Carson M R, Winter M C, Travis S M, Welsh M J
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242, USA.
J Biol Chem. 1995 Sep 1;270(35):20466-72. doi: 10.1074/jbc.270.35.20466.
A unique feature of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is regulation by ATP through the two cytoplasmic nucleotide-binding domains (NBDs). To better understand this process, we asked how channel activity is affected by inorganic pyrophosphate (PPi), a compound that binds to NBDs in other proteins. PPi and three nonhydrolyzable PPi analogs reversibly stimulated the activity of phosphorylated channels. Kinetic modeling of single channel data demonstrated that PPi affected two distinct steps in channel regulation. First, PPi increased the rate at which channels opened. Second, once channels were open, PPi delayed their closure. PPi could only stimulate channels when it was applied in the presence of ATP. PPi also increased the photolabeling of CFTR by an ATP analog. These two findings suggest that PPi modifies the activity of ATP-dependent CFTR channel gating. Based on these and previous data, we speculate that the effects of PPi are mediated by binding of PPi to NBD2 where it regulates channel opening by NBD1, and then, because it is not hydrolyzed, it slows the rate of NBD2-mediated channel closing. Because PPi stimulated wild-type channels, we tested its effect on CFTR containing the cystic fibrosis mutations: delta F508, R117H, and G551S. PPi stimulated all three. PPi also stimulated endogenous CFTR in the apical membrane of permeabilized T-84 epithelia. These results suggest that PPi or an analog might be of value in the development of new approaches to the treatment of cystic fibrosis.
囊性纤维化跨膜传导调节因子（CFTR）氯离子通道的一个独特特征是通过两个胞质核苷酸结合结构域（NBDs）受ATP调节。为了更好地理解这一过程，我们研究了无机焦磷酸（PPi）对通道活性的影响，PPi是一种能与其他蛋白质中的NBDs结合的化合物。PPi和三种不可水解的PPi类似物可逆地刺激磷酸化通道的活性。单通道数据的动力学建模表明，PPi影响通道调节的两个不同步骤。首先，PPi增加通道开放的速率。其次，一旦通道开放，PPi会延迟其关闭。只有在ATP存在的情况下应用PPi时，它才能刺激通道。PPi还增加了ATP类似物对CFTR的光标记。这两个发现表明，PPi改变了ATP依赖的CFTR通道门控的活性。基于这些以及之前的数据，我们推测PPi的作用是通过其与NBD2结合介导的，在NBD2处它调节NBD1介导的通道开放，然后，由于它不被水解，它减缓了NBD2介导的通道关闭速率。因为PPi刺激野生型通道，我们测试了它对含有囊性纤维化突变（ΔF508、R117H和G551S）的CFTR的影响。PPi对这三种突变体均有刺激作用。PPi还刺激了透化的T - 84上皮细胞顶端膜中的内源性CFTR。这些结果表明，PPi或其类似物可能在开发治疗囊性纤维化的新方法中具有价值。
