Fig. 6.
AMPK activation inhibits basolateral KCNQ1 currents in polarized mpkCCDc14 cells. A: representative Western blot of cell lysates showing 24-h time course of AMPK activity (pThr172-α1-AMPK; top) with β-actin as loading control (bottom) following treatment of polarized mpkCCDc14 cells with 1 mM metformin. B: summary of means ± SE relative AMPK activities (normalized to control at time 0) following treatment for up to 24 h with 2 mM AICAR (○), 1 mM metformin (●), or vehicle control (◊). *P < 0.05, unpaired t-test compared with control at time 0; n = 3 replicate experiments for each drug treatment data point; n = 6 for controls. C: representative short-circuit current (Isc) tracings of mpkCCDc14 cells pretreated for 16 h with 1 mM metformin (dark gray trace) or vehicle (light gray trace) in the presence of an apical-to-basolateral K+ gradient, as described in materials and methods. Cell monolayers were mounted in Ussing chambers and treated first with 20 μM amiloride apically to inhibit ENaC, then 20 μM amphotericin B apically to isolate conductance at the basolateral membrane, then 50 μM of the KCNQ1 activator L-364,373 basolaterally, then 50 μM chromanol 293B basolaterally, and finally 2 mM BaCl2 basolaterally at the indicated times. One-second transepithelial voltage pulses of ±2 mV were performed each minute throughout the recordings but were removed from the displayed current trace for clarity. D: summary of means ± SE basolateral KCNQ1-mediated Isc (defined as difference between peak current following L-364,373 treatment and the current measured after chromanol 293B treatment) with or without 1 mM metformin pretreatment for 16 h. E: summary of means ± SE basolateral KCNQ1-mediated Isc with or without 2 mM AICAR pretreatment for 16 h. *P < 0.05, unpaired t-test, compared with control; n = 8 filters analyzed in 4 separate experiments for all conditions. Inset: representative immunoblots of cell lysates for pThr172-α1-AMPK and β-actin following the AICAR treatment experiment.