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: 6271807
• 负责人: VESNA Ana ETEROVIC
• 金额: $ 7.99万
• 依托单位: UNIVERSIDAD CENTRAL DEL CARIBE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6271807
• 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.

来自加州鱼雷的烟碱乙酰胆碱受体 (AChR) 电器官的亚基化学计量为 alpha2 beta gamma 三角洲。 它的两个乙酰胆碱结合域位于 alpha gamma 和 alpha delta 子单元接口和显示不同 对某些竞争性拮抗剂（如锥螺）的亲和力 毒液衍生的肽，称为α-芋螺毒素。 长期来看 本研究的目的是更好地了解分子 这些α-芋螺毒素结合位点的结构。 假设为 在此项目中进行测试的状态：gamma 和 delta 的段 显示有助于 α-芋螺毒素 MI 结合位点的亚基 在鱼雷 AChR 上，其中残基为 gamma K34/deltaS36， gammaY111/deltaR113 和 gammaH172/deltaI178。 α 的差异 芋螺毒素亲和力是由这些残留物的差异造成的 职位。 该项目将结合放射性标记的芋螺毒素结合 对表达细胞系的研究和电生理学研究 加州鱼雷 AchR。 该项目的具体目标是： A. 合成放射性碘标记的 α-芋螺毒素 GI 和 EI 在直接结合研究中用作两种特异性的新型配体 分别是αγ和αδ位点。 B. 表征 125I- 的平衡结合和结合动力学 从电器官组织制备的 GI 和 125I-EI 到 AchR。 C. 表征 125I-GI 和 125I-EI 与细胞表达的结合 使用野生的天然（alpha2 betaamma2 和 alpha2 betaamma2）AchR 型和突变型γ和δ亚基。 伽玛/德尔塔 要研究的互换突变体包括：gammaK34S、deltaS36K、 gammaY111R、deltaR113Y、gammaH172I 和 deltaI178H。 D. 使用膜片钳方法来表征激动剂结合和 芋螺毒素 GI 和 EI 对同一细胞表达的抑制作用 通过直接放射性配体结合来研究 AchR 分子。 这项研究应该有助于我们更好地理解结构 类似的脊椎动物肌肉 AchR 和神经肌肉疾病过程 涉及到这个分子。