ETHANOL ACTION ON ION CHANNELS IN ARTIFICIAL BILAYERS

乙醇对人工双层离子通道的作用

• 批准号: 6371518
• 负责人: STEVEN N TREISTMAN
• 金额: $ 24.11万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6371518
• 关键词: artificial membranes; calcium flux; cell line; electrophysiology; ethanol; membrane lipids; membrane proteins; potassium channel; tissue /cell culture; voltage /patch clamp; voltage gated channel

Previous work from our laboratory has established the importance of ethanol (EtOH) inhibition of voltage-gated calcium channels and the potentiation of large conductance Ca++-activated K+ (BK) channels, to the inhibition of peptide release from nerve terminals in the posterior pituitary. Here, we propose to examine the action of ethanol on BK channels in an extremely simplified preparation, in which we can control the identity of the channel protein and the composition of the lipid environment, to explore the potential role of membrane lipids in modulating the actions of ethanol on the protein. Single channel recording techniques will be used to monitor four parameters of alcohol action on BK function: a) channel open probability; b) influence of intracellular Ca++ on EtOH action; c) channel open and closed dwell-time distribution; and d) acute tolerance to ethanol-induced BK channel activation. For each parameter of alcohol action to be measured, we will first collect data on the BK channel (mslo) transfected into HEK293 cells. This data will comprise a baseline representing alcohol effects in native membrane. BK channels will then be harvested from the HEK293 cell and reconstituted into a series of planar lipid bilayers chosen for either known effects on channel function, or for previous findings that particular lipids are involved in responses to chronic ethanol exposure. The major hypothesis to be tested is that the actions of ethanol on an identified channel are modified by the lipid environment of the channel. A sub-hypothesis which derives from this is that the effects of specific features of the lipid environment (e.g. charge, acyl chain, etc.) translate into differential modulation of specific EtOH actions on the channel (e.g. closed-time distribution; Ca++ antagonism of EtOH action, etc.). The establishment of this experimental system will also allow future studies of a number of aspects of ethanol action by providing the means to examine the actions of the drug on diverse naturally-occurring and mutagenized proteins in identical lipid environments.

我们实验室之前的工作已经确定了乙醇 (EtOH) 抑制电压门控钙通道和增强大电导 Ca++ 激活的 K+ (BK) 通道对抑制垂体后叶神经末梢肽释放的重要性。 在这里，我们建议以极其简化的制备方法检查乙醇对 BK 通道的作用，其中我们可以控制通道蛋白的身份和脂质环境的组成，以探索膜脂在调节作用中的潜在作用乙醇对蛋白质的影响。 单通道记录技术将用于监测酒精对 BK 功能作用的四个参数： a) 通道开放概率； b) 细胞内Ca++对EtOH作用的影响； c) 通道开放和封闭停留时间分布； d) 对乙醇诱导的 BK 通道激活的急性耐受性。对于要测量的每个酒精作用参数，我们将首先收集转染至 HEK293 细胞的 BK 通道 (mslo) 的数据。 该数据将包括代表酒精对天然膜的影响的基线。 然后将从 HEK293 细胞中收获 BK 通道，并重构为一系列平面脂质双层，这些平面脂质双层的选择要么是针对通道功能的已知影响，要么是针对先前发现的特定脂质参与对慢性乙醇暴露的反应的发现。 要测试的主要假设是乙醇对已识别通道的作用是由通道的脂质环境改变的。 由此衍生的一个子假设是，脂质环境的特定特征（例如电荷、酰基链等）的影响转化为通道上特定 EtOH 作用的差异调节（例如闭合时间分布；Ca++ 拮抗作用）。乙醇作用等）。 该实验系统的建立还将通过提供检查药物对相同脂质环境中多种天然存在和诱变蛋白质的作用的方法，允许未来对乙醇作用的许多方面进行研究。