Receptor Species-Dependent Signaling of Gqpcrs Controls IKs Activity

Abstract

The membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) interacts with signaling molecules at the plasma membrane and positively modulates KCNQ1/KCNE1 (IKs) ion channels. Membrane PIP2 is reduced by activity of phospholipase C (PLC), which in turn is controlled by Gq protein-coupled receptors (GqPCRs). Our study compared the muscarinic M1 receptor (M1-R) and the α1B adrenergic receptor (α1B-AR) in their ability to modulate IKs. We analyzed temporal aspects of receptor signaling in single HEK 293 cells and CHO cells stably expressing KCNQ1/KCNE1 using FRET-based biosensors for the Gq cycle and PLC activation. IKs was measured using voltage-clamp. Both receptors activate PLC and cause PIP2 depletion. Activation of M1-R caused robust inhibition of IKs with a kinetics that resembled the time course of PIP2-depletion and recovery of IKs upon removal of the agonist. In contrast, the α1B-AR-induced inhibition of IKs was transient and turned into activation after agonist withdrawal. This delayed response exceeded basal activation levels and occurred at a point when the PIP2 pool was completely restored. Furthermore, the α1B-AR, but not the M1-R, displayed acute desensitization in the presence of agonist that was evident on the levels of Gq and PLC activation. This indicates a feedback modulation by kinases. Indeed, global inhibition of all protein kinase C (PKC) isoforms with staurosporine removed receptor desensitization and abolished the delayed facilitation of IKs. A more specific inhibition of Ca2+-sensitive PKCs with Go6976 reduced α1B-AR desensitization but not facilitation of IKs, which was rather mediated by diacylglycerol-activated kinases. Notably, activation of M1-R and its influence on IKs were not dependent on PKC activation. We conclude from these data that fine-tuning of IKs activity via Gq is controlled by recruitment of kinases downstream of PIP2 depletion in a receptor species-specific fashion.