ALPHA CONOTOXIN BINDING SITES ON ACETYLCHOLINE RECEPTOR

乙酰胆碱受体上的α芋螺毒素结合位点

• 批准号: 6395885
• 负责人: VESNA Ana ETEROVIC
• 金额: $ 13.96万
• 依托单位: UNIVERSIDAD CENTRAL DEL CARIBE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6395885
• 关键词: acetylcholine; alternatives to animals in research; animal poison; calcium channel blockers; cell line; chemical binding; chimeric proteins; electrodes; electrophysiology; fish electric organ; neuromuscular blocking agents; neuromuscular function; neurotoxins; nicotinic receptors; protein structure function; radionuclides; receptor binding; receptor expression; voltage /patch clamp

The nicotinic acetylcholine receptor (AChR) from Torpedo californica electric organ has a subunit stoichiometry of alpha2 beta gamma delta. Its two acetylcholine-binding domains are located at the alpha gamma and alpha delta subunit interfaces and display differing affinities for certain competitive antagonists such as the cone snail venom-derived peptides known as alpha-conotoxins. The long-term objective of this research is to better understand the molecular structure of these alpha-conotoxin binding sites. The hypothesis to be tested in this project states: The segments of the gamma and delta subunits shown to contribute to the alpha-conotoxin MI binding sites on the Torpedo AChR where the residues are gamma K34/deltaS36, gammaY111/deltaR113 and gammaH172/deltaI178. Differences in alpha- conotoxin affinities result from residue differences at those positions. This project will combine radiolabelled conotoxin binding studies and electrophysiological studies on a cell line expressing Torpedo californica AchR. The specific aims of the project are: A. To synthesize radioiodinated alpha-conotoxins GI and EI to be utilized in direct binding studies as two novel ligands specific for the alpha gamma and alpha delta sites, respectively. B. To characterize equilibrium binding and binding kinetics of 125I- GI and 125I-EI to AchR prepared fromelectric organ tissue. C. To characterize binding of 125I-GI and 125I-EI to cell-expressed native (alpha2 betagamma2 and alpha2 betagamma2) AchR using both wild type and mutant gamma and delta subunits. The gamma/delta interchange mutants to be studied will include: gammaK34S, deltaS36K, gammaY111R, deltaR113Y, gammaH172I and deltaI178H. D. To use patch-clamp methodology to characterize agonist binding and its inhibition by conotoxins GI and EI to the same cell-expressed AchR molecules to be studied with direct radioligand binding. This research should lad to a better understanding of the sructurally similar vertebrate muscle AchR and of neuromuscular disease processes involving this molecule.

来自Torpedo Californica的烟碱乙酰胆碱受体（ACHR） 电气器官具有α2β伽玛的亚基化学计量法 三角洲。 它的两个乙酰胆碱结合域位于 Alpha Gamma和Alpha Delta亚基界面和显示不同的 某些竞争性拮抗剂（例如锥蜗牛）的亲和力 毒液衍生的肽称为α-抗毒素。 长期 这项研究的目的是更好地了解分子 这些α-抗毒素结合位点的结构。 假设 在此项目中进行测试：伽马和三角洲的细分市场 显示有助于α-抗毒素MI结合位点的亚基 在鱼雷ACHR上，残留物为gamma k34/deltas36， gammay111/deltar113和gammah172/deltai178。 α-的差异 结合毒素亲和力是由于残留的差异而导致的 位置。 该项目将结合放射性标记的结合毒素结合 对表达细胞系的研究和电生理研究 鱼雷加利福尼亚ACHR。 该项目的具体目的是： 答：合成放射性施工的α-偶联毒素GI和EI为 在直接结合研究中用作两个特有的新型配体 α伽马和阿尔法三角洲站点分别。 B.表征125i的平衡结合和结合动力学 gi和125i-ei到ACHR，制备了电源器官组织。 C.表征125i-GI和125I-EI与细胞表达的结合 使用两个野生 类型和突变伽玛和三角洲亚基。 伽玛/三角洲 要研究的互换突变体将包括：gammak34s，deltas36k， gammay111r，deltar113y，gammah172i和deltai178h。 D.使用斑块钳方法来表征激动剂结合和 蛋白毒素GI和EI对同一细胞表达的抑制作用 用直接放射性结合研究的ACHR分子。 这项研究应该在更好地理解s ructurural 类似的脊椎动物肌肉ACHR和神经肌肉疾病过程 涉及该分子。