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 电压门控性钙通道与大电导钙激活钾(BK)的增强作用 抑制垂体后叶神经末梢的多肽释放。在这里，我们 建议在一种极其简化的制备方法中研究乙醇对BK通道的作用 我们可以控制通道蛋白的特性和脂质环境的组成， 探讨膜脂在调节乙醇对蛋白质作用中的潜在作用。 将使用单通道记录技术来监测酒精对BK作用的四个参数 功能：a)通道开放概率；b)细胞内钙离子对乙醇作用的影响；c)通道 开放和封闭的停留时间分布；以及d)对乙醇诱导的BK通道的急性耐受性 激活。对于要测量的酒精行为的每个参数，我们将首先收集关于BK的数据 通道基因(MSLO)导入HEK293细胞。该数据将包括代表酒精的基线 在天然膜中的作用。然后从HEK293细胞中采集BK通道，并 重组成一系列选择用于对通道功能的已知影响的平面脂质双层， 或者之前的发现，即特定的脂类参与了对慢性酒精暴露的反应。 需要检验的主要假设是乙醇在已识别的通道上的作用被修改 受经络脂质环境的影响。由此衍生出的一个次要假设是， 脂类环境的特定特征(如电荷、酰基链等)转化为差异化 对通道的特定乙醇作用的调制(例如，关闭时间分布；钙离子拮抗 Etoh动作等)。这一实验系统的建立还将使未来能够对 通过提供检查药物对酒精的作用的手段，乙醇作用的一些方面 在相同的脂质环境中，不同的自然产生和诱变的蛋白质。

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.