ETHANOL ACTIONS ON SLO CHANNELS FROM ARTERIES VS BRAIN

大电导，Ca++激活的K+通道（BK）活性， 参与动脉张力和中枢神经元的调节 兴奋性，已被发现由急性乙醇（EtOH）调制， 相关浓度。为了了解控制 EtOH与其靶标中特定区域的相互作用， 离子通道蛋白，在酒精领域是至关重要的，因为，与一些 最近的例外情况，EtOH在相关的特定相互作用 浓度与蛋白质的选择性区域，一个要求， 从经典药理学的“受体”，仍然在很大程度上难以捉摸。我们 已经发现BK通道活性不被动脉中的EtOH增强， 平滑肌中，而来自神经的BK通道中显着增加 终端和PC 12细胞。这种二分法仍然存在时，乙醇的行动是 研究了两个99%相同的克隆通道， 插入相同的蛋白质-脂质环境中：50 mM EtOH抑制 通道从动脉平滑肌（β-alpha亚单位），而它 当两者都是时，激活大脑通道（mslo，α亚基） 在卵母细胞中表达。因此，主要假设是， EtOH对这些通道的作用是由于在这些通道中的特定差异。 两个通道蛋白之间的序列。使用单声道 无细胞贴片的记录，EtOH的浓度依赖性 作用于bmos通道，以及由EtOH修饰的通道特性，将 被确定。将结果与来自多通道的结果进行比较。以来 通道特性与蛋白质中的限定区域相关联， 不同的EtOH改性EtOH改性的具体性质， 这两个克隆将引导我们到蛋白质中的定义区域， EtOH的推定识别位点。然后，研究了乙醇对 构建的突变通道的电生理学特性， 交换这两种蛋白质的非保守区， 基因将证实我们对蛋白质中哪些区域 确定对EtOH的敏感性并确定其差异效应。研究中心- 在两个克隆之间的非保守区中的定向诱变将 解决哪些氨基酸参与BK通道诱变， 两个克隆之间的非保守区将针对哪个氨基酸 酸参与Bk通道-EtOH相互作用。结果BK 克隆将帮助我们专注于特定的通道区域和属性 当评价药物对BK通道的作用时， 在脑动脉细胞中，药物直接产生 血管收缩，可能是由于抑制BK通道。 阐明相互作用的分子机制， 来自动脉的EtOH和BK通道将有助于理解直接 药物对动脉张力的作用，并且，也许，导致 临床上有用的药剂。

Large conductance, Ca++-activated K+ channel (BK) channel activity, involved in the regulation of arterial tone and central neuron excitability, has been found to be modulated by acute ethanol (EtOH) at relevant concentrations. To understand the mechanisms which govern the interaction of EtOH with specific regions in its targets, in particular ion channel proteins, is critical in the alcohol field since, with a few recent exceptions, the specific interaction of EtOH at relevant concentrations with selective regions of a protein, a requirement for a "receptor" from classical pharmacology, has remained largely elusive. We have found that BK channel activity is not potentiated by EtOH in arterial smooth muscle, while it markedly increased in BK channels from nerve terminals and PC12 cells. This dichotomy remained when EtOH action was studied on two 99% identical cloned channels encoded by slo genes, inserted in the same proteo-lipid environment: 50 mM EtOH inhibits channels from arterial smooth muscle (bslo alpha subunit) whereas it activates channels from brain (mslo, alpha subunit) when both are expressed in oocytes. Thus, the main hypothesis is that the differential action of EtOH on these channels is due to specific differences in the sequences between the two channel proteins. Using single channel recordings from cell-free patches, the concentration-dependence of EtOH action on bslo channels, and the channel properties modified by EtOH, will be determined. Results will be compared to those from mslo channels. Since channel properties are linked to defined regions in the proteins, differential EtOH-modification EtOH-modification of specific properties in the two clones will direct us to defined regions in the protein as putative recognition sites for EtOH. Then, the study of EtOH action on electrophysiological properties of mutated channels constructed by exchanging non-conserved regions of these two proteins encoded by slo genes will confirm our predictions of which regions in the protein determine sensitivity to EtOH and underlie its differential effect. Site- directed mutagenesis in non-conserved regions between the two clones will address which amino acids are involved in the BK channel-mutagenesis in non-conserved regions between the two clones will address which amino acids are involved in the Bk channel-EtOH interaction. Results from BK clones will help us to focus on specific channel regions and properties targeted by EtOH when the action of the drug is evaluated on BK channels in cerebral arterial cells, where the drug produces direct vasoconstriction, probably due to an inhibition of BK channels. Elucidating the molecular mechanisms underlying the interaction between EtOH and BK channels from arteries will help in understanding direct actions of the drug on arterial tone, and, perhaps, lead to development of clinically useful agents.