Regulation of atrial G-protein activated (GIRK) channels by phosphatidylinositol 4,5-bisphosphate and proteinkinase C isoforms
Final Report Abstract
Signalling 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 proteinkinase C (PKC). The contribution of different PKC isoforms to phenylephrine- and angiotensin-induced GIRK channel inhibition was analyzed in voltage-clamp experiments in rat atrial myocytes and in CHO or HEK 293 cells. GIRK inhibition during stimulation of wildtype α1B-adrenergic receptors (α1B-ARs) was specifically induced by the cPKC isoform PKCα. In contrast, AT1-receptor-induced GIRK inhibition is independent of cPKC activation but determined by the PKCε isoform. Expression of the dominant negative PKC isoform DN PKCε significantly prolonged the onset of GIRK inhibition and reduced AT1-R desensitization, indicating that PKCε regulates both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism. To obtain information about the PKC phosphorylation sites interfering with receptor-specific GIRK channel inhibition, we analyzed the activity of phosphorylation-deficient (GIRK4 (S191A)) GIRK4 channel mutants during stimulation of α1B-ARs or AT1-receptors in rat atrial myocytes. Stimulation of α1B-ARs induced pronounced GIRK4 (S191A) channel inhibition, suggesting that S191 within the GIRK4 subunit is not subject to PKCα-induced phosphorylation. In contrast, GIRK4 (S191A) channel inhibition was significantly reduced during AT1-R stimulation, indicating that the inhibitory effect of angiotensin comprises at least phosphorylation of S191. Mutation of S418, an important phosphorylation site for PKCε in the GIRK4 subunit, did not impede angiotensin-mediated GIRK 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 phosphorylation-deficient GIRK4 (S418A) mutant. To summarize, we identified receptor-specific PKC isoforms that participate in the regulation of atrial GIRK channel activity by wildtype α1B-ARs and AT1-receptors. Our data indicate that the receptorspecies dependent modulation of GIRK channel activity depends on the integrity of different phosphorylation sites within the GIRK4 subunit. The receptor-dependent phosphorylation pattern differentially regulates GIRK channel activity and determines the extent of GIRK reduction by modulating the balance between inhibitory and facilitative PKC effects.
Publications
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PKC-isoform specific regulation of receptor desensitization and KCNQ1/KCNE1 K+ channel activity by mutant α1B-adrenergic receptors. Cellular Signalling, 91, 110228.
Renkhold, Lina; Kollmann, Rike; Inderwiedenstraße, Leonie & Kienitz, Marie-Cecile
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Receptor-specific regulation of phosphorylation-deficient (GIRK4S191A) G protein-activated K+ channels, 11th Symposium of the Young Physiologists 31.03- 01.04.2022, Essen
Kienitz, Marie-Cécile
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Receptor-specific regulation of G protein-activated K+ channels, 12th Symposium of the Young Physiologists 30.03- 31.03.2023, Kiel
Kienitz, Marie-Cécile
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Angiotensin receptors and α1B-adrenergic receptors regulate native IK(ACh) and phosphorylation-deficient GIRK4 (S418A) channels through different PKC isoforms. Pflügers Archiv - European Journal of Physiology, 476(7), 1041-1064.
Inderwiedenstraße, Leonie & Kienitz, Marie-Cécile
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Functional impact of the serine residue S418 within the GIRK4 subunit for Angiotensininduced GIRK current modulation, 13th Symposium of the Young Physiologists, 11.3.-12.3.2024, Mannheim
Kienitz, Marie-Cécile
