Regulation of channel gating by AMP-activated protein kinase modulates cystic fibrosis transmembrane conductance regulator activity in lung submucosal cells.
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., Witters, L. A. & Foskett, J. K. (2000) J. Clin. Invest. 105, 1711-1721). Here we demonstrate that this AMPK-CFTR 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.
Hallows
Kenneth R
KR
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
McCane
Jill E
JE
Kemp
Bruce E
BE
Witters
Lee A
LA
Foskett
J Kevin
JK
D055372:AMP-Activated Protein Kinases; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D016764:Cell Polarity; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D008168:Lung / Q000378:metabolism; D009097:Multienzyme Complexes / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D012265:Ribonucleotides / Q000494:pharmacology
C505032:CFTR protein, human; D009097:Multienzyme Complexes; D012265:Ribonucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000620:Aminoimidazole Carboxamide; C526222:PRKAG1 protein, human; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide