Molecular Mechanisms of Retinal cGMP-Activated Ion Channels

视网膜cGMP激活离子通道的分子机制

• 批准号: 7994761
• 负责人: MICHAEL D. VARNUM
• 金额: $ 35.09万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-04 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7994761
• 关键词: Address; Apoptosis; Area; Binding; Biochemical; Blindness; Brain; Calcium; Calmodulin; Cell Death; Cell Survival; Cells; Cessation of life; Code; Con-fer; Cyclic GMP; Data; Defect; Development; Disease; Event; Functional disorder; Genes; Goals; Health; Homeostasis; Human; In Vitro; Ion Channel; Ion Channel Protein; Knowledge; Lead; Link; Macular degeneration; Molecular; Mutation; Outcome; Phosphatidylinositols; Photoreceptors; Phototransduction; Predisposition; Prevention approach; Process; Production; Progress Reports; Proteins; Public Health; Publishing; Regulation; Regulatory Pathway; Research; Research Design; Research Methodology; Resources; Retinal; Retinal Cone; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Role; Signal Transduction; Testing; Transgenes; Transgenic Organisms; Vision; Vision Disorders; Work; Xenopus oocyte; Zebrafish; achromatopsia; base; cyclic-nucleotide gated ion channels; disease-causing mutation; endoplasmic reticulum stress; gain of function mutation; in vivo; innovation; insight; mutant; photoreceptor degeneration; prevent; retinal rods; success; trafficking; visual information

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.

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导致人类全色盲、视锥细胞营养不良和黄斑变性，在知识上存在根本性差距。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锥体 CNG 通道门控或运输突变对细胞活力影响的细胞机制，以及磷酸肌醇控制通道的关键结构特征。最近，我们对锥状 CNG 通道的 CNGA3 和 CNGB3 亚基中的几种与疾病相关的突变进行了功能性表征，并发现对通道门控、调节和/或运输的显着影响，但这些缺陷的细胞后果尚未确定。中心假设是锥体 CNG 通道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 (Ca2+) 稳态紊乱以及随后的 Ca2+ 依赖性细胞凋亡导致光感受器死亡。相反，运输缺陷预计会通过内质网（ER）应激损害细胞活力。拟议研究的基本原理是，了解与异常 CNG 通道活动相关的光感受器功能障碍和损失，将为几种相关视锥细胞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亚基赋予磷酸肌醇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、生化方法和/或电生理学研究，对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的人类 CNG 通道进行研究，并作为斑马鱼视锥光感受器中的转基因。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，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，并为预防光感受器损失的潜在方法提供见解。公共健康相关性：拟议的研究与公共健康相关，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了解视力，以及编码对视力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。我们研究负责产生最终被大脑解释为视觉信息的电信号的离子通道蛋白。这个新项目的主要目标是阐明通道功能或控制的致病性变化与细胞死亡之间的联系的具体机制。