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: 6296736
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6296736
• 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.

加利福尼亚鱼雷的烟碱乙酰胆碱受体