GATING MECHANISMS OF RETINAL ROD CGMP ACTIVATED CHANNELS

环核苷酸门控（CNG）离子通道在几个方面发挥关键作用， 视网膜中信号处理的各个阶段。感光细胞外 它们检测cGMP浓度的下降并发出信号， 由视紫红质吸收光引起的;在内部节中 在锥状光感受器上，它们调节递质释放到 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管道高度专业化 在信号处理中的作用。我们研究的长期目标是 是去了解它们的分子机制 专业化。cGMP通过直接结合到一个 环核苷酸结合结构域的羧基末端区域的每个 通道亚单位cAMP是这些通道的非常差的激活剂。这 环核苷酸的选择性是由环核苷酸的构象变化引起的。 与离子开放有关的环核苷酸结合位点 导电孔此外，这些通道由Ca/2 +- 钙调素和磷酸化。这种调制被认为涉及一种 蛋白质之间的相互作用的氨基末端区域的每一个 亚基和环核苷酸结合结构域。在本提案中，我们 将研究构象变化背后的分子机制 以及它是如何受此调控的 蛋白质相互作用渠道将被研究 电生理学通过外源表达cDNA克隆， 非洲爪蟾卵母细胞，和生化通过表达氨基末端 区域和环核苷酸结合结构域的通道作为融合 细菌中的蛋白质我们将探讨在构象的变化， 单通道记录的环核苷酸结合位点， 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结构域，以及它们如何解释 通过Ca/2 +-钙调蛋白和磷酸化调节通道。 这些实验应该提供洞察CNG通道的作用 在信号传导中。

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.