Identification of functionally important residues of the rat P2X4 receptor by alanine scanning mutagenesis of the dorsal fin and left flipper domains.
Identification of functionally important residues of the rat P2X4 receptor by alanine scanning mutagenesis of the dorsal fin and left flipper domains.
复制标题
DOI：
10.1371/journal.pone.0112902
复制
发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Zemkova H
中科院分区：
文献类型：
--
作者：
Tvrdonova V;Rokic MB;Stojilkovic SS;Zemkova H
斑马鱼 P2X4 受体在打开和关闭状态下的结晶揭示了胞外域结构的构象差异，包括背鳍和左鳍结构域。在这里，我们重点关注这些结构域在受体激活、对正位 ATP 类似物激动剂的反应和脱敏中的作用。大鼠 P2X4 受体的 R203-L214（背鳍）和 D280-N293（左鳍）序列的丙氨酸扫描诱变表明，26 个丙氨酸突变体中有 15 个的 ATP 效力/功效降低。 R203A、N204A 和 N293A 突变体基本上没有功能，但受体功能被变构调节剂伊维菌素恢复。 I205A、T210A、L214A、P290A、G291A 和 Y292A 突变体对正位类似物激动剂 2-(甲硫基)腺苷 5'-三磷酸、腺苷 5'-(γ-硫代)三磷酸、 2'(3'-O-(4-苯甲酰基苯甲酰基)腺苷5'-三磷酸和α,β-亚甲基腺苷5'-三磷酸。相比之下，L206A、N208A、D280A、T281A、R282A和H286A突变体对类似物激动剂的反应性与野生型相当 受体。在这些突变体中，D280A、T281A、R282A、H286A、G291A和Y292A也表现出脱敏电流响应的时间常数增加。这些实验与同源建模一起表明，位于背鳍和左鳍结构域上部的残基，相对于距通道孔的距离，有助于 ATP 结合袋的组织 并启动向两个结构域下部残基的信号传输。深埋在蛋白质中的 R203 和 N204 残基可以将这两个结构域的输出信号整合到门上。此外，左鳍状肢残基主要负责控制通道从开放状态到脱敏状态的转变。
Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. Here, we focused on the role of these domains in receptor activation, responsiveness to orthosteric ATP analogue agonists, and desensitization. Alanine scanning mutagenesis of the R203-L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor showed that ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists 2-(methylthio)adenosine 5′-triphosphate, adenosine 5′-(γ-thio)triphosphate, 2′(3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate, and α,β-methyleneadenosine 5′-triphosphate. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. Among these mutants, D280A, T281A, R282A, H286A, G291A, and Y292A also exhibited increased time-constant of the desensitizing current response. These experiments, together with homology modeling, indicate that residues located in the upper part of the dorsal fin and left flipper domains, relative to distance from the channel pore, contribute to the organization of the ATP binding pocket and to the initiation of signal transmission towards residues in the lower part of both domains. The R203 and N204 residues, deeply buried in the protein, may integrate the output signal from these two domains towards the gate. In addition, the left flipper residues predominantly account for the control of transition of channels from an open to a desensitized state.
登录
查看更多内容
影响因子：
4.7
作者：
Digby, HR;Roberts, JA;Evans, RJ
通讯作者：
Evans, RJ
影响因子：
4.8
作者：
Ennion, S;Hagan, S;Evans, RJ
通讯作者：
Evans, RJ
DOI：
10.1073/pnas.1102170108
发表时间：
2011-05-31
影响因子：
11.1
作者：
Jiang, Ruotian;Lemoine, Damien;Grutter, Thomas
通讯作者：
Grutter, Thomas
影响因子：
4.8
作者：
Adriouch, Sahil;Bannas, Peter;Koch-Nolte, Friedrich
通讯作者：
Koch-Nolte, Friedrich
影响因子：
5.3
作者：
Haines, WR;Voigt, MM;Egan, TM
通讯作者：
Egan, TM