State-dependent modulation of CFTR gating by pyrophosphate
State-dependent modulation of CFTR gating by pyrophosphate
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DOI：
10.1085/jgp.200810186
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发表时间：
2009-04-01
影响因子：
3.8
通讯作者：
Hwang, Tzyh-Chang
中科院分区：
文献类型：
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作者：
Tsai, Ming-Feng;Shimizu, Hiroyasu;Hwang, Tzyh-Chang
囊性纤维化跨膜电导调节器(CFTR)是一种三磷酸腺苷(ATP)门控氯离子通道。ATP诱导CFTR2个核苷酸结合域(NBD)的二聚化反映了通道的开放状态，而ATP的水解与通道的关闭有关。焦磷酸盐(PPI)与非水解性ATP类似物一样，在与ATP一起使用时，已知可锁定CFTR通道数十秒。在这里，我们证明了PPI本身以一种依赖于镁离子的方式开放CFTR通道，在ATP从离体膜补片的细胞质一侧移除很长时间之后。然而，由MgPPI引起的短暂的开放状态(tau类似于1.5 S)表明，单独的MgPPI并不支持稳定的NBD二聚体构型。令人惊讶的是，当在三磷酸腺苷开放的通道关闭后不久给药时，镁PPI会引起长期的开放事件(与30岁的S相似)。这些结果表明，在通道关闭时，存在两种不同的闭合状态(C-1和C-2)，并且MgPPI在CFTR门控中存在状态依赖的效应。在ATP冲刷阶段，进入由MgPPI诱导的长开放突发的通道的相对量随着时间的推移而减少，这表明可以被MgPPI锁定打开的闭合状态(C-2)随时间的消散。当通道最初被高亲和力的ATP类似物N-6-苯乙基-ATP开放时，C-2状态的稳定性增强，但被W401G突变减弱，这可能会削弱ATP与NBD1的结合，这表明ATP分子在C-2状态下仍然与NBD1结合。利用Y1219G-CFTR的慢开放速率，我们能够识别出在处于C-1状态的通道被ATP打开之前存在的C-2等效态(C-2*)。这种闭合状态对MgPPI的响应比C-2状态低得多。最后，我们证明了MGAMP-PNP通过与MGPPI类似的机制对CFTR门控起作用。讨论了我们的发现在结构和功能上的意义。
Cystic fibrosis transmembrane conductance regulator (CFTR) is an adenosine triphosphate (ATP)-gated chloride channel. ATP-induced dimerization of CFTR's two nucleotide-binding domains (NBDs) has been shown to reflect the channel open state, whereas hydrolysis of ATP is associated with channel closure. Pyrophosphate (PPi), like nonhydrolytic ATP analogues, is known to lock open the CFTR channel for tens of seconds when applied with ATP. Here, we demonstrate that PPi by itself opens the CFTR channel in a Mg2+-dependent manner long after ATP is removed from the cytoplasmic side of excised membrane patches. However, the short-lived open state (tau similar to 1.5 s) induced by MgPPi suggests that MgPPi alone does not support a stable NBD dimer configuration. Surprisingly, MgPPi elicits long-lasting opening events (tau similar to 30 s) when administrated shortly after the closure of ATP-opened channels. These results indicate the presence of two different closed states (C-1 and C-2) upon channel closure and a state-dependent effect of MgPPi on CFTR gating. The relative amount of channels entering MgPPi-induced long-open bursts during the ATP washout phase decreases over time, indicating a time-dependent dissipation of the closed state (C-2) that can be locked open by MgPPi. The stability of the C-2 state is enhanced when the channel is initially opened by N-6-phenylethyl-ATP, a high affinity ATP analogue, but attenuated by W401G mutation, which likely weakens ATP binding to NBD1, suggesting that an ATP molecule remains bound to the NBD1 site in the C-2 state. Taking advantage of the slow opening rate of Y1219G-CFTR, we are able to identify a C-2-equivalent state (C-2*), which exists before the channel in the C-1 state is opened by ATP. This closed state responds to MgPPi much more inefficiently than the C-2 state. Finally, we show that MgAMP-PNP exerts its effects on CFTR gating via a similar mechanism as MgPPi. The structural and functional significance of our findings is discussed.