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）离子通道的基因中的突变如何在人类中产生色盲、视锥细胞营养不良和黄斑变性，存在根本的知识空白.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。本申请的核心目标是确定负责锥体CNG通道门控或运输突变对细胞活力的影响的细胞机制，以及对磷酸肌醇控制通道至关重要的结构特征。最近，我们在功能上表征了锥CNG通道的CNGA 3和CNGB 3亚基中的几种疾病相关突变，并发现了对通道门控、调节和/或运输的显著影响，但这些缺陷的细胞后果尚未确定。核心假设是，锥CNG通道中的功能获得性突变通过增强或不受控制的通道活性、细胞内钙（Ca 2+）稳态的干扰和随后的Ca 2+依赖性细胞凋亡导致感光细胞死亡。相反，预计运输缺陷通过内质网（ER）应激损害细胞活力。拟议研究的基本原理是，了解与异常CNG通道活性相关的感光细胞功能障碍和损失，将为几种相关的视锥细胞营养不良提供可能的治疗方法。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）确定锥CNG通道中疾病相关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CNGB 3亚基赋予磷酸肌醇对通道控制敏感性的能力的机制和相互作用。这些研究将利用分子和细胞操作，生物化学方法和/或电生理学研究的人CNG通道中表达的锥光感受器衍生的661 W细胞或非洲爪蟾卵母细胞，并作为转基因在斑马鱼锥光感受器。拟议的研究是创新的，在体外研究的信息将扩展到在体内的突变CNG通道的转基因表达。总的来说，拟议的工作是重要的，因为它有望提高我们对导致视网膜变性和失明的机制的理解，并提供深入了解潜在的方法来预防感光细胞损失。公共卫生相关性：拟议的研究与公共卫生有关，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，以及编码对视觉至关重要的蛋白质的基因突变如何导致功能障碍和视网膜变性。我们研究负责产生电信号的离子通道蛋白，这些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性变化与细胞死亡联系起来的特定机制。

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.