Receptor-specific regulation of atrial GIRK channel activity by different Ca2+-dependent PKC isoforms.

G Protein-activated K+ channels (GIRK) channels are inhibited by depletion of PtdIns(4,5)P2(PIP2), and/or channel phosphorylation by proteinkinase C (PKC). By using FRET-based biosensors, expressed in HEK293 cells or in atrial myocytes, we quantified receptor-specific Gq-coupled receptor (GqPCR) signalling on the level of phospholipase C (PLC) activation by monitoring PIP2-depletion and diacylglycerol (DAG) formation. Simultaneous voltage-clamp experiments on GIRK channel activity were performed as a functional readout for Gq-coupled α1B- and ET-receptor-induced signalling. GqPCR-induced fast inhibition of GIRK channel activity is mediated by depletion of PIP2, whereas phosphorylation of GIRK channels results in delayed, but effective GIRK current inhibition. We demonstrate a receptor-induced inhibitory component on GIRK activity that is independent of PIP2-depletion, but attributed to the activation of Ca2+-dependent PKC isoforms. As a novel finding, we demonstrate receptor-dependent differences in GIRK inhibition according to receptor-specific activation of the Ca2+-dependent PKC isoforms PKCα and PKCß. Pharmacological inhibition of PKCα, but not of PKCß, abolishes GIRK inhibition induced by stimulation of α1B-receptors. In contrast, ET-R-induced reduction of GIRK activity is sensitive to pharmacological block of PKCß, but not of PKCα. Coexpression of α1B-receptors (or ETB-R) and PKCα (or PKCß) in HEK 293 cells increased homologous receptor desensitization as indicated by a rapid decline of the CKAR FRET signal monitoring receptor activity. These data suggest that receptor-species dependent differences in PKC isoform activation regulate both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism.

Biased signaling of Ca2+-sensing receptors in cardiac myocytes regulates GIRK channel activity.

Ca2+-sensing receptors (CaSRs) belong to the class C of G protein-coupled receptors and are activated by extracellular Ca2+. CaSRs display biased G protein signaling by coupling to different classes of heterotrimeric G proteins depending on agonist and cell type. In this study we used fluorescent biosensors to directly analyze G protein coupling to CaSRs and downstream signaling in living cells. In HEK 293 cells, CaSRs displayed biased signaling: elevation of extracellular Ca2+ or application of the alternative agonist spermine caused activation of Gi- and Gq-proteins. Adult cardiac myocytes express endogenous CaSRs, which have been implicated in regulating Ca2+ signaling and contractility. Biased signaling of CaSRs has not been investigated in these cells. To evaluate efficiencies of Gi- and Gq-signaling via CaSRs in rat atrial myocytes, we measured G protein-activated K+ (GIRK) channels. Activation of GIRK requires binding of Gßγ subunits released from Gi proteins, whereas Gq-signaling results in inhibition of GIRK channel activity. Stimulation of CaSRs by Ca2+ or spermine failed to directly activate Gi and GIRK channels. When GIRK channels were pre-activated via endogenous M2 receptors, stimulation of CaSRs caused pronounced inhibition of GIRK currents. This effect was specific to CaSR activation: GIRK current inhibition was sensitive to NPS-2143, a negative allosteric modulator of CaSRs, and abrogated by FR900359, a direct inhibitor of Gq. GIRK current inhibition was also sensitive to the PKC inhibitor chelerythrine, suggesting that following activation of CaSR and Gq, GIRK currents are modulated by PKC phosphorylation. We conclude from this data that cardiac CaSRs do not activate Gi and affect GIRK currents preferentially via the Gq/PKC pathway.