Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells.

Klein H, Garneau L, Trinh NT, Prive A, Dionne F, Goupil E, Thuringer D, Parent L, Brochiero E, Sauve R. Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells. Am J Physiol Cell Physiol 296: C285-C295, 2009. First published December 3, 2008; doi:10.1152/ajpcell.00418.2008.-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 mu M AMP in 1 mM ATP. Evidence for an interaction between the COOH-terminal region of KCa3.1 and the gamma 1-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-gamma 1/KCa3.1. Inside-out experiments on cells coexpressing KCa3.1 with the dominant negative AMPK-gamma 1-R299G mutant showed a reduced sensitivity of KCa3.1 to AMP, arguing for a functional link between KCa3.1 and the gamma 1-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-gamma 1 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 gamma 1-subunit of AMPK.