Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells.
The vectorial transport of ions and water across epithelial cells depends to a large extent on the coordination of the apical and basolateral ion fluxes with energy supply. In this work we provide the first evidence for a regulation by the 5'-AMP-activated protein kinase (AMPK) of the calcium-activated potassium channel KCa3.1 expressed at the basolateral membrane of a large variety of epithelial cells. Inside-out patch-clamp experiments performed on human embryonic kidney (HEK) cells stably transfected with KCa3.1 first revealed a decrease in KCa3.1 activity following the internal addition of AMP at a fixed ATP concentration. This effect was dose dependent with half inhibition at 140 muM AMP in 1 mM ATP. Evidence for an interaction between the COOH-terminal region of KCa3.1 and the gamma1-subunit of AMPK was next obtained by two-hybrid screening and pull-down experiments. Our two-hybrid analysis confirmed in addition that the amino acids extending from Asp(380) to Ala(400) in COOH-terminal were essential for the interaction AMPK-gamma1/KCa3.1. Inside-out experiments on cells coexpressing KCa3.1 with the dominant negative AMPK-gamma1-R299G mutant showed a reduced sensitivity of KCa3.1 to AMP, arguing for a functional link between KCa3.1 and the gamma1-subunit of AMPK. More importantly, coimmunoprecipitation experiments carried out on bronchial epithelial NuLi cells provided direct evidence for the formation of a KCa3.1/AMPK-gamma1 complex at endogenous AMPK and KCa3.1 expression levels. Finally, treating NuLi monolayers with the membrane permeant AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) caused a significant decrease of the KCa3.1-mediated short-circuit currents, an effect reversible by coincubation with the AMPK inhibitor Compound C. These observations argue for a regulation of KCa3.1 by AMPK in a functional epithelium through protein/protein interactions involving the gamma1-subunit of AMPK.
Klein
Hélène
H
Groupe d'étude sur les protéines membranaires, Département de physiologie, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, Québec H3C3J7, Canada.
Garneau
Line
L
Trinh
Nguyen Thu Ngan
NT
Privé
Anik
A
Dionne
François
F
Goupil
Eugénie
E
Thuringer
Dominique
D
Parent
Lucie
L
Brochiero
Emmanuelle
E
Sauvé
Rémy
R
D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D016764:Cell Polarity; D002478:Cells, Cultured; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D047468:Immunoprecipitation; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects; D017136:Ion Transport; D008564:Membrane Potentials; D009154:Mutation; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D020545:Respiratory Mucosa / Q000187:drug effects / Q000201:enzymology*; D012265:Ribonucleotides / Q000494:pharmacology; D014162:Transfection; D020798:Two-Hybrid System Techniques
D020536:Enzyme Activators; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; D011994:Recombinant Proteins; D012265:Ribonucleotides; D000620:Aminoimidazole Carboxamide; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; C526222:PRKAG1 protein, human; D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide