EFFECT OF ETHANOL AND GENERAL ANESTHETICS ON PROTEIN KINASE C IN BRAIN

乙醇和全身麻醉药对脑内蛋白激酶 C 的影响

• 批准号: 6267082
• 负责人: CHRISTOPHER D STUBBS
• 金额: $ 13.07万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6267082
• 关键词: SDS polyacrylamide gel electrophoresis; anesthetics; biological signal transduction; brain; chimeric proteins; enzyme activity; enzyme inhibitors; enzyme structure; ethanol; fluorescent dye /probe; general anesthesia; laboratory rat; lipid bilayer membrane; membrane activity; membrane lipids; membrane model; membrane proteins; molecular site; nuclear magnetic resonance spectroscopy; physical chemical interaction; polymerase chain reaction; protein binding; protein kinase C; protein purification; synapses; tissue /cell culture

Protein kinase C [PKC] is a key regulatory element in synaptic signal transduction. During the previous period of support it was found that alcohols and general anesthetics inhibit PKC with a potency that is a linear function of the octanol/buffer partition coefficient indicating a hydrophobic binding site. The effect follows the Meyer-Overton "rule" of alcohol and anesthetic action which requires that anesthetic effects be a linear function of hydrophobicity. The measurements were made in the absence of membrane lipids suggesting the site is on the molecule itself. This observation has also been obtained at the single isoform level with purified recombinant PKCalpha. Evidence obtained so far points to the phorbol ester binding site as the site of alcohol interaction. While membrane lipid-associated PKCalpha is also inhibited by short chain alcohols, by contrast, long chain alcohols and general anesthetics potentiate activity. To further understand the effects of alcohols and general anesthetics on PKC and its role in ethanol intoxication and general anesthesia it is proposed to extend and broaden the study to encompass the three major PKC isoforms classes since they possess widely differing properties. Also since PKC has been found to target cytoskeletal elements involved in neurotransmission, we propose to investigate this process following preliminary findings showing ethanol effects. To accomplish this the cDNA encoding the major PKC isoforms that occur in brain, has been obtained and inserted into baculovirus vectors and the PKC expressed in Sf9 cells and purified. It is proposed to investigate the effect of alcohols with respect to activator-and substrate-type differences that have been observed in preliminary experiments. The site of alcohol interaction within PKC and its molecular details will be determined using a series of full length and fusion-protein mutants along with the effects of alcohols on the increased proteolytic sensitivity of activated-PKC that leads to down regulation. Overall the results should contribute to understanding of the mechanism and consequences of ethanol and general anesthetics on synaptic signaling and neurotransmission.

蛋白激酶 C [PKC] 是突触信号的关键调节元件 转导。在之前的支持期间发现 酒精和全身麻醉剂会抑制 PKC，其效力是 辛醇/缓冲液分配系数的线性函数表示 疏水性结合位点。该效果遵循迈耶-奥弗顿“规则”： 酒精和麻醉作用，要求麻醉效果是 疏水性的线性函数。测量是在 不存在膜脂表明该位点位于分子本身上。 这一观察结果也在单一亚型水平上获得 纯化的重组 PKCα。迄今为止获得的证据表明 佛波酯结合位点作为醇相互作用的位点。尽管 膜脂相关的 PKCα 也受到短链的抑制 相比之下，醇类、长链醇和全身麻醉剂 增强活性。为了进一步了解酒精的影响 全身麻醉剂对 PKC 及其在乙醇中毒和酒精中毒中的作用 全身麻醉 建议将研究延伸和扩大到 涵盖三个主要的 PKC 同工型类别，因为它们具有广泛的 不同的属性。此外，由于 PKC 被发现可以靶向细胞骨架 涉及神经传递的元素，我们建议对此进行研究 根据显示乙醇影响的初步结果进行的过程。到 实现这一目标的是编码主要 PKC 同种型的 cDNA 脑，已获得并插入杆状病毒载体和 PKC 在Sf9细胞中表达并纯化。建议调查 醇对活化剂和底物类型的影响 初步实验中观察到的差异。现场 PKC 内的酒精相互作用及其分子细节将是 使用一系列全长和融合蛋白突变体来确定 酒精对蛋白水解敏感性增加的影响 激活的 PKC 导致下调。总体结果应该 有助于理解乙醇的机制和后果 以及对突触信号传导和神经传递的全身麻醉剂。