Physiological modulation of CFTR activity by AMP-activated protein kinase in polarized T84 cells
Physiological modulation of CFTR activity by AMP-activated protein kinase in polarized T84 cells
复制标题
DOI：
10.1152/ajpcell.00227.2002
复制
发表时间：
2003-05-01
影响因子：
5.5
通讯作者：
Foskett, JK
中科院分区：
文献类型：
--
作者：
Hallows, KR;Kobinger, GP;Foskett, JK
囊性纤维化跨膜传导调节因子（CFTR）是cAMP激活的ATP门控Cl-通道和细胞传导调节因子，但CFTR调节的详细机制及其对其他转运蛋白的调节仍不清楚。我们以前确定的代谢传感器AMP激活的蛋白激酶（AMPK）作为一种新的蛋白质与CFTR相互作用，并发现AMPK磷酸化CFTR和抑制CFTR依赖的全细胞电导时，与CFTR在非洲爪蟾卵母细胞共表达。为了解决CFTR-AMPK相互作用的生理相关性，我们现在已经研究了极化上皮细胞，并评估了内源性AMPK和CFTR的定位，并测量了CFTR活性与AMPK活性的调节。通过免疫荧光成像，AMPK和CFTR在几种大鼠上皮组织中共享重叠的顶端分布，包括鼻咽、下颌下腺、胰腺和回肠。CFTR依赖的短路电流（I-SC）测量极化T84细胞生长在可渗透的支持，和几个独立的方法被用来调节内源性AMPK活性。用细胞渗透性腺苷类似物5-氨基-4-咪唑甲酰胺-1-β-D-呋喃核糖苷（AICAR）激活内源性AMPK，可抑制基底外侧膜非透化单层和制霉菌素透化单层中毛喉素刺激的CFTR依赖性I-sc。提高细胞内AMP浓度的单层与α-毒素渗透基底外侧膜也抑制CFTR，这是一种与腺苷受体无关的效果。最后，激酶死亡突变体AMPK-alpha 1亚基（alpha 1-K45 R）的过表达增强了极化T84单层中毛喉素刺激的I-SC，这与内源性AMPK对CFTR抑制的显性负性减少一致。这些结果表明AMPK在极化上皮细胞中调节CFTR活性中起生理作用，并提出了离子转运与细胞代谢耦合的新范例。
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-gated Cl- channel and cellular conductance regulator, but the detailed mechanisms of CFTR regulation and its regulation of other transport proteins remain obscure. We previously identified the metabolic sensor AMP-activated protein kinase (AMPK) as a novel protein interacting with CFTR and found that AMPK phosphorylated CFTR and inhibited CFTR-dependent whole cell conductances when coexpressed with CFTR in Xenopus oocytes. To address the physiological relevance of the CFTR-AMPK interaction, we have now studied polarized epithelia and have evaluated the localization of endogenous AMPK and CFTR and measured CFTR activity with modulation of AMPK activity. By immunofluorescent imaging, AMPK and CFTR share an overlapping apical distribution in several rat epithelial tissues, including nasopharynx, submandibular gland, pancreas, and ileum. CFTR-dependent short-circuit currents (I-sc) were measured in polarized T84 cells grown on permeable supports, and several independent methods were used to modulate endogenous AMPK activity. Activation of endogenous AMPK with the cell-permeant adenosine analog 5-amino-4-imidazolecarboxamide-1-beta-D-ribofuranoside (AICAR) inhibited forskolin- stimulated CFTR-dependent I-sc in nonpermeabilized monolayers and monolayers with nystatin permeabilization of the basolateral membrane. Raising intracellular AMP concentration in monolayers with basolateral membranes permeabilized with alpha-toxin also inhibited CFTR, an effect that was unrelated to adenosine receptors. Finally, overexpression of a kinase-dead mutant AMPK-alpha1 subunit (alpha1-K45R) enhanced forskolin- stimulated I-sc in polarized T84 monolayers, consistent with a dominant-negative reduction in the inhibition of CFTR by endogenous AMPK. These results indicate that AMPK plays a physiological role in modulating CFTR activity in polarized epithelia and suggest a novel paradigm for the coupling of ion transport to cellular metabolism.