Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating.
Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating.
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DOI：
10.1085/jgp.200308798
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发表时间：
2003-09
期刊：
影响因子：
--
通讯作者：
Gadsby DC
中科院分区：
文献类型：
--
作者：
Basso C;Vergani P;Nairn AC;Gadsby DC
Cftr是囊性纤维化中的缺陷蛋白，其功能是受cAMP依赖的蛋白激酶调节的氯离子−通道。CFTR也是一种ATPase，由两个核苷酸结合域(NbD)组成，被认为是结合和水解三磷酸腺苷。在可水解的核苷三磷酸盐中，PKA磷酸化的CFTR通道从闭合的(间歇的)一秒或更长的时间间隔开放为突发式，持续约一秒。为了研究通道门控的核苷酸相互作用，我们研究了[α32P]8-N3ATP或[γ32P]8-N3ATP光标记在HEK293T细胞或非洲爪哇卵母细胞中表达的完整cftr通道。我们还利用分裂的CFTR通道来区分Nbd1和Nbd2的光标记。为了在没有水解和门控反应的情况下检测核苷酸的简单结合，我们在0°C孵育后进行了光标记，无需洗涤。门控条件下的核苷酸相互作用在30°C孵育后用光标记法检测，同时也是在30°C广泛洗涤。PKA对CFTR的磷酸化仅对两种方案中的光标记法影响很小。值得注意的是，在30℃时，核苷酸以非水解型核苷三磷酸的形式在NBD1上紧密结合了许多分钟。由于cftr通道的核苷酸依赖的门控在可比条件下发生在秒的时间尺度上，这表明核苷酸相互作用，包括水解，cftr通道的开放和关闭时间主要发生在Nbd2。钒酸盐似乎也作用于Nbd2，可能中断了它的水解循环，并显著延迟了通道开放突发的终止。钒酸盐在一定程度上增加了Nbd1上紧密结合的核苷酸的缓慢丢失的幅度，但没有改变其速度。对镁离子通道门控的动力学分析表明，在饱和[核苷酸]时CFTR通道开放的速率限制步骤是在核苷酸与两个NBD结合之后进行的。我们认为，ATP在CFTR的NBD1上保持紧密结合或长时间闭塞，在Nbd2与ATP的结合导致通道开放，然后它的水解促使通道关闭，磷酸化就像汽车离合器一样，参与NBD事件，驱动跨膜离子孔的门控。
CFTR, the protein defective in cystic fibrosis, functions as a Cl− channel regulated by cAMP-dependent protein kinase (PKA). CFTR is also an ATPase, comprising two nucleotide-binding domains (NBDs) thought to bind and hydrolyze ATP. In hydrolyzable nucleoside triphosphates, PKA-phosphorylated CFTR channels open into bursts, lasting on the order of a second, from closed (interburst) intervals of a second or more. To investigate nucleotide interactions underlying channel gating, we examined photolabeling by [α32P]8-N3ATP or [γ32P]8-N3ATP of intact CFTR channels expressed in HEK293T cells or Xenopus oocytes. We also exploited split CFTR channels to distinguish photolabeling at NBD1 from that at NBD2. To examine simple binding of nucleotide in the absence of hydrolysis and gating reactions, we photolabeled after incubation at 0°C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30°C, with extensive washing, also at 30°C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30°C nucleotide remained tightly bound at NBD1 for many minutes, in the form of nonhydrolyzed nucleoside triphosphate. As nucleotide-dependent gating of CFTR channels occurred on the time scale of seconds under comparable conditions, this suggests that the nucleotide interactions, including hydrolysis, that time CFTR channel opening and closing occur predominantly at NBD2. Vanadate also appeared to act at NBD2, presumably interrupting its hydrolytic cycle, and markedly delayed termination of channel open bursts. Vanadate somewhat increased the magnitude, but did not alter the rate, of the slow loss of nucleotide tightly bound at NBD1. Kinetic analysis of channel gating in Mg8-N3ATP or MgATP reveals that the rate-limiting step for CFTR channel opening at saturating [nucleotide] follows nucleotide binding to both NBDs. We propose that ATP remains tightly bound or occluded at CFTR's NBD1 for long periods, that binding of ATP at NBD2 leads to channel opening wherupon its hydrolysis prompts channel closing, and that phosphorylation acts like an automobile clutch that engages the NBD events to drive gating of the transmembrane ion pore.
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影响因子：
16
作者：
Junop, MS;Obmolova, G;Yang, W
通讯作者：
Yang, W
影响因子：
3.8
作者：
Dousmanis, AG;Nairn, AC;Gadsby, DC
通讯作者：
Gadsby, DC
影响因子：
3.5
作者：
Aleksandrov, AA;Riordan, JR
通讯作者：
Riordan, JR
DOI：
10.1073/pnas.77.12.7487
发表时间：
1980-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
KACZMAREK, LK;JENNINGS, KR;GREENGARD, P
通讯作者：
GREENGARD, P
影响因子：
4.8
作者：
Gao, M;Cui, HR;Deeley, RG
通讯作者：
Deeley, RG