ALPHA CONOTOXIN BINDING SITES ON ACETYLCHOLINE RECEPTOR

加州鱼雷的烟碱型乙酰胆碱受体(AChR) 电子器官的亚单位化学计量比为α2β伽马 达美航空。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 某些竞争性对手的亲和力，如锥螺 蛇毒衍生的多肽被称为α-芋螺毒素。长期的 这项研究的目的是更好地了解分子 这些α-芋螺毒素结合部位的结构。假设是为了 在这个项目中被测试的状态：伽马和三角洲的片段 与α-芋螺毒素MI结合位点有关的亚基 在残基为伽马K34/增量S36的鱼雷AChR上， GammaY111/deltaR113和GammaH172/deltaI178。阿尔法的差异- 芋螺毒素的亲和力是由于它们之间的残基差异 各就各位。该项目将结合放射性标记的芋螺毒素结合 一株表达基因的细胞系的研究和电生理研究 鱼雷卡利福尼卡ACHR。该项目的具体目标是： A.合成放射性碘化α-芋螺毒素GI和EI为 在直接结合研究中用作两种新的特异性配体 分别是阿尔法伽马和阿尔法三角洲地点。 B.表征~(125)I-的平衡结合和结合动力学 GI和~(125)I-EI对AchR的抑制作用。 C.鉴定125I-GI和125I-EI与细胞表达的结合 原生(Alpha2 Betagamma2和Alpha2 Betagamma2)Achr 类型和突变的伽马和德尔塔亚基。伽马/三角洲 待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 用直接放射性配基结合来研究ACHR分子。 这项研究应该有助于更好地理解 类似脊椎动物的肌肉ACHR和神经肌肉疾病的过程 涉及到这种分子。

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.