LIPID INTERACTIONS ALTER ACETYLCHOLINE RECEPTOR

描述(改编自申请人的摘要)：第一种方法将 进一步研究大鼠乙酰胆碱受体(AChR)的分离 雏鸡心肌细胞中胆固醇的增加。在雏鸡的肌细胞中， 胆固醇升高引起两个AchR通道电导(51和39pS)。 一种可行的假说是，胆固醇导致横向时相分离。 会改变胆固醇的局部浓度和相互作用程度 与AChR一起。局部的胆固醇环境施加了变构 对ACHR信道属性的影响。温度对温度的影响 我们将研究胆固醇诱导的乙酰胆碱受体的异质性分布。 将进行胆固醇消耗研究以检查AChR通道 在降低胆固醇水平的膜中发挥作用。全麻药 将用于探测56ps和39ps AChR的疏水环境 通道存在于富含胆固醇的心肌细胞中。 第二种方法是扩展定点突变分析。 鱼雷口径AChR对M3的脂质暴露残留量 跨膜段。M3有类似程度的接触 与M4相比，脂肪含量更高。将对M3给予特别关注 四个亚基(α、a、β、b、Gamma、g和Delta、d)上的位置 相当于之前的M4突变产生了显著的影响 频道选通。苯丙氨酸和色氨酸取代是第一个 由于在以前的M4取代中，这些侧链具有 已被证明在改变离子通道门控方面特别有效。 在非洲爪哇卵母细胞中，将使用电压钳和 膜片钳电生理学。 第三种方法是将非天然氨基酸加入到 使用无意义抑制技术确定脂质暴露位置 脂类暴露残留物与血管紧张素转换酶之间的结构和功能联系 ACHR通道门控(特定目标3)。一个可行的假设是吲哚 偶极矩在对AChR产生这样的影响方面特别有效 频道选通。苯丙氨酸和色氨酸类似物将在 M3和M4跨膜节段的敏感位置以定义 脂类暴露残基与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.