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

• Published in: Pflügers Archiv Vol. 476; no. 7; pp. 1041 - 1064
• Main Authors: Inderwiedenstraße, Leonie, Kienitz, Marie-Cécile
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-07-2024; Springer Nature B.V
• Subjects: Adrenergic receptors; Angiotensin; Angiotensin receptors; Biomedical and Life Sciences; Biomedicine; Cell Biology; Channel gating; Excitability; G protein-coupled receptors; Heart rate; Human Physiology; Ion Channels; Isoforms; Kinases; Molecular Medicine; Mutants; Myocytes; Neurosciences; Parasympathetic nervous system; Phenylephrine; Phosphatidylinositol 4,5-diphosphate; Phosphorylation; Potassium channels (inwardly-rectifying); Protein kinase C; Receptors; Receptors and Transporters; Serine; GIRK channel; α; receptor; AT; Angiotensin; PKCε; GIRK4 (S418A) mutant
• ISSN: 0031-6768; 1432-2013; 1432-2013
• DOI: 10.1007/s00424-024-02966-5

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 G
q
-coupled receptors (G
q
PCRs) by depletion of phosphatidyl-4,5-bisphosphate (PIP
2
) and/or channel phosphorylation by protein kinase C (PKC). The G
q
PCR-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 α
1B
-adrenergic receptors (α
1B
-ARs). Deletion of the α
1B
-AR serine residues S
396
and S
400
results in a preferential regulation of GIRK activity by PKCβ. As a novel finding, we report that the AT
1
-receptor-induced GIRK inhibition depends on the activation of the nPKC isoform PKCε whereas PKCα and PKCβ 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 AT
1
-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 S
418
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 α
1B
-ARs or AT
1
-receptors. Mutation of S
418
did not impede α
1B
-AR-mediated GIRK inhibition, suggesting that S
418
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 AT
1
-R-induced GIRK reduction. Instead, phosphorylation of S
418
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 G
q
PCR-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.