On the mechanism of CFTR inhibition by a thiazolidinone derivative.
On the mechanism of CFTR inhibition by a thiazolidinone derivative.
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DOI：
10.1085/jgp.201010518
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发表时间：
2010-12
期刊：
影响因子：
--
通讯作者：
Hwang TC
中科院分区：
文献类型：
--
作者：
Kopeikin Z;Sohma Y;Li M;Hwang TC
噻唑烷酮衍生物，3-[（3-三氟甲基）苯基]-5-[（4-羧基苯基）亚甲基]-2-硫代-4-噻唑烷酮（或CFTRinh-172），对囊性纤维化跨膜传导调节因子（CFTR）门控的影响进行了研究，在中国仓鼠卵巢细胞瞬时表达野生型和突变型CFTR的由内而外切除的膜补丁。我们发现CFTRinh-172的应用导致通道的平均关闭时间增加和平均开放时间减少。闭合速率和[CFTRinh-172]之间的双曲线关系表明CFTRinh-172不作为简单的孔阻断剂。有趣的是，抑制效力随着通道开放时间的增加而增加，对于锁定在开放状态数十秒的CFTR通道，IC 50在低纳摩尔范围内。我们的研究还提供了CFTRinh-172可以结合到开放状态和闭合状态的证据。然而，至少需要一个额外的步骤，大概反映了通道诱导的构象变化，关闭后的抑制剂的结合通道的电导。使用水解缺陷突变体E1371 S作为工具，因为该突变体的关闭速率显著降低，我们发现CFTRinh-172依赖性抑制CFTR通道门控，在两个方面，模拟电压依赖性阳离子通道的失活。首先，与电压门控通道失活的恢复类似，一旦CFTR被CFTRinh-172抑制，在三磷酸腺苷（ATP）不存在的情况下去除抑制剂后，可以看到通道重新开放。第二，ATP诱导的双相电流反应的通道结合的封闭通道，如果ATP开放的通道“关闭”，尽管连续存在的ATP。简化的六态动力学方案可以很好地描述我们的数据，至少在定性上是这样。将讨论CFTRinh-172作用的几种可能的结构机制。
The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitor-induced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172–dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels “inactivate” despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.
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DOI：
10.1085/jgp.201010443
发表时间：
2010-05
期刊：
The Journal of general physiology
影响因子：
--
作者：
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DOI：
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发表时间：
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影响因子：
56.9
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DOI：
10.1085/jgp.58.4.413
发表时间：
1971-10
期刊：
The Journal of general physiology
影响因子：
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Hollenstein, Kaspar;Frei, Dominik C.;Locher, Kaspar P.
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