ELECTROPHYSIOLOGICAL STUDIES OF VOLTAGE GATED CHANNELS

描述（改编自研究者摘要）：长期目标 这个项目的一个重要部分是了解 电压依赖性通道的门控。 在本提案中， 设计实验以更详细地描述动力学 Na和K通道的性质，并将Na和K通道的功能与Na和K通道的功能相关联。 已知一级结构发生变化的通道。 渠道将 在非洲爪蟾卵母细胞中表达，其功能将通过 门控电流测量，宏观离子电流，单通道 记录、门控电流波动和膜导纳。 有四个具体目标。 1)振动筛性能的研究 K通道、克隆鱿鱼K通道和Na大鼠脑IIA型通道 以完成通道的动力学状态的描述。 （二） 结构与功能的相关性。 这将由以下人员处理： 研究通过中和碱性和碱性的功能产物的变化， 已知酸性残基会产生电压依赖性的变化， 电导。 门控电荷将在同一膜中测定 其中，将使用信道的波动分析来对信道进行计数。 冷冻断裂显微照片中的离子电流或颗粒计数。 门控电流的波动分析将用于确定 涉及门控的基本电荷的变化。 3)介电 膜蛋白的性质。 通过测量 卵母细胞膜中存在已知量的蛋白质（如通过 颗粒计数）表达，研究者将尝试评估如何 电场使蛋白质内的偶极结构定向。 这 研究开始时，振荡器K通道处于未激活状态（保持 在0 mV）和膜蛋白，其不是电压门控的， 包括其结构已知为约9分辨率视紫红质。 四、 建模--提案的这一部分将把其他三部分放在一起 目的，并将有助于指导类型的实验，根据 基于先前假设的模型预测 实验 最初将探索动力学模型， 结构-功能相关性的结果出现，更多的物理模型 将被提议。 预计这些实验将提供 研究人员对电压的分子起源有了更多的了解 依赖性是许多膜蛋白的基本性质 与细胞内稳态和兴奋性基本相关。

DESCRIPTION (Adapted from investigator's abstract): The long term aim of this project is the understanding of the molecular events underlying the gating of voltage dependent channels. In the present proposal, experiments are designed to describe in more detail the kinetic properties of Na and K channels and to correlate the function of the channel with known changes in the primary structure. Channels will be expressed in Xenopus oocytes and the function will be probed by measuring gating currents, macroscopic ionic currents, single channel recordings, fluctuation of gating currents and membrane admittance. There are four specific aims. 1) Studies on the function of the Shaker K channel, cloned squid K channels and Na rat brain type IIA channels to complete the description of the kinetic states of the channels. 2) Correlation between structure and function. This will be approached by studying changes in the function product by neutralization of basic and acidic residues known to produce changes in the voltage dependence of the conductance. Gating charge will be determined in the same membrane where the channels will be counted using fluctuation analysis of the ionic currents or particle counting in freeze fracture micrographs. Fluctuation analysis of gating currents will be used to determine changes in the elementary charge involved in gating. 3) Dielectric properties of membrane proteins. By measuring complex capacitance of the oocyte membrane where a known amount of protein (as determined by particle counting) is expressed, the investigator will try to assess how the electric field orients dipolar structures within the protein. This study will begin with Shaker K channel in the inactivated state (holding at O mV) and membrane proteins which are not voltage gated and it will include rhodopsin whose structure is known to about 9A resolution. 4) Modeling -- this part of the proposal will put together the other three aims and will help in directing the type of experiments according to predictions of models based on the hypothesis generated by previous experiments. Initially kinetic models will be explored and as the results of structure-function correlation emerge, more physical models will be proposed. These experiments are expected to give the investigators more insights into the molecular origin of voltage dependence which is a fundamental property of many membrane proteins with basic relevance in cell homeostasis and excitability.