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 电压门控钙通道和大电导增强，Ca+* 激活的K*（BK） 通道，抑制肽释放神经末梢在垂体后叶。这里我们 我建议在一个非常简单的制备过程中研究乙醇对BK通道的作用， 我们可以控制通道蛋白的身份和脂质环境的组成， 探索膜脂在调节乙醇对蛋白质作用中的潜在作用。 单通道记录技术将用于监测酒精对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通道的急性耐受性 activation.对于每个待测量的酒精作用参数，我们将首先收集BK上的数据 通道（mGlutamine）转染到HEK 293细胞中。该数据将包括代表酒精的基线 对原生膜的影响然后从HEK 293细胞收获BK通道， 重构成一系列平面脂质双层， 或先前的发现，即特定的脂质参与对慢性乙醇暴露的反应。 待检验的主要假设是乙醇对已识别通道的作用被修改 通过通道的脂质环境。由此产生的一个子假设是， 脂质环境的特定特征（例如电荷、酰基链等）转化为微分 调节特定EtOH对通道的作用（例如，闭合时间分布; EtOH作用等）。这一实验系统的建立也将使未来的研究， 通过提供检查药物作用的方法， 在相同的脂质环境中多种天然存在和突变蛋白质。

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.