca2+依赖性凋亡导致光受体死亡。相反，运输缺陷预计会通过内质网（ER）应激损害细胞活力。该研究的基本原理是了解与异常CNG通道活性相关的光感受器功能障碍和损失，将为几种相关锥体营养不良的可能治疗提供见解。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)确定锥体CNG通道中与疾病相关的功能变化与导致光感受器功能障碍和死亡的细胞机制之间的联系；(2)确定CNGB3亚基对磷酸肌苷通道控制的敏感性的机制和相互作用。这些研究将利用分子和细胞操作、生化方法和/或电生理学研究人类CNG通道在锥体光感受器衍生的661W细胞或爪虫卵母细胞中表达，以及斑马鱼锥体光感受器中的转基因。该研究的创新之处在于，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。总的来说，这项工作是重要的，因为它有望增强我们对导致视网膜变性和失明的机制的理解，并为预防光感受器丧失的潜在方法提供见解。公共卫生相关性：拟议的研究与公共卫生相关，因为这些研究的完成将为预防和治疗视力丧失提供重要的见解。我们的工作重点是在分子和细胞水平上理解视觉，以及对视觉至关重要的蛋白质编码基因的突变如何导致功能障碍和视网膜变性。我们研究离子通道蛋白，它们负责产生电信号，最终被大脑解释为视觉信息。这个新项目的主要目标是阐明将通道功能或控制的致病变化与细胞死亡联系起来的具体机制。

DESCRIPTION (provided by applicant): 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 humans. 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 gating, 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 understanding 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 associated functional changes in cone CNG channels and the cellular mechanisms leading to photoreceptor dysfunction 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 significant because it is expected to enhance our understanding of the mechanisms that lead to retinal degeneration and blindness, and to provide insight into potential approaches for prevention of photoreceptor loss. PUBLIC HEALTH RELEVANCE: The proposed research has relevance to public health, because completion of these studies will provide important insight for preventing and treating vision loss. Our work is focused on understanding vision at the molecular and cellular levels and how mutations in genes coding for proteins critical for vision can lead to dysfunction and retinal degeneration. We study ion channel proteins that are responsible for generating electrical signals ultimately interpreted by the brain as visual information. The major goal for this new project is to elucidate specific mechanisms linking pathogenic changes in channel function or control to cell death.