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 G_q-coupled receptor (G_qPCR) 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 G_q-coupled α_1B- and ET-receptor-induced signalling. G_qPCR-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 PKCβ. Pharmacological inhibition of PKCα, but not of PKCβ, abolishes GIRK inhibition induced by stimulation of α_1B-receptors. In contrast, ET-R-induced reduction of GIRK activity is sensitive to pharmacological block of PKCβ, but not of PKCα. Coexpression of α_1B-receptors (or ET_B-R) and PKCα (or PKCβ) 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.