Journal: American Journal of Physiology - Cell Physiology
Article Title: Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells
doi: 10.1152/ajpcell.00418.2008
Figure Lengend Snippet: Impact of AMPK activity on KCa3.1currents. Short-circuit currents ( I sc ) measured in Ussing chamber on Nuli cell monolayers cultured for 4 to 6 wk, at air-liquid-interface. A : applying the KCa3.1 potentiator 1-EBIO (1 mM) to the basolateral side of the monolayer caused an increase in I sc , which was inhibited by a basolateral application of the KCa3.1 inhibitor Tram-34 (5 μM). This result confirms the presence of KCa3.1 at the basolateral membrane of Nuli cell monolayers. B : similarly, applying 1-EBIO (1 mM) to the apical side of the monolayer caused an increase in I sc , which was inhibited by apical application of Tram-34 (5 μM), confirming the presence of KCa3.1 at the apical membrane of Nuli cell monolayers. C : basolateral addition of Tram-34 following apical stimulation of KCa3.1 by 1-EBIO did not result in a significant I sc decrease, confirming the side specificity of the action of Tram-34 seen in B. D – F : impact of AMPK activity on KCa3.1 currents was evaluated by I sc measured in nontreated (Ctl), AICAR-treated (1 mM, 1 h, apical and basolateral side), or AICAR + Compound-C treated (10 μM, 1 h, apical and basolateral side) monolayers. AICAR is used to activate AMPK via the internal production of 5-amino-4-imidazole-carboxamide ribotide (ZMP). Basal short-circuit currents ( I basal , D ; n = 11), 1-EBIO-induced currents ( I 1-EBIO , E ; n = 11), and 1-EBIO-induced, Tram-34 inhibited currents ( I Tram-34 , F ; n = 11) were then compared in control, AICAR, and AICAR + Compound-C-treated monolayers. The presence of Compound-C succeeded to reverse the effect of AICAR on the KCa3.1-dependent short-circuit currents. These results argue for a control of the KCa3.1 activity by AMPK in Nuli monolayers.
Article Snippet: For immunoprecipitation of endogenous KCa3.1 or AMPK-γ1 proteins, precleared soluble lysates were incubated for 1–2 h with a rabbit anti-KCa3.1 antibody (1:100, Alomone Labs, Jerusalem, Israel) or with a rabbit anti-AMPK-γ1 antibody (1:100 or 1:150, Abcam, Cambridge, MA).
Techniques: Activity Assay, Cell Culture, CTL Assay