Angiotensin receptors and α1B-adrenergic receptors regulate native IK(ACh) and phosphorylation-deficient GIRK4 (S418A) channels through different PKC isoforms.

Signaling of G protein-activated inwardly rectifying K+ (GIRK) channels is an important mechanism of the parasympathetic regulation of the heart rate and cardiac excitability. GIRK channels are inhibited during stimulation of Gq-coupled receptors (GqPCRs) by depletion of phosphatidyl-4,5-bisphosphate (PIP2) and/or channel phosphorylation by protein kinase C (PKC). The GqPCR-dependent modulation of GIRK currents in terms of specific PKC isoform activation was analyzed in voltage-clamp experiments in rat atrial myocytes and in CHO or HEK 293 cells. By using specific PKC inhibitors, we identified the receptor-activated PKC isoforms that contribute to phenylephrine- and angiotensin-induced GIRK channel inhibition. We demonstrate that the cPKC isoform PKCα significantly contributes to GIRK inhibition during stimulation of wildtype α1B-adrenergic receptors (α1B-ARs). Deletion of the α1B-AR serine residues S396 and S400 results in a preferential regulation of GIRK activity by PKCß. As a novel finding, we report that the AT1-receptor-induced GIRK inhibition depends on the activation of the nPKC isoform PKCε whereas PKCα and PKCß do not mainly participate in the angiotensin-mediated GIRK reduction. Expression of the dominant negative (DN) PKCε prolonged the onset of GIRK inhibition and significantly reduced AT1-R desensitization, indicating that PKCε regulates both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism. The serine residue S418 represents an important phosphorylation site for PKCε in the GIRK4 subunit. To analyze the functional impact of this PKC phosphorylation site for receptor-specific GIRK channel modulation, we monitored the activity of a phosphorylation-deficient (GIRK4 (S418A)) GIRK4 channel mutant during stimulation of α1B-ARs or AT1-receptors. Mutation of S418 did not impede α1B-AR-mediated GIRK inhibition, suggesting that S418 within the GIRK4 subunit is not subject to PKCα-induced phosphorylation. Furthermore, activation of angiotensin receptors induced pronounced GIRK4 (S418A) channel inhibition, excluding that this phosphorylation site contributes to the AT1-R-induced GIRK reduction. Instead, phosphorylation of S418 has a facilitative effect on GIRK activity that was abolished in the GIRK4 (S418A) mutant. To summarize, the present study shows that the receptor-dependent regulation of atrial GIRK channels is attributed to the GqPCR-specific activation of different PKC isoforms. Receptor-specific activated PKC isoforms target distinct phosphorylation sites within the GIRK4 subunit, resulting in differential regulation of GIRK channel activity with either facilitative or inhibitory effects on GIRK currents.

PKC-isoform specific regulation of receptor desensitization and KCNQ1/KCNE1 K+ channel activity by mutant α1B-adrenergic receptors.

Activation of a specific protein kinase C (PKC) isoform during stimulation of Gq protein-coupled receptors (GqPCRs) is determined by homologous receptor desensitization that controls the spatiotemporal formation of downstream Gq signalling molecules. Furthermore, GqPCR-activated PKC isoforms specifically regulate receptor activity via a negative feedback mechanism. In the present study, we investigated the contribution of several phosphorylation sites in the α1B-adrenergic receptor (α1B-AR) for PKC and G protein coupled receptor kinase 2 (GRK2) to homologous receptor desensitization and effector modulation. We analyzed signalling events downstream to human wildtype α1B-ARs and α1B-ARs lacking PKC or GRK2 phosphorylation sites (Δ391-401, α1B-ΔPKC-AR and Δ402-520, α1B-ΔGRK-AR) by means of FRET-based biosensors in HEK293 that served as online-assays of receptor activity. K+ currents through KCNQ1/KCNE1 channels (IKs), which are regulated by both phosphatidylinositol 4,5-bisphosphate (PIP2)-depletion and/or phosphorylation by PKC, were measured as a functional readout of wildtype and mutant α1B-AR receptor activity. As a novel finding, we provide evidence that deletion of PKC and GRK2 phosphorylation sites in α1B-ARs abrogates the contribution of PKCα to homologous receptor desensitization. Instead, the time course of mutant receptor activity was specifically modulated by PKCß. Mutant α1B-ARs displayed pronounced homologous receptor desensitization that was abolished by PKCß-specific pharmacological inhibitors. IKs modulation during stimulation of wildtype and mutant α1B-ARs displayed transient inhibition and current facilitation after agonist withdrawal with reduced capability of mutant α1B-ARs to induce IKs inhibition. Pharmacological inhibition of the PKCß isoform did not augment IKs reduction by mutant α1B-ARs, but shifted IKs modulation towards current facilitation. Coexpression of an inactive (dominant-negative) PKCδ isoform (DN-PKCδ) abolished IKs facilitation in α1B-ΔGRK-AR-expressing cells, but not in α1B-ΔPKC-AR-expressing cells. The data indicate that the differential modulation of IKs activity by α1B-ΔGRK- and α1B-ΔPKC-receptors is attributed to the activation of entirely distinct novel PKC isoforms. To summarize, specific phosphorylation sites within the wildtype and mutant α1B-adrenergic receptors are targeted by different PKC isoforms, resulting in differential regulation of receptor desensitization and effector function.