ETHANOL ACTION ON ION CHANNELS IN ARTIFICIAL BILAYERS

我们实验室以前的工作已经证实，乙醇(Etoh)抑制电压门控性钙通道和增强大电导钙激活的K+(BK)通道对抑制垂体后叶神经末梢释放多肽的重要性。在这里，我们建议用一种极其简单的制备方法来研究乙醇对BK通道的作用，在这种制备中，我们可以控制通道蛋白的同一性和脂环境的组成，以探索膜脂在调节乙醇对蛋白质的作用中的潜在作用。单通道记录技术将被用来监测酒精对BK功能作用的四个参数：a)通道开放概率；b)细胞内钙离子对无水乙醇作用的影响；c)通道开放和关闭停留时间分布；d)对乙醇诱导的BK通道激活的急性耐受性。对于要测量的酒精作用的每个参数，我们首先收集关于BK通道(MSLO)的数据，该通道被导入HEK293细胞。这些数据将包括代表天然膜中酒精影响的基线。然后，将从HEK293细胞中获取BK通道，并将其重组为一系列平面脂质双层，以选择对通道功能的已知影响，或先前发现的特定脂类参与对慢性乙醇暴露的反应。需要检验的主要假设是，乙醇对已识别的通道的作用被该通道的脂质环境改变。由此衍生出的一个子假说是，脂质环境的特定特征(如电荷、酰基链等)的影响。转化为对通道上特定的乙醇作用的差异化调制(例如，关闭时间分布；乙醇作用的钙离子拮抗等)。这一实验系统的建立还将使未来对乙醇作用的多个方面的研究成为可能，因为它提供了检查药物在相同脂质环境中对各种自然产生的和诱变的蛋白质的作用的手段。

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.