ELECTROPHYSIOLOGICAL STUDIES OF VOLTAGE GATED CHANNELS

这个项目的目的是描述和表征分子 电压依赖过程的基础。 特别是，目标是 理解对电压依赖性负责的物理事件 钠和钾通道以及钠/钙交换。 这些实验使用电生理技术来描述 电流通过大量的通道（宏观电流），通过 几个通道（噪声测量），通过单通道和 通道带电基团重排产生的电流 大分子 通道将被修改（BTX用于钠，ATP用于 钾），试图获得更多关于他们的物理信息， states. 结果将被解释使用动力学模型与能量 物理状态之间的障碍， 大分子中的带电粒子。 参数将与这些参数相匹配 模型使用来自电气测量的所有信息， 将根据拟合结果进行修改。 此外，本发明还提供了一种方法， 标记的ATP将用于标记K通道并尝试其提取 和特征化。 Na/Ca交换的特征在于： 在膜电位控制下测量同位素通量和电流 由交换产生。 我们将尝试确定其 化学计量和电压依赖性。 实验将使用鱿鱼的巨大轴突进行， 灌注、透析或切开配置。 后一种技术 允许从膜的内侧进行膜片钳。 的 将进行需要大量膜材料的实验 从乌贼的视网膜神经中提取的膜中。 本研究 预计将提供新的信息，详细的机制， 电压门控通道和钠/钙交换的操作 这将有助于理解电压的分子机制 依赖过程。 预计这些研究将与 神经、肌肉、可兴奋组织和细胞的生理学。

The object of this project is to describe and characterize the molecular basis of voltage dependent processes. In particular, the aim is an understanding of the physical events responsible for the voltage dependence of sodium and potassium channels and the sodium/calcium exchange. The experiments use electrophysiological techniques to describe the currents through a large number of channels (macroscopic currents), through a few channels (noise measurements), through single channels and the currents produced by the rearrangement of the charged groups of the channel macromolecule. Channels will be modified (BTX for sodium and ATP for potassium) in an attempt to gain more information about their physical states. Results will be interpreted using kinetic models with energy barriers between the physical states represented by the positions of the charged particles in the macromolecule. Parameters will be fitted to these models using all the information from the electrical measurements and they will be modified according to the results of the fitting. In addition, labelled ATP will be used to mark the K channel and attempt its extraction and characterization. The Na/Ca exchange will be characterized by measuring isotopic fluxes under membrane potential control and the currents produced by the exchange. We will attempt the determination of its stoichiometry and voltage dependence. Experiments will be performed using the giant axon of the squid in perfused, dialyzed or cut-open configurations. This latter technique allows patch clamping from the internal side of the membrane. The experiments requiring larger amounts of membrane material will be performed in membranes extracted from the retinal nerve of the squid. This research is expected to provide new information on the detailed mechanisms of voltage gated channels and on the operation of the sodium/calcium exchange which will help in the understanding of the molecular mechanisms of voltage dependent processes. These studies are expected to have relevance in the physiology of nerve, muscle, excitable tissues and cells in general.