LIPID INTERACTIONS ALTER ACETYLCHOLINE RECEPTOR

脂质相互作用改变乙酰胆碱受体

• 批准号: 2385688
• 负责人: JOSE Antonio LASALDE-DOMINICCI
• 金额: $ 17.69万
• 依托单位: UNIVERSITY OF PUERTO RICO RIO PIEDRAS
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2385688
• 关键词: LIPID; INTERACTIONS; ALTER; ACETYLCHOLINE; RECEPTOR

DESCRIPTION (Adapted from applicant's abstract): The first approach will examine further the segregation of acetylcholine receptors (AChR) induced by cholesterol enrichment in chick myocytes. In the chick myocyte, the cholesterol enrichment induced two ACHR channel conductances (51 & 39 pS). A working hypothesis is that cholesterol induces lateral phase separations that alter the local concentration and degree of interaction of cholesterol with the AChR. The local cholesterol environment exerts an allosteric effect on the ACHR channel properties. The effect of temperature on the heterogeneous distribution of AChRs induced by cholesterol will be examined. Cholesterol depletion studies will be performed to examine AChR channel function in a membrane with reduced cholesterol levels. General anesthetics will be used to probe the hydrophobic environment of the 56 and 39 pS AChR channel in the cholesterol enriched myocyte. The second approach is to extend the site-directed mutagenesis analysis of the lipid exposed residues of the Torpedo californica AChR to the M3 transmembrane segment. The M3 has a similar degree of contact with the lipid when compared to the M4. Particular attention will be given to M3 positions on the four subunits (alpha, a, beta, b, gamma, g, and delta, d) equivalent to the previous M4 mutations that produced significant effects on channel gating. Phenylalaline and tryptophan substitutions are the first to be introduced since in previous M4 substitutions, these side chains have been shown to be especially effective in altering ion channel gating. Mutants will be examined in Xenopus laevis oocytes using voltage-clamp and patch clamp electrophysiology. The third approach to be used is to incorporate unnatural amino acids into lipid exposed positions using the nonsense suppressor technique to define the structural and functional linkage between lipid exposed residues and the AChR channel gating (specific aim 3). A working hypothesis is that indole dipole moments are specially effective in producing such effects on the AChR channel gating. Phenylalanine and tryptophan analogs will be introduced at sensitive positions of the M3 and M4 transmembrane segments to define the structural linkage between lipid exposed residues and AChR channel function.

描述（改编自申请人的摘要）：第一种方法将 进一步检查乙酰胆碱受体（AChR）诱导的分离 鸡肌细胞中胆固醇富集。 在鸡肌细胞中， 胆固醇富集诱导两个 ACHR 通道电导（51 和 39 pS）。 一个可行的假设是胆固醇引起横向相分离 改变胆固醇的局部浓度和相互作用程度 与乙酰胆碱受体。 局部胆固醇环境发挥变构作用 对 ACHR 通道属性的影响。 温度对的影响 将检查胆固醇诱导的 AChR 的异质分布。 将进行胆固醇消耗研究以检查 AChR 通道 在膜中发挥作用，降低胆固醇水平。 全身麻醉药 将用于探测 56 和 39 pS AChR 的疏水环境 富含胆固醇的肌细胞中的通道。 第二种方法是扩展定点突变分析 加州鱼雷 AChR 与 M3 的脂质暴露残基 跨膜片段。 M3 与 M3 具有类似的接触程度 与 M4 相比，脂质。 M3将受到特别关注 四个亚基上的位置（alpha、a、beta、b、gamma、g 和 delta、d） 相当于之前的 M4 突变，对 通道选通。 苯丙氨酸和色氨酸取代是第一个 由于在之前的 M4 替代中引入，这些侧链已经 已被证明在改变离子通道门控方面特别有效。 将使用电压钳和电压钳在非洲爪蟾卵母细胞中检查突变体 膜片钳电生理学。 第三种方法是将非天然氨基酸掺入 使用无意义抑制技术来定义脂质暴露位置 暴露于脂质的残基和 AChR 通道门控（具体目标 3）。 一个可行的假设是吲哚 偶极矩对 AChR 产生这种效应特别有效 通道选通。 苯丙氨酸和色氨酸类似物将在 M3 和 M4 跨膜片段的敏感位置来定义 脂质暴露残基和 AChR 通道功能之间的结构联系。