Ligand Binding Domains in the 5-HT3 Receptor

超出提供的空间。5-羟色胺3(5-HT3)受体是配体门控离子通道烟碱型乙酰胆碱受体家族中研究最少的成员，它可以由A亚基的同质异构体或A、B亚基的异构体组成。除了在神经系统中调节突触传递的作用外，它还调节胃肠运动和呕吐反射。最近发表的乙酰胆碱结合蛋白的晶体结构(Brejc等人，2001)已经确定了这个超家族N-末端结构域的一般结构，但参与介导离子通道激活的配体识别位点和残基的精确三维构型尚不清楚。这项建议的目标是绘制有助于配体识别的氨基酸残基图并对其空间构型进行建模。小鼠和人的5-HT3A受体在氨基酸水平上有84%的同源性，但对许多与配体识别位点结合的药物有不同的敏感性，如d-管胡萝卜碱(Curare)和3-(2-羟基，4-甲氧基亚甲基)-花生碱(2-OHMBA)。B亚基与A亚基的结合进一步改变了这些化合物的效价。将构建鼠-人和人-鼠嵌合体，并在非洲爪哇卵母细胞中表达。负责药物作用变化的区域将通过双电极电压钳电生理记录进行评估。然后，将确定个别氨基酸在赋予敏感性方面的作用。将评估取代的3-亚甲基-花生碱类似物中关键部分与已确定残基的结构-活性关系。热力学突变环分析将确定关键药物部分与配体结合域中已识别的氨基酸残基的特定接触点。带有C-末端六组氨酸标签的5-HTaA受体将被表达和纯化。然后用[~3H]-5-HT和[14C]-2-OHMBA对受体进行光标记，有无叠氮侧链，并通过测序鉴定放射性标记残基。通过光亲和标记和电生理实验鉴定的氨基酸，以及5-羟色胺、咖喱和亚苄叉麻黄碱类似物的结构，将作为激动剂识别位点的分子模拟研究的模板。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. The 5-Hydroxytryptamine3 (5-HT3) receptor, which may be composed of homomers of A subunits or heteromers of A and B subunits, is the least studied member of the nicotinic acetylcholine receptor family of ligand-gated ion channels. In addition to its role in mediating synaptic transmission in the nervous system, it also regulates gastrointestinal motility and the vomiting reflex. The recently published crystal structure of the :acetylcholine binding protein (Brejc et al., 2001) has identified the generalized structure of the N-terminal domains of this superfamily, but the precise three-dimensional configuration of the ligand recognition site and the residues involved in mediating ion channel activation are unknown. The goal of this proposal is to map amino acid residues that contribute to ligand recognition and model their spatial configuration. Mouse and human 5-HT3A receptors possess 84% identity at the amino acid level, yet have differential sensitivities to i numerous drugs that bind to the ligand recognition site, such as d-tubocurarine (curare) and 3-(2-hydroxy, 4- methoxy-benzylidene)-anabaseine (2-OHMBA). The addition of the B subunit to the A subunit further alters ipotency of these compounds. Mouse-human and human-mouse chimeras will be constructed and expressed in Xenopus oocytes. Domains responsible for the change in drug action will be assessed with two-electrode voltage clamp electrophysiological recordings. Individual amino acids will then be identified for their roles in conferring sensitivity. Structure-activity relationships of key moieties in the substituted 3-benzylidene- anabaseine analogs with identified residues will be assessed. Thermodynamic mutant cycle analysis will pinpoint the specific point of contact of the key drug moiety with the identified amino acid residue in the ligand binding domain. 5-HTaAreceptors with C-terminal hexa-histidine tags will be expressed and purified. Receptor will then be photolabeled with [3H]-5-HT and [14C]-2-OHMBA, with and without azido side-chains, and radiolabeled residues will be identified through sequencing. Amino acids identified with photoaffinity labeling and electrophysiological experiments, along with the structures of 5-HT, curare, and benzylidene-anabaseine analogs, will serve as a template for molecular modeling studies of the agonist-recognition site. PERFORMANCE SITE ========================================Section End===========================================