On the contribution of the first transmembrane domain to whole-cell current through an ATP-gated ionotropic P2X receptor
On the contribution of the first transmembrane domain to whole-cell current through an ATP-gated ionotropic P2X receptor
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DOI：
10.1523/jneurosci.21-16-05885.2001
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发表时间：
2001-08-15
影响因子：
5.3
通讯作者：
Egan, TM
中科院分区：
文献类型：
--
作者：
Haines, WR;Voigt, MM;Egan, TM
扫描半胱氨酸诱变用于识别离子型 P2X(2) 受体亚基的第一个跨膜结构域内和周围的潜在成孔残基。通过脂转染在 HEK293 细胞中单独表达 28 个独特的半胱氨酸取代突变体 (R28C-Y55C)。通过将 ATP 应用于转染的电压钳制细胞来检测，其中 23 个具有功能。各个突变体对 ATP 的敏感性各不相同；除此之外，通过功能性突变受体的电流与同聚野生型（WT）受体的电流相似。在 23 个功能突变体中的 5 个（H33C、R34C、I50C、K53C 和 S54C）中，共同应用 30 muM ATP 和 500 nM Ag+ 不可逆地抑制随后单独应用 ATP 引起的内向电流。这些抑制不会导致激动剂浓度-反应曲线的横向移动，并且不太可能涉及激动剂结合位点的修饰。当通道门控时，在存在 ATP 的情况下应用 Ag+ 修饰的五个残基中的两个（K53C 和 S54C）也通过在通道关闭时不存在 ATP 的情况下应用的 1 mM (2-氨乙基)甲硫磺酸盐进行修饰。这些数据表明，靠近第一个跨膜结构域两端的结构域影响通过 P2X(2) 受体孔的离子传导。
Scanning cysteine mutagenesis was used to identify potential pore-forming residues in and around the first transmembrane domains of ionotropic P2X(2) receptor subunits. Twenty-eight unique cysteine-substituted mutants (R28C-Y55C) were individually expressed in HEK293 cells by lipofection. Twenty-three of these were functional as assayed by application of ATP to transfected voltage-clamped cells. Individual mutants varied in their sensitivity to ATP; otherwise, currents through functional mutant receptors resembled those of the homomeric wild-type (WT) receptor. In five (H33C, R34C, I50C, K53C, and S54C) of 23 functional mutants, coapplication of 30 muM ATP and 500 nM Ag+ irreversibly inhibited inward current evoked by subsequent applications of ATP alone. These inhibitions did not result in a lateral shift in the agonist concentration-response curve and are unlikely to involve a modification of the agonist binding site. Two (K53C and S54C) of the five residues modified by Ag+ applied in the presence of ATP when the channels were gating were also modified by 1 mM (2-aminoethyl)methanethiosulfonate applied in the absence of ATP when the channels were closed. These data suggest that domains near either end of the first transmembrane domain influence ion conduction through the pore of the P2X(2) receptor.