LIPID INTERACTIONS ALTER ACETYLCHOLINE RECEPTOR

描述（改编自申请人的摘要）：第一种方法将 进一步研究乙酰胆碱受体（AChR）的分离诱导 鸡心肌细胞胆固醇富集。 在鸡心肌细胞中， 胆固醇富集诱导两个ACHR通道电导（51和39pS）。 一个工作假设是胆固醇诱导横向相分离 改变胆固醇的局部浓度和相互作用程度 与AChR 局部胆固醇环境发挥变构作用， 对ACHR通道特性的影响。 温度对 将检查由胆固醇诱导的AChR的不均匀分布。 将进行胆固醇耗竭研究，以检查AChR通道 胆固醇水平降低的膜功能。 全身麻醉药 将用于探测56和39 pS AChR的疏水环境 通道中的胆固醇丰富的心肌细胞。 第二种方法是扩展定点突变分析， 加州电鳐AChR的脂质暴露残基与M3 跨膜片段 M3的接触程度与 与M4相比， 将特别关注M3 四个亚基（α、a、β、B、γ、g和δ、d）上的位置 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 由于在先前的M4取代中，这些侧链具有 已显示在改变离子通道门控方面特别有效。 突变体将在非洲爪蟾卵母细胞中使用电压钳和 膜片钳电生理学 使用的第三种方法是将非天然氨基酸掺入 脂质暴露位置使用无义抑制技术来定义 暴露于脂质的残基与脂质之间的结构和功能连接 AChR通道门控（具体目标3）。 一个工作假设是，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，以确定 脂质暴露残基与AChR通道功能之间的结构联系。

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.