Ligand Binding Domains in the 5-HT3 Receptor

描述（由申请人提供）：5-羟色胺3 （5-HT3）受体可能由A亚基的同型异构体或A和B亚基的异型异构体组成，是配体门控离子通道中烟碱乙酰胆碱受体家族中研究最少的成员。它除了在神经系统中介导突触传递外，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已经确定了该超家族n端结构域的一般结构，但配体识别位点的精确三维构型以及介导离子通道激活的残基尚不清楚。本提案的目标是绘制有助于配体识别的氨基酸残基并模拟其空间构型。小鼠和人类5-HT3A受体在氨基酸水平上具有84%的一致性，但对结合配体识别位点的许多药物具有不同的敏感性，例如d-管curare （curare）和3-（2-羟基，4-甲氧基苄基）-anabaseine （2-OHMBA）。在A亚基上加入B亚基进一步改变了这些化合物的效力。鼠-人嵌合体和人-鼠嵌合体将在非洲爪蟾卵细胞中构建和表达。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。然后将确定单个氨基酸在赋予敏感性方面的作用。在取代的3-苄基苯基碱基类似物中，关键部分的结构-活性关系将被评估。热力学突变循环分析将精确定位关键药物片段与配体结合域中鉴定的氨基酸残基的具体接触点。带c端六组氨酸标签的5-HT3A受体将被表达和纯化。然后用[3H]-5-HT和[14C]-2-OHMBA（带或不带叠氮侧链）对受体进行光标记，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-HT、curare和苄基苯胺-苯基苯胺类似物的结构，将作为激动剂识别位点分子模型研究的模板。

DESCRIPTION (provided by applicant): 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 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 percent identity at the amino acid level, yet have differential sensitivities to 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 potency 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-benzylideneanabaseine 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-HT3A receptors 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.