ALPHA CONOTOXIN BINDING SITES ON ACETYLCHOLINE RECEPTOR

加利福尼亚电鳐烟碱型乙酰胆碱受体（AChR） 电子器官的亚单位化学计量为α 2 β γ δ的 它的两个乙酰胆碱结合结构域位于 α-γ和α-δ亚基界面， 对某些竞争性拮抗剂如锥螺的亲和力 称为α-芋螺毒素的毒液衍生肽。 长期 这项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， 在这个项目中进行测试的国家：伽马和三角洲的部分 显示有助于α-芋螺毒素MI结合位点的亚基 在鱼雷AChR上，其中残基是γ K34/δ S36， γ Y111/deltaR 113和γ H172/deltaI 178。 阿尔法的差异- 芋螺毒素的亲和力是由那些 岗位 本项目将联合收割机放射性标记芋螺毒素结合 研究和电生理学研究的细胞系表达 加利福尼亚鱼雷 该项目的具体目标是： A.合成放射性碘标记α-芋螺毒素GI和EI， 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德尔塔的位置。 B。为了表征125 I-的平衡结合和结合动力学， GI和~（125）I-EI对电子器官组织制备的AchR的影响。 C.为了表征125 I-GI和125 I-EI与细胞表达的 天然（α 2 β γ 2和α 2 β γ 2）AchR，使用两种野生型 型和突变的γ和δ亚基。 Gamma/Delta 待研究的交换突变体将包括：γ K34 S，δ S36 K， γ Y111 R、δ R113 Y、γ H172 I和δ I178 H。 D.使用膜片钳方法来表征激动剂结合， 芋螺毒素GI和EI对同一细胞表达的抑制作用 用直接放射性配体结合研究AchR分子。 这项研究应该有助于更好地理解结构上的 类似脊椎动物肌肉AchR和神经肌肉疾病过程 涉及到这个分子。

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.