Amino Acid Residues Constituting the Agonist Binding Site of the Human P2X3 Receptor
Amino Acid Residues Constituting the Agonist Binding Site of the Human P2X3 Receptor
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DOI：
10.1074/jbc.m110.167437
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发表时间：
2011-01-28
影响因子：
4.8
通讯作者：
Rubini, Patrizia
中科院分区：
文献类型：
--
作者：
Bodnar, Mandy;Wang, Haihong;Rubini, Patrizia
同源P2 X3受体存在于感觉神经节中并参与疼痛感知。将人（h）P2 X3受体的4个假定的核苷酸结合片段（NBS）中的氨基酸（AA）残基替换为丙氨酸，并在HEK 293细胞和非洲爪蟾卵母细胞中表达。膜片钳和双电极电压钳测量以及钙离子成像技术被用来比较的浓度-反应曲线的选择性P2 X1，3激动剂α，β-亚甲基ATP在野生型P2 X3受体和其NBS突变体。在这些NBS中，某些Gly（Gly-66）、Lys（Lys-63、Lys-176、Lys-284、Lys-299）、Asn（Asn-177、Asn-279）、Arg（Arg-281、Arg-295）和Thr（Thr-172）残基对于完全激动剂反应非常重要。然而，在NBS中用Ala替换进一步的AA也似乎修改了电流和/或[Ca 2 +] i响应的幅度，尽管有时在较小的程度上。在用Ala同时替换两个相邻的AA（K65 A/G66 A、F171 A/T172 A、N279 A/F280 A、F280 A/R281 A）后，激动剂效力降低是累加的，但在用Ala替换同一NBS内的两个不相邻的AA（F171 A/N177 A）后，激动剂效力没有改变。Cy 5细胞表面标记形式的SDS-PAGE证明突变体出现在卵母细胞的细胞表面。因此，在NBS而不是单个氨基酸中组织的AA组似乎负责hP 2X 3受体的激动剂结合。这些NBS位于形成功能性受体的三个亚基的界面处。
Homomeric P2X3 receptors are present in sensory ganglia and participate in pain perception. Amino acid (AA) residues were replaced in the four supposed nucleotide binding segments (NBSs) of the human (h) P2X3 receptor by alanine, and these mutants were expressed in HEK293 cells and Xenopus laevis oocytes. Patch clamp and two-electrode voltage clamp measurements as well as the Ca2+ imaging technique were used to compare the concentration-response curves of the selective P2X1,3 agonist alpha, beta-methylene ATP obtained at the wild-type P2X3 receptor and its NBS mutants. Within these NBSs, certain Gly (Gly-66), Lys (Lys-63, Lys-176, Lys-284, Lys-299), Asn (Asn-177, Asn-279), Arg (Arg-281, Arg-295), and Thr (Thr-172) residues were of great importance for a full agonist response. However, the replacement of further AAs in the NBSs by Ala also appeared to modify the amplitude of the current and/or [Ca2+] i responses, although sometimes to a minor degree. The agonist potency decrease was additive after the simultaneous replacement of two adjacent AAs by Ala (K65A/G66A, F171A/T172A, N279A/F280A, F280A/R281A) but was not altered after Ala substitution of two non-adjacent AAs within the same NBS (F171A/N177A). SDS-PAGE in the Cy5 cell surface-labeled form demonstrated that the mutants appeared at the cell surface in oocytes. Thus, groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the hP2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor.