ALPHA CONOTOXIN BINDING SITES ON NICOTINIC ACH RECEPTOR

α - conotoxin EI是已知的唯一一种与电器官乙酰胆碱受体（AChR）的α δ亚基界面上的激动剂位点结合的亲和力远高于α γ界面上的其他激动剂位点的肽。本研究项目的目的是确定delta亚基和EI上的哪些氨基酸相互作用产生这种独特的选择性。实现这一目标的三个具体目标是：1)通过制造delta和gamma嵌合体以及delta和gamma点突变体，并测量EI高亲和力在细胞中与野生型ACHR亚基共表达时的损失或获得，来识别EI高亲和力的δ链决定残基；2)通过制作EI类似物替代非半胱氨酸位置的丙氨酸来鉴定EI上的高亲和力决定残基，并观察其如何影响野生型ACHR的肽亲和力；3)通过双突变循环分析来检验目的1和目的2中鉴定的高亲和力决定残基对是否相互作用。肽的亲和力将通过其与achr特异性放射配体125i - α - bungarotoxin的竞争来估计。这些研究将使我们更清楚地了解这种受体的结构以及α -conotoxins是如何与它结合的。这将有助于更好地了解人类同源受体，如肌肉和神经元achr，以及它们在各种神经系统疾病过程中的作用。像EI这样的α - conotoxins作为潜在的神经肌肉和神经元阻断剂以及作为帮助开发此类药物的部位特异性探针具有临床前景。

Alpha-Conotoxin EI is the only peptide known to bind with much higher affinity to the agonist site at the alphadelta subunit interface of the electric organ acetylcholine receptor (AChR) than to the other agonist site of the alphagamma interface. The objective of this research proj4ect is to determine which amino acids on the delta subunit and on EI interact to produce this unique selectivity. The three specific aims to achieve this are 1) to identify the EI high-affinity determinant residues on the delta chain by making deltagamma and gammadelta chimeras and delta and gamma point mutants and measuring the loss or gain of EI high affinity when these are co-expressed in cells along with the wild-type ACHR subunits, 2) to identify the high-affinity determinant residues on EI by making EI analogues which substitute an alanine in which non-cysteine position and observing how this affects peptide affinity for wild-type ACHR and 3) to test if the high-affinity determinant residue pairs identified in Aims 1 and 2 interact with each other by using double- mutant cycle analysis. Peptide affinity will be estimated from its competition with the AChR-specific radioligand, 125I-alpha- bungarotoxin. These studies will give a clearer understanding of the structure of this receptor and how alpha-conotoxins bind to it. This should lead to a better understanding of homologous human receptors, such as the muscle and neuronal AChRs, and their involvement in various neurological disease processes. Alpha-Conotoxins like EI show clinical promise as potential neuromuscular and neuronal blocking agents and as site-specific probes to aid in the development of such agents.