Strict coupling between CFTR's catalytic cycle and gating of its Cl- ion pore revealed by distributions of open channel burst durations
Strict coupling between CFTR's catalytic cycle and gating of its Cl- ion pore revealed by distributions of open channel burst durations
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DOI：
10.1073/pnas.0911061107
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发表时间：
2010-01-19
影响因子：
11.1
通讯作者：
Gadsby, David C.
中科院分区：
文献类型：
--
作者：
Csanady, Laszlo;Vergani, Paola;Gadsby, David C.
CFTR是囊性纤维化中有缺陷的ABC蛋白，其功能是阴离子通道。一旦被蛋白激酶A磷酸化，CFTR通道通过其两个胞质核苷酸结合结构域（NBD）处的事件打开和关闭。已经提出通过ATP结合在一个NBD的保守步行者A和B基序与另一个的ABC特异性特征序列之间的两个界面复合位点中形成头-尾NBD 1/NBD 2异二聚体来触发通道开放。ATP水解在唯一的催化活性界面网站建议，然后不稳定的NBD二聚体和提示通道关闭。但是这种门控机制，以及CFTR通道的开放和关闭与其催化循环的紧密程度，仍然存在争议。在这里，我们确定了个别CFTR通道的开放爆发持续时间的分布，并使用最大似然法来评估适合平衡和非平衡机制，并估计速率常数，管理通道关闭。我们研究了部分和完全磷酸化的野生型CFTR通道，和两个突变CFTR通道，每个通道在一个或其他复合ATP结合位点中具有有害突变。我们表明，野生型CFTR通道门控循环基本上是不可逆的，并紧密耦合到ATP酶循环，这种耦合是完全破坏的NBD 2沃克B突变D1370 N，但只有部分破坏的NBD 1步行者A突变K464 A。
CFTR, the ABC protein defective in cystic fibrosis, functions as an anion channel. Once phosphorylated by protein kinase A, a CFTR channel is opened and closed by events at its two cytosolic nucleotide binding domains (NBDs). Formation of a head-to-tail NBD1/NBD2 heterodimer, by ATP binding in two interfacial composite sites between conserved Walker A and B motifs of one NBD and the ABC-specific signature sequence of the other, has been proposed to trigger channel opening. ATP hydrolysis at the only catalytically competent interfacial site is suggested to then destabilize the NBD dimer and prompt channel closure. But this gating mechanism, and how tightly CFTR channel opening and closing are coupled to its catalytic cycle, remains controversial. Here we determine the distributions of open burst durations of individual CFTR channels, and use maximum likelihood to evaluate fits to equilibrium and nonequilibrium mechanisms and estimate the rate constants that govern channel closure. We examine partially and fully phosphorylated wild-type CFTR channels, and two mutant CFTR channels, each bearing a deleterious mutation in one or other composite ATP binding site. We show that the wild-type CFTR channel gating cycle is essentially irreversible and tightly coupled to the ATPase cycle, and that this coupling is completely destroyed by the NBD2Walker B mutation D1370N but only partially disrupted by the NBD1 Walker A mutation K464A.