PHOTORECEPTOR LIGHT MODULATED CHANNEL--MOLECULAR STUDIES

这项研究的重点是确定单个离子群如何 通道，由cGMP选通，传递外层的光化学。 分段为传输所有经过预处理的可视信息的电流。 通过这些通道的电流是由两个钠离子的流入产生的 和钙离子。由于细胞内钙水平的变化需要调整 感光器对背景光强度变化的敏感度， 关于光转导的一个基本问题是， 外段电流由钙携带。该频道可以是 维持固定的钠和钙内流比率或改变比率作为回应 通过胞浆cGMP或Ca的变化来改变背景光的水平 级别。因此，我们研究的目的之一是确定流入比率。 因为离子不能从电生理上区分，所以我们将 测量培养的哺乳动物细胞中22Na和45Ca的内流 快递渠道。 细胞内cGMP水平的连续监测 通道的表面导致以下概率的改变 频道开通。因此，另一个根本问题是， 蛋白质监测这些浓度的变化，并随后转化为 将信息转化为电导变化。通过表达cDNAfor 在卵母细胞中处于低水平的通道，我们将从单通道记录 并检查各cGMP占有率之间的关系 具有通道开口的结合部位。 最后，为了解释cGMP结合特异性和 与通道的二价相互作用，我们将研究 突变形式的通道中的核苷酸激活电流。 与cGMP相互作用的氨基酸侧链的变化可能 提供核苷酸特异性的线索，而基因的改变 推测的钙结合谷氨酸和天冬氨酸残基可能有助于确定 通道拓扑。

The focus of this research is to determine how a single population of ion channels, gated by cGMP, transduces the photochemistry of the outer segment into a current that conveys all pre-processed visual information. The current through these channels is generated by the influx of both Na and Ca ions. Since changes in cytosolic Ca levels are required to adjust the sensitivity of photoreceptors to changes in background light levels, a fundamental question about phototransduction is what fraction of the outer segment current is carried by Ca. The channel could either maintain fixed influx ratios of Na and Ca or alter the ratio in response to varying levels of background light via changes in cytosolic cGMP or Ca levels. Thus, one aim of our research is to determine the influx ratios. Because the ions cannot be distinguished electrophysiologically, we will measure 22Na and 45Ca influx in cultured mammalian cells that transiently express the channels. Continuous surveillance of cytosolic cGMP levels through binding sites on the surface of the channel results in alterations of the probability of channel opening. Another fundamental question, therefore, is how the protein monitors these concentration changes and subsequently converts the information into an conductance change. By expressing the cDNA for the channel at low levels in oocytes, we will record from single channel patches and examine the relationship between occupancy of each cGMP binding site with channel opening. Finally, to explain the molecular basis of cGMP binding specificity and divalent interactions with the channel, we will examine nucleotide-activated currents in mutant forms of the channel. Alterations of the amino acid side chains that interact with cGMP might provide clues to the nucleotide specificity, while alterations in putative Ca binding Glu and Asp residues might be useful in defining the channel topology.