Molecular Mechanisms of Retinal cGMP-Activated Ion Channels

项目摘要/摘要 关于编码环核苷酸的基因突变是如何发生的，在知识上存在一个根本性的缺口。 门控(CNG)离子通道可导致人黄斑变性、视锥细胞营养不良和黄斑变性。 曼斯。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 这一应用是为了确定锥体CNG通道效应的细胞机制 门控或运输突变对细胞活性的影响，以及对控制通道至关重要的结构特征 磷脂酰肌醇。最近，我们已经在功能上表征了几个与疾病相关的突变 CNG锥体通道的CNGA3和CNGB3亚基，并发现对通道门有显著影响。 ING、管制和/或贩运，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，细胞内钙稳态的紊乱和 随后的钙依赖性细胞凋亡。相反，运输缺陷预计会损害细胞的生存能力。 通过内质网(ER)胁迫。拟议研究的理由是，开发一种不充分的- 与CNG通道活动异常相关的光感受器功能障碍和丢失的标准将提供 洞察几种相关视锥细胞营养不良的可能治疗方法。在强劲的初步数据指引下，我们 将通过追求两个具体目标来解决这些问题：(1)确定疾病之间的联系- 视锥CNG通道的相关功能改变及其导致光感受器功能障碍的细胞机制 功能和死亡；以及(2)确定CNGB3能力的机制和相互作用 亚基赋予磷脂酰肌醇对通道控制的敏感性。这些研究将利用分子 以及人CNG的细胞操作、生化方法和/或电生理研究 通道在锥光感受器来源的661W细胞或非洲爪哇卵母细胞中表达，并作为转基因在 斑马鱼视锥感光器。拟议的研究是创新的，因为信息丰富的体外研究将 可推广到体内突变CNG通道的转基因表达。总体而言，拟议的工作是有意义的- ICANT是因为它有望增强我们对导致视网膜退化的机制的理解- 为预防光感受器丢失的潜在方法提供洞察力。

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.