ALPHA CONOTOXIN BINDING SITES ON NICOTINIC ACH RECEPTOR

α-芋螺毒素EI是目前已知的唯一与电器官乙酰胆碱受体(AChR)α-β亚基界面激动剂部位结合的多肽，其亲和力远高于α-乙酰胆碱受体(AChR)另一个激动剂部位。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来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)通过使delagamma和Gammadelta嵌合体以及Delta和Gamma点突变并测量当这些EI与野生型AchR亚单位在细胞中共同表达时，EI高亲和力决定簇的损失或获得，以鉴定三角洲链上的EI高亲和力决定簇；2)通过制作取代非半胱氨酸位置的丙氨酸的EI类似物，并观察这对野生型AchR的多肽亲和力的影响，来鉴定EI上的高亲和力决定簇残基；3)通过双突变循环分析来测试AIMS 1和2中确定的高亲和力决定簇残基对是否相互作用。多肽亲和力将通过它与AChR特异性放射性配基125I-α-银环蛇毒素的竞争来估计。这些研究将使人们更清楚地了解这种受体的结构，以及α-芋螺毒素是如何与其结合的。这应该会导致更好地了解同源人类受体，如肌肉和神经元AChRs，以及它们参与各种神经疾病过程。像EI这样的甲芋毒素显示出作为潜在的神经肌肉和神经元阻滞剂的临床前景，以及作为位置特异性探针来帮助此类药物的开发。

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.