ETHANOL ACTION ON ION CHANNELS IN ARTIFICIAL BILAYERS

我们实验室以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，我们建议检查的行动，乙醇对BK通道在一个非常简化的准备，在其中我们可以控制的身份的通道蛋白和脂质环境的组成，探索膜脂质在调节乙醇对蛋白质的行动的潜在作用。 将使用单通道记录技术监测酒精对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）通道开放和闭合停留时间分布;和d）对乙醇诱导的BK通道激活的急性耐受性。对于待测量的酒精作用的每个参数，我们将首先收集转染到HEK 293细胞中的BK通道（mRNA）的数据。 该数据将包括代表天然膜中酒精效应的基线。 然后从HEK 293细胞中收获BK通道，并将其重建为一系列平面脂质双层，这些脂质双层是根据对通道功能的已知影响或先前的发现（特定脂质参与对慢性乙醇暴露的响应）而选择的。 待检验的主要假设是乙醇对已识别通道的作用被通道的脂质环境修饰。 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; EtOH作用的Ca++拮抗作用等）。 该实验系统的建立也将允许通过提供检查药物在相同脂质环境中对多种天然存在的和诱变的蛋白质的作用的手段来进一步研究乙醇作用的许多方面。

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.