Dual effects of ADP and adenylylimidodiphosphate on CFTR channel kinetics show binding to two different nucleotide binding sites.
Dual effects of ADP and adenylylimidodiphosphate on CFTR channel kinetics show binding to two different nucleotide binding sites.
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ADP 和腺苷酰亚胺二磷酸对 CFTR 通道动力学的双重影响显示出与两个不同的核苷酸结合位点的结合。
DOI：
10.1085/jgp.114.1.55
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Nagel,G
中科院分区：
文献类型：
--
作者：
Weinreich,F;Riordan,JR;Nagel,G
来自*马克斯普朗克生物物理研究所，德国法兰克福60596；‡SC Johnson医学研究中心，梅奥诊所，斯科茨代尔，亚利桑那州85259；和？约翰-沃尔夫冈-歌德大学，生物中心，60439法兰克福/M，德国摘要cftr氯通道受蛋白激酶，特别是PKA的磷酸化以及与两个核苷酸结合域NbD-A和NbD-B相互作用的核苷酸调节。以表达人上皮囊性纤维化跨膜电导调节因子(CFTR)的非洲爪哇卵母细胞为材料，研究了PKA和核苷酸对其氯离子电导的影响。ATP、其非水解性类似物腺苷二磷酸(AMP-PNP)及其耐光性衍生物ATP-P3-[1-(2-硝基苯基)乙酯]或ADP浓度的快速变化导致氯电导随特征时间常数的变化，这反映了CFTR与这些核苷酸的相互作用。强磷酸化通道的电导变化慢于部分磷酸化的cftr。AMP-PNP减缓了电导增加和衰减的松弛，而ATP-P3-[1-(2-硝基苯基)乙酯]酯仅减缓了加入ATP后电导的增加。ADP使加入ATP时电导增加的速度减慢，而在撤除ATP时电导下降的速度加快。这些结果首次直接证明AMP-PNP与CFTR上的两个位点结合。由于观察到的两种不同的抑制模式，ADP的作用还提示了两个不同的结合部位：它与ATP竞争闭合通道上的结合(与NBD-A)，但它也与ATP开放的通道结合，这可能反映了与NBD-A的结合(即在水解循环中的产物抑制)或变构与NBD-B的结合，从而加速了NBD-A的水解循环。
From the* Max-Planck-Institut für Biophysik, 60596 Frankfurt/M., Germany;‡ SC Johnson Medical Research Center, Mayo Clinic, Scottsdale, AZ 85259; and § Johann-Wolfgang-Goethe-Universität, Biozentrum, 60439 Frankfurt/M., Germany abstract The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl) ethyl] ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay, whereas ATP-P3-[1-(2-nitrophenyl) ethyl] ester only decelerated the conductance increase upon ATP addition. ADP decelerated the conductance increase upon ATP addition and accelerated the conductance decay upon ATP withdrawal. The results present the first direct evidence that AMP-PNP binds to two sites on the CFTR. The effects of ADP also suggest two different binding sites because of the two different modes of inhibition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either reflect binding to NBD-A (ie, product inhibition in the hydrolysis cycle) or allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NBD-A.