Thompson Andrew J, Padgett Claire L, Lummis Sarah C R
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.
J Biol Chem. 2006 Jun 16;281(24):16576-82. doi: 10.1074/jbc.M601265200. Epub 2006 Apr 4.
The 5-HT(3) receptor is a member of the Cys-loop family of ligand-gated ion channels. The extracellular domains of these proteins contain six amino acid loops (A-F) that converge to form the ligand binding site. In this study we have mutated 21 residues in or close to the 5-HT(3) receptor F-loop (Ile(192) to Gly(212)) to Ala or to a residue with similar chemical properties. Mutant receptors were expressed in HEK293 cells, and binding affinity was measured using [(3)H]granisetron. Two regions displayed decreases in binding affinity when mutated to Ala (Ile(192)-Arg(196) and Asp(204)-Ser(206)), but only one region was sensitive when mutated to chemically similar residues (Ile(192)-Val(201)). Homology modeling using acetylcholine-binding protein crystal structures with a variety of different bound ligands suggests there may be distinct movements of Trp(195) and Asp(204) upon ligand binding, indicating that these residues and their immediate neighbors have the ability to interact differently with different ligands. The models suggest predominantly lateral movement around Asp(204) and rotational movement around Trp(195), indicating the former is in a more flexible region. Overall our results are consistent with a flexible 5-HT(3) receptor F-loop with two regions that have specific but distinct roles in ligand binding.
5-羟色胺（3）受体是半胱氨酸环家族配体门控离子通道的成员。这些蛋白质的细胞外结构域包含六个氨基酸环（A-F），它们汇聚形成配体结合位点。在本研究中，我们将5-羟色胺（3）受体F环（Ile（192）至Gly（212））内或其附近的21个残基突变为丙氨酸或具有相似化学性质的残基。突变受体在HEK293细胞中表达，并使用[³H]格拉司琼测量结合亲和力。当突变为丙氨酸时，两个区域的结合亲和力下降（Ile（192）-Arg（196）和Asp（204）-Ser（206）），但当突变为化学性质相似的残基时，只有一个区域敏感（Ile（192）-Val（201））。使用结合了各种不同配体的乙酰胆碱结合蛋白晶体结构进行同源建模表明，配体结合后Trp（195）和Asp（204）可能会有不同的移动，这表明这些残基及其紧邻的残基与不同配体相互作用的方式可能不同。模型表明，主要是围绕Asp（204）的横向移动和围绕Trp（195）的旋转移动，这表明前者处于一个更灵活的区域。总体而言，我们的结果与灵活的5-羟色胺（3）受体F环一致，该环有两个在配体结合中具有特定但不同作用的区域。
