Altering intracellular pH reveals the kinetic basis of intraburst gating in the CFTR Cl- channel
Altering intracellular pH reveals the kinetic basis of intraburst gating in the CFTR Cl- channel
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改变细胞内 pH 值揭示了 CFTR Cl-通道内突发门控的动力学基础。
DOI：
10.1113/jp273205
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发表时间：
2017-02-01
影响因子：
5.5
通讯作者：
Sheppard, David N.
中科院分区：
文献类型：
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作者：
Chen, Jeng-Haur;Xu, Weiyi;Sheppard, David N.
囊性纤维化跨膜传导调节因子（CFTR）是遗传性疾病囊性纤维化（CF）中的ATP门控Cl-通道缺陷。CFTR的门控行为的特征在于通道开口的突发被短暂的、闪烁的闭合中断，突发之间被长的闭合分开。开放爆发的进入和退出由ATP与CFTR中的两个ATP结合位点（位点1和2）的相互作用控制。为了更好地理解CFTR爆发内门控的动力学基础，我们研究了人CFTR在不同细胞内pH（pH（i））值下的单通道活性。当与对照（pH（i）7.3）相比时，将pH（i）酸化至6.3或将pH（i）碱化至8.3和8.8引起野生型CFTR的开放时间常数（（o））的小幅降低。相比之下，快速闭合时间常数（（cf））在pH（i）5.8和6.3时大大增加，该常数描述了中断开放爆发的短暂闭合。为了分析爆发内动力学，我们使用线性三态门控方案。所有的数据都令人满意地模拟的C-1 O C-2动力学方案。改变细胞内ATP浓度对（o）、（cf）及其对pH（i）变化的反应没有影响。然而，破坏ATP与ATP结合位点1相互作用的突变，包括K464 A、D572 N和CF相关突变G1349 D，都消除了pH（i）6.3时（cf）的延长。两者合计，我们的数据表明，CFTR爆发内门控的调节是不同的ATP依赖的机制，控制通道的开放和关闭。然而，我们的数据也表明，ATP结合位点1调制内脉冲门控。
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated Cl- channel defective in the genetic disease cystic fibrosis (CF). The gating behaviour of CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures, separated by long closures between bursts. Entry to and exit from an open burst is controlled by the interaction of ATP with two ATP-binding sites, sites 1 and 2, in CFTR. To understand better the kinetic basis of CFTR intraburst gating, we investigated the single-channel activity of human CFTR at different intracellular pH (pH(i)) values. When compared with the control (pH(i) 7.3), acidifying pH(i) to 6.3 or alkalinizing pH(i) to 8.3 and 8.8 caused small reductions in the open-time constant ((o)) of wild-type CFTR. By contrast, the fast closed-time constant ((cf)), which describes the short-lived closures that interrupt open bursts, was greatly increased at pH(i) 5.8 and 6.3. To analyse intraburst kinetics, we used linear three-state gating schemes. All data were satisfactorily modelled by the C-1 O C-2 kinetic scheme. Changing the intracellular ATP concentration was without effect on (o), (cf) and their responses to pH(i) changes. However, mutations that disrupt the interaction of ATP with ATP-binding site 1, including K464A, D572N and the CF-associated mutation G1349D all abolished the prolongation of (cf) at pH(i) 6.3. Taken together, our data suggest that the regulation of CFTR intraburst gating is distinct from the ATP-dependent mechanism that controls channel opening and closing. However, our data also suggest that ATP-binding site 1 modulates intraburst gating.