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: 6342623
• 负责人: William N Zagotta
• 金额: $ 16.08万
• 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/6342623
• 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.

循环核苷酸门控（CNG）离子通道在多个 视网膜中信号处理的阶段。在光感受器外部 段，他们检测并发出CGMP浓度下降的信号 由视紫红质吸收的光吸收；在内部段 在锥形光感受器上，它们调节发射机释放到 双极细胞；在一个双极细胞中，它们负责 对谷氨酸的抑制作用。 CNG渠道高度专业 因为它们在信号处理中的作用。我们研究的长期目标 是要了解其基础的分子机制 专业。 CGMP通过直接结合到A来激活通道 循环核苷酸结合结构域的每个羧基末端区域 通道亚基。 CAMP是这些频道的非常差的激活因子。这 环状核苷酸选择性来自于构象变化 与离子开放有关的环状核苷酸结合位点 进行孔。此外，这些通道由CA/2+ - 调节 钙调蛋白和磷酸化。人们认为这种调节涉及 每个氨基末端区域之间的蛋白质蛋白质相互作用 亚基和环状核苷酸结合结构域。在这个建议中，我们 将检查构象变化的分子机制 在环状核苷酸结合位点以及如何调节它 蛋白质蛋白质相互作用。频道将研究 通过外源表达cDNA克隆的电生理学 爪蟾卵母细胞和生化通过表达氨基终端 通道的区域和环状核苷酸结合结构域作为融合 细菌中的蛋白质。我们将探测构象的变化 带有单通道记录的循环核苷酸结合位点 定向诱变和状态依赖性半胱氨酸的修饰。在 此外，使用蛋白质相互作用测定，我们将检查 氨基末端相互作用的结构决定因素 区域和环状核苷酸结合域以及它们如何解释 通过Ca/2+ - 钙统治蛋白和磷酸化对通道的调节。 这些实验应洞悉CNG通道的作用 在信号转导中。