Regulation of the desensitization and ion selectivity of ATP-gated P2X2 channels by phosphoinositides
Regulation of the desensitization and ion selectivity of ATP-gated P2X2 channels by phosphoinositides
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DOI：
10.1113/jphysiol.2006.115246
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发表时间：
2006-10-01
影响因子：
5.5
通讯作者：
Kubo, Yoshihiro
中科院分区：
文献类型：
--
作者：
Fujiwara, Yuichiro;Kubo, Yoshihiro
磷脂酰肌醇（PIP(n)s）已知可调节某些离子通道的活性。在此我们确定ATP门控的P2X(2)通道也受PIP(n)s调节，并研究了这种调节的结构基础和独特特征。我们首先使用双电极电压钳分析在非洲爪蟾卵母细胞中表达的P2X(2)通道的电生理特性，并观察到用渥曼青霉素或LY294002（两种PI3K抑制剂）预孵育会加速通道脱敏。细胞质C末端结构域近端区域中保守的带正电荷的氨基酸残基K365Q或K369Q突变也会加速脱敏，而K365R或K369R突变则不会。我们观察到在应用ATP后，通道对N -甲基 - D -葡萄糖胺（NMDG）的通透性先短暂增加然后降低，并且K365Q或K369Q突变或PI3K抑制会加速降低的速度。然后我们使用跨越近端C末端区域的带有谷胱甘肽S -转移酶（GST）标签的重组蛋白，利用涂有PIP(n)s的硝酸纤维素膜分析它们的P2X(2)细胞质结构域与阴离子脂质的结合情况。我们发现包含带正电荷区域的重组蛋白与PIPs和PIP(2)s结合，并且这种结合因K365Q和K369Q突变而消除。我们还使用荧光测定法证实，在COS - 7细胞中表达的包含P2X(2)近端C末端区域与增强型绿色荧光蛋白（EGFP）的融合蛋白与细胞膜紧密相关。综上所述，这些结果表明，膜结合的PIP(n)s通过与P2X(2)细胞质C末端结构域近端区域的静电相互作用，在维持通道活性和调节孔扩张方面起着关键作用。
Phosphoinositides (PIP(n)s) are known to regulate the activity of some ion channels. Here we determined that ATP-gated P2X(2) channels also are regulated by PIP(n)s, and investigated the structural background and the unique features of this regulation. We initially used two-electrode voltage clamp to analyse the electrophysiological properties of P2X(2) channels expressed in Xenopus oocytes, and observed that preincubation with wortmannin or LY294002, two PI3K inhibitors, accelerated channel desensitization. K365Q or K369Q mutation of the conserved, positively charged, amino acid residues in the proximal region of the cytoplasmic C-terminal domain also accelerated desensitization, whereas a K365R or K369R mutation did not. We observed that the permeability of the channel to N-methyl-D-glucamine (NMDG) transiently increased and then decreased after ATP application, and that the speed of the decrease was accelerated by K365Q or K369Q mutation or PI3K inhibition. Using GST-tagged recombinant proteins spanning the proximal C-terminal region, we then analysed their binding of the P2X(2) cytoplasmic domain to anionic lipids using PIP(n)s-coated nitrocellulose membranes. We found that the recombinant proteins that included the positively charged region bound to PIPs and PIP(2)s, and that this binding was eliminated by the K365Q and K369Q mutations. We also used a fluorescence assay to confirm that fusion proteins comprising the proximal C-terminal region of P2X(2) with EGFP expressed in COS-7 cells closely associated with the membrane. Taken together, these results show that membrane-bound PIP(n)s play a key role in maintaining channel activity and regulating pore dilation through electrostatic interaction with the proximal region of the P2X(2) cytoplasmic C-terminal domain.