RÉSUMÉ
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.
Sujet(s)
Cricetulus , Canaux potassiques rectifiants entrants couplés aux protéines G , Protéine kinase C , Canaux potassiques rectifiants entrants couplés aux protéines G/métabolisme , Animaux , Phosphorylation , Cellules HEK293 , Humains , Rats , Protéine kinase C/métabolisme , Cellules CHO , Récepteurs alpha-1 adrénergiques/métabolisme , Myocytes cardiaques/métabolisme , Mâle , Rat Wistar , Protein kinase C-alpha/métabolisme , Isoenzymes/métabolismeRÉSUMÉ
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.