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Heterologous Facilitation of G Protein-Activated K(+) Channels by β-Adrenergic Stimulation via Camp-Dependent Protein Kinase
To investigate possible effects of adrenergic stimulation on G protein–activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise...
Autores principales: | , , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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The Rockefeller University Press
2000
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2217221/ https://www.ncbi.nlm.nih.gov/pubmed/10779313 |
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author | Müllner, Carmen Vorobiov, Dimitry Bera, Amal Kanti Uezono, Yasuhito Yakubovich, Daniel Frohnwieser-Steinecker, Bibiane Dascal, Nathan Schreibmayer, Wolfgang |
author_facet | Müllner, Carmen Vorobiov, Dimitry Bera, Amal Kanti Uezono, Yasuhito Yakubovich, Daniel Frohnwieser-Steinecker, Bibiane Dascal, Nathan Schreibmayer, Wolfgang |
author_sort | Müllner, Carmen |
collection | PubMed |
description | To investigate possible effects of adrenergic stimulation on G protein–activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0.4 ± 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 ± 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 ± 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 μM H89 {N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA}. This heterologous facilitation of GIRK current by β-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing β(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30–60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the β(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the β-adrenergic receptor (β(2)AR) by PKA was excluded by using a mutant β(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the α subunit of G proteins (Gα(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Gα(s), GIRK currents were inhibited, presumably due to sequestration of the β/γ subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, β-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from β(2)AR. |
format | Text |
id | pubmed-2217221 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2000 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-22172212008-04-21 Heterologous Facilitation of G Protein-Activated K(+) Channels by β-Adrenergic Stimulation via Camp-Dependent Protein Kinase Müllner, Carmen Vorobiov, Dimitry Bera, Amal Kanti Uezono, Yasuhito Yakubovich, Daniel Frohnwieser-Steinecker, Bibiane Dascal, Nathan Schreibmayer, Wolfgang J Gen Physiol Original Article To investigate possible effects of adrenergic stimulation on G protein–activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0.4 ± 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 ± 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 ± 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 μM H89 {N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA}. This heterologous facilitation of GIRK current by β-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing β(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30–60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the β(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the β-adrenergic receptor (β(2)AR) by PKA was excluded by using a mutant β(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the α subunit of G proteins (Gα(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Gα(s), GIRK currents were inhibited, presumably due to sequestration of the β/γ subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, β-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from β(2)AR. The Rockefeller University Press 2000-05-01 /pmc/articles/PMC2217221/ /pubmed/10779313 Text en © 2000 The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Original Article Müllner, Carmen Vorobiov, Dimitry Bera, Amal Kanti Uezono, Yasuhito Yakubovich, Daniel Frohnwieser-Steinecker, Bibiane Dascal, Nathan Schreibmayer, Wolfgang Heterologous Facilitation of G Protein-Activated K(+) Channels by β-Adrenergic Stimulation via Camp-Dependent Protein Kinase |
title | Heterologous Facilitation of G Protein-Activated K(+) Channels by β-Adrenergic Stimulation via Camp-Dependent Protein Kinase |
title_full | Heterologous Facilitation of G Protein-Activated K(+) Channels by β-Adrenergic Stimulation via Camp-Dependent Protein Kinase |
title_fullStr | Heterologous Facilitation of G Protein-Activated K(+) Channels by β-Adrenergic Stimulation via Camp-Dependent Protein Kinase |
title_full_unstemmed | Heterologous Facilitation of G Protein-Activated K(+) Channels by β-Adrenergic Stimulation via Camp-Dependent Protein Kinase |
title_short | Heterologous Facilitation of G Protein-Activated K(+) Channels by β-Adrenergic Stimulation via Camp-Dependent Protein Kinase |
title_sort | heterologous facilitation of g protein-activated k(+) channels by β-adrenergic stimulation via camp-dependent protein kinase |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2217221/ https://www.ncbi.nlm.nih.gov/pubmed/10779313 |
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