ALPHA CONOTOXIN BINDING SITES ON NICOTINIC ACH RECEPTOR

烟碱 ACH 受体上的α芋螺毒素结合位点

• 批准号: 6564579
• 负责人: VESNA Ana ETEROVIC
• 金额: $ 14.53万
• 依托单位: UNIVERSIDAD CENTRAL DEL CARIBE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-15 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564579
• 关键词: acetylcholine; alternatives to animals in research; aminoacid; binding sites; calcium channel blockers; cell line; chemical binding; chemical structure; chimeric proteins; conotoxin; electrodes; electrophysiology; fish electric organ; marine toxins; neuromuscular blocking agents; neuromuscular function; neurotoxins; nicotinic receptors; protein purification; protein structure function; radionuclides; receptor binding; receptor expression; site directed mutagenesis; voltage /patch clamp

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.

α-芋螺毒素 EI 是已知的唯一一种肽，它与电器官乙酰胆碱受体 (AChR) αδ 亚基界面上的激动剂位点的结合亲和力比与 αγ 界面上其他激动剂位点的结合亲和力高得多。该研究项目的目的是确定 δ 亚基上的哪些氨基酸和 EI 上的哪些氨基酸相互作用以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) 通过制备 deltaamma 和 gammadelta 嵌合体以及 delta 和 gamma 点突变体来鉴定 Delta 链上的 EI 高亲和力决定残基，并测量当它们与野生型 ACHR 亚基在细胞中共表达时 EI 高亲和力的损失或增加，2) 通过制作 EI 来鉴定 EI 上的高亲和力决定残基 替代非半胱氨酸位置的丙氨酸的类似物，并观察这如何影响野生型 ACHR 的肽亲和力，以及 3) 通过使用双突变体循环分析来测试目标 1 和 2 中鉴定的高亲和力决定残基对是否彼此相互作用。肽亲和力将根据其与AChR特异性放射性配体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该受体的结构以及α-芋螺毒素如何与其结合。这应该有助于更好地了解同源人类受体，例如肌肉和神经元 AChR，以及它们在各种神经系统疾病过程中的参与。像 EI 这样的α-芋螺毒素显示出作为潜在的神经肌肉和神经元阻断剂以及作为有助于开发此类药物的位点特异性探针的临床前景。