GATING MECHANISMS OF RETINAL ROD CGMP ACTIVATED CHANNELS

环核苷酸门控(CNG)离子通道在几个 视网膜中信号处理的各个阶段。在光感受器外部 片段，它们检测并发出cGMP浓度下降的信号 由视紫红质吸收光引起的；内节的 在视锥感光器上，它们将发射器的释放调制到 在一个双极细胞中，它们负责 对谷氨酸的抑制反应。CNG渠道高度专业化 因为他们在信号处理中扮演的角色。我们研究的长期目标是 就是了解它们背后的分子机制 专业化认证。CGMP通过直接绑定到 三者羧基末端区域的环核苷酸结合区 通道子单元。坎普是这些渠道的一个非常糟糕的激活者。这 环核苷酸选择性是由环核苷酸的构象变化引起的 与离子开放相关的环核苷酸结合位点 传导毛孔。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一种 各氨基酸末端区域之间的蛋白质相互作用 亚基和环核苷酸结合区。在这份提案中，我们 将研究构象变化背后的分子机制 在环核苷酸结合部位，以及它是如何受此调节的 蛋白质-蛋白质相互作用。将对渠道进行研究 外源表达基因克隆的电生理学研究 非洲爪哇卵母细胞，并通过表达氨基末端进行生化 AS融合通道的区域和环核苷酸结合域 细菌中的蛋白质。我们将探索分子中的构象变化 单通道记录的环核苷酸结合位点 定向突变和状态依赖的半胱氨酸修饰。在……里面 此外，使用蛋白质相互作用分析，我们将检查 氨基末端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 钙/2-钙调素和磷酸化对通道的调节作用。 这些实验应该为CNG通道的作用提供洞察力 在信号转导方面。

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.