Regulation of channel gating by AMP-activated protein kinase modulates cystic fibrosis transmembrane conductance regulator activity in lung submucosal cells
Regulation of channel gating by AMP-activated protein kinase modulates cystic fibrosis transmembrane conductance regulator activity in lung submucosal cells
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DOI：
10.1074/jbc.m210621200
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发表时间：
2003-01-10
影响因子：
4.8
通讯作者：
Foskett, JK
中科院分区：
文献类型：
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作者：
Hallows, KR;McCane, JE;Foskett, JK
囊性纤维化跨膜电导调节器(CFTR)氯通道活性对许多上皮细胞的液体和电解质运输非常重要，包括肺，囊性纤维化相关的发病率最高的部位。在离子通道中，cftr是独一无二的，它的门控需要ATP水解酶，这表明它的活性与细胞代谢状态有关。代谢感受器AMP激活的激酶(AMPK)与CFTR结合并磷酸化，与其在不同的组织中共定位，并抑制非洲爪哇卵母细胞中的CFTR电流(Hlowers，K.R.，Raghuram，V.，Kemp，B.E.9Witters，L.A.&Foskett，J.K.(2000)J.Clin)。投资。105、1711-1721)。在这里，我们证明了这种AMPKCFTR相互作用在人肺上皮细胞中具有功能意义。药物激活AMPK可抑制Forskolin刺激的CALU-3细胞单层短路电流。在全细胞膜片钳实验中，内源性AMPK的激活，无论是药理上的激活还是通过AMPK激活的非催化亚单位突变体(AMPK-Gamma1-R70Q)的过表达，都显著抑制了Forsklin刺激的CALU-3和CFTR表达的中国仓鼠卵巢细胞的CFTR电导。通过表面生物素化来评估CFTR的质膜表达不受AMPK激活的影响。相反，在细胞附着式膜片钳测量中，在转染AMPK激活突变体的CALU-3细胞中，CFTR的单通道开放概率显著降低，这主要是由于通道的平均关闭时间显著延长。作为细胞中的代谢感受器，AMPK可能在调节CFTR活性与细胞能量电荷，从而将跨上皮运输和维持细胞离子梯度与细胞代谢联系起来方面发挥重要作用。
Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity is important for fluid and electrolyte transport in many epithelia including the lung, the site of most cystic fibrosis-associated morbidity. CFTR is unique among ion channels in requiring ATP hydrolysis for its gating, suggesting that its activity is coupled to cellular metabolic status. The metabolic sensor AMP-activated kinase (AMPK) binds to and phosphorylates CFTR, co-localizes with it in various tissues, and inhibits CFTR currents in Xenopus oocytes (Hallows, K. R., Raghuram, V., Kemp, B. E.9 Witters, L. A. & Foskett, J. K. (2000) J. Clin. Invest. 105, 1711-1721). Here we demonstrate that this AMPKCFTR interaction has functional implications in human lung epithelial cells. Pharmacologic activation of AMPK inhibited forskolin-stimulated CFTR short circuit currents in polarized Calu-3 cell monolayers. In whole-cell patch clamp experiments, the activation of endogenous AMPK either pharmacologically or by the overexpression of an AMPK-activating non-catalytic subunit mutant (AMPK-gamma1-R70Q) dramatically inhibited forskolin-stimulated CFTR conductance in Calu-3 and CFTR-expressing Chinese hamster ovary cells. Plasma membrane expression of CFTR, assessed by surface biotinylation, was not affected by AMPK activation. In contrast, the single channel open probability of CFTR was strongly reduced in cell-attached patch clamp measurements of Calu-3 cells transfected with the AMPK-activating mutant, an effect due primarily to a substantial prolongation of the mean closed time of the channel. As a metabolic sensor in cells, AMPK may be important in tuning CFTR activity to cellular energy charge, thereby linking transepithelial transport and the maintenance of cellular ion gradients to cellular metabolism.