Evidence for activation of BKCa channels by a known inhibitor of focal adhesion kinase, PF573228

AimsPF573228 is an inhibitor of focal adhesion kinase (FAK) and recognized to affect cell adhesion and migration in many types of cells. Its effects on ion currents and membrane potential have been investigated in this study.Main methodElectrophysiological studies of PF573228 actions on ion currents were performed in pituitary tumor (GH3) cells, in GH3 cells transfected with KCa1.1 siRNAs and in human embryonic kidney (HEK) cells expressing α-human slowpoke (α-hSlo).Key findingsIn whole-cell experiments, PF573228 reversibly increased the amplitude of Ca2+-activated K+ currents (IK(Ca)) in GH3 cells. In inside-out recordings, this compound added to the bath did not modify single-channel conductance but stimulated large-conductance Ca2+-activated K+ (BKCa) channels with an EC50 value of 3.2 μM. As BKCa-channel activity was stimulated by PF573228 (3 μM), subsequent application of BMS191011 (3 μM) did not further increase channel activity. PF573228 shifted the activation curve of BKCa channels to less positive membrane potential. Change in the kinetic behavior of BKCa channels caused by this compound is a result of the increased backward rate constants between closed states. PF573228 depressed the firing of action potentials in GH3 cells. However, in GH3 cells transfected with KCa1.1 siRNAs, PF573228-stimulated IK(Ca) was abolished. In HEK293T cells expressing α-hSlo, PF573228 enhanced BKCa-channel activity.SignificanceIn addition to an inhibition of FAK phosphorylation, PF573228 is effective in activating BKCa channels. The direct stimulation of these channels by this compound may contribute to the underlying mechanism through which it influences cell behavior.