GATING MECHANISMS OF RETINAL ROD CGMP ACTIVATED CHANNELS

视网膜杆 CGMP 激活通道的门控机制

• 批准号: 6489805
• 负责人: William N Zagotta
• 金额: $ 16.56万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6489805
• 关键词: SDS polyacrylamide gel electrophoresis; Xenopus; Xenopus oocyte; allosteric site; alternatives to animals in research; aminoacid; binding proteins; binding sites; conformation; cyclic GMP; electrophysiology; membrane channels; molecular cloning; phosphorylation; polymerase chain reaction; protein protein interaction; protein structure function; rod cell; single cell analysis; site directed mutagenesis; surface plasmon resonance; visual photoreceptor; visual phototransduction; voltage /patch clamp; yeast two hybrid system

Cyclic nucleotide-gated (CNG) ion channels play a key role at several stages of signal processing in the retina. In photoreceptor outer segments, they detect and signal the drop in cGMP concentration resulting from the absorption of light by rhodopsin; in inner segments on the cone photoreceptors they modulate transmitter release onto the bipolar cells; and in one-bipolar cells they are responsible for the inhibitory response to glutamate. CNG channels are highly specialized for their role in signal processing. The long-term goal of our research is to understand the molecular mechanisms that underlie their specializations. cGMP activates the channel by binding directly to a cyclic nucleotide-binding domain in the carboxyl-terminal region of each channel subunit. cAMP is a very poor activator of these channels. This cyclic nucleotide selectivity arises from a conformational change in the cyclic nucleotide-binding site associated with opening of the ion conducting pore. Furthermore, these channels are modulated by Ca/2+- calmodulin and phosphorylation. This modulation is thought to involve a protein-protein interaction between the amino-terminal region of each subunit and the cyclic nucleotide-binding domain. In this proposal we will examine the molecular mechanism underlying the conformation change in the cyclic nucleotide-binding site and how it is regulated by this protein-protein interaction. The channels will be studied electrophysiologically by exogenously expressing the cDNA clones in Xenopus oocytes, and biochemically by expressing the amino terminal region and cyclic nucleotide-binding domain of the channel as fusion proteins in bacteria. We will probe the conformation change in the cyclic nucleotide-binding site with single-channel recording, site- directed mutagenesis, and state-dependent cysteine modification. In addition, using protein interaction assays, we will examine the structural determinants for the interaction between the amino-terminal region and the cyclic nucleotide-binding domain and how they account for modulation of the channels by Ca/2+-calmodulin and phosphorylation. These experiments should provide insight into the role of CNG channels in signal transduction.

环核苷酸门控 (CNG) 离子通道在多个方面发挥着关键作用 视网膜信号处理的阶段。在感光器外层 它们检测并发出 cGMP 浓度下降信号 由于视紫红质吸收光而产生；在内段 在视锥细胞上，它们调节发射机释放到 双极细胞；在单双极细胞中，它们负责 对谷氨酸的抑制反应。 CNG渠道高度专业化 以表彰它们在信号处理中的作用。我们研究的长期目标 是了解其背后的分子机制 专业化。 cGMP 通过直接结合到 每个的羧基末端区域都有环核苷酸结合域 通道子单元。 cAMP 是这些通道的非常差的激活剂。这 环核苷酸的选择性来自于构象的变化 与离子打开相关的环核苷酸结合位点 导电孔。此外，这些通道受 Ca/2+- 调制 钙调蛋白和磷酸化。这种调制被认为涉及 每个氨基酸末端区域之间的蛋白质-蛋白质相互作用 亚基和环核苷酸结合结构域。在这个提案中我们 将检查构象变化背后的分子机制 在环核苷酸结合位点以及它是如何受其调节的 蛋白质-蛋白质相互作用。渠道将被研究 通过在电生理学中外源表达 cDNA 克隆 非洲爪蟾卵母细胞，通过表达氨基末端进行生化分析 区域和通道的环核苷酸结合域融合 细菌中的蛋白质。我们将探究构象变化 具有单通道记录的环核苷酸结合位点，位点 定向诱变和状态依赖性半胱氨酸修饰。在 此外，使用蛋白质相互作用测定，我们将检查 氨基末端之间相互作用的结构决定因素 区域和环核苷酸结合域以及它们如何解释 Ca/2+-钙调蛋白和磷酸化对通道的调节。 这些实验应该可以深入了解 CNG 通道的作用 在信号转导中。