ETHANOL ACTION ON ION CHANNELS IN ARTIFICIAL BILAYERS

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，我们建议在一个极其简单的制备中检测乙醇对BK通道的作用，在这个制备中，我们可以控制通道蛋白的身份和脂质环境的组成，以探索膜脂在调节乙醇对蛋白质的作用中的潜在作用。单通道记录技术将用于监测酒精作用于BK函数的四个参数：a)通道打开概率；b)细胞内Ca++对EtOH作用的影响；C)通道开闭停留时间分布；d)对乙醇诱导的BK通道激活的急性耐受。对于要测量的酒精作用的每个参数，我们将首先收集转染到HEK293细胞中的BK通道（mslo）的数据。这些数据将包括一个代表酒精在天然膜中的作用的基线。然后从HEK293细胞中收集BK通道并重组成一系列平面脂质双分子层，这些双分子层要么是已知的对通道功能的影响，要么是先前发现的特定脂质参与慢性乙醇暴露的反应。要验证的主要假设是乙醇在已识别通道上的作用被通道的脂质环境所改变。由此产生的一个子假设是，脂质环境的特定特征（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（如封闭时间分布、EtOH作用的ca++拮抗作用等）。该实验系统的建立还将通过提供方法来检查药物在相同脂质环境中对多种自然发生和诱变的蛋白质的作用，从而允许未来对乙醇作用的许多方面进行研究。

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.