Identification of the structural determinant responsible for the phosphorylation of G‐protein activated potassium channel 1 by cAMP‐dependent protein kinase

Müllner, Carmen; Steinecker, Bibiane; Gorischek, Astrid; Schultze-Seemann, Wolfgang

doi:10.1111/j.1742-4658.2009.07325.x

Cited by 9 publications

(11 citation statements)

References 36 publications

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“…3). The archetypal G s -adenylyl cyclase-PKA cascade enhances GIRK's P o through phosphorylation of the channel subunits on defined C-terminal amino acids residues (Medina et al, 2000;Mullner, Steinecker, Gorischek, & Schreibmayer, 2009;Mullner et al, 2000;Mullner, Yakubovich, Dessauer, Platzer, & Schreibmayer, 2003;Rusinova et al, 2009). Phosphorylation by PKA has been proposed to serve as an "on-switch" for channel activation by Gβγ itself, with dephosphorylation by protein phosphatase 2A turning off the channel (Medina et al, 2000).…”

Section: Regulation Of Girks By Activation Of Ptx-insensitive Gαmentioning

confidence: 99%

The Roles of Gβγ and Gα in Gating and Regulation of GIRK Channels

Dascal

Kahanovitch

2015

International Review of Neurobiology

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Section: Regulation Of Girks By Activation Of Ptx-insensitive Gαmentioning

confidence: 99%

The Roles of Gβγ and Gα in Gating and Regulation of GIRK Channels

Dascal

Kahanovitch

2015

International Review of Neurobiology

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“…Rabbit antisera were raised against the recombinant protein comprising the rat GIRK1 (aa 183-501; 98.8% identity to the human sequence) and human GIRK4 (aa 178-374) C-termini that had been produced in bacteria as described [Mü llner et al, 2009]. Secondary antibody.…”

Section: Antibodiesmentioning

confidence: 99%

Cloning and characterisation of GIRK1 variants resulting from alternative RNA editing of the KCNJ3 gene transcript in a human breast cancer cell line

Wagner

Stadelmeyer

Riederer

et al. 2010

J of Cellular Biochemistry

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The aim of this study was to investigate the impact of increased mRNA levels encoding GIRK1 in breast tumours on GIRK protein expression. mRNA levels encoding hGIRK1 and hGIRK4 in the MCF7, MCF10A and MDA-MB-453 breast cancer cell lines were assessed and the corresponding proteins detected using Western blots. cDNAs encoding for four hGIRK1 splice variants (hGIRK1a, 1c, 1d and 1e) were cloned from the MCF7 cell line. Subcellular localisation of fluorescence labelled hGIRK1a-e and hGIRK4 and of endogenous GIRK1 and GIRK4 subunits was monitored in the MCF7 cell line. All hGIRK1 splice variants and hGIRK4 were predominantly located within the endoplasmic reticulum. Heterologous expression in Xenopus laevis oocytes and two electrode voltage clamp experiments together with confocal microscopy were performed. Only the hGIRK1a subunit was able to form functional GIRK channels in connection with hGIRK4. The other splice variants are expressed, but exert a dominant negative effect on heterooligomeric channel function. Hence, alternative splicing of the KCNJ3 gene transcript in the MCF7 cell line leads to a family of mRNA's, encoding truncated versions of the hGIRK1 protein. The very high abundance of mRNA's encoding GIRK1 together with the presence of GIRK1 protein suggests a pathophysiological role in breast cancer.

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“…cRNA was synthesised according to [28]. Phosphorylation deficient mutations of heterooligomeric rGIRK1 WT were produced using the identical primers as described for the homooligomeric rGIRK1 ⁎ construct [25]. The primers used for site directed mutagenesis of potential S/Ts within the hGIRK4 sequence are listed in supplementary Table S1.…”

Section: Methodsmentioning

confidence: 99%

“…The primers used for site directed mutagenesis of potential S/Ts within the hGIRK4 sequence are listed in supplementary Table S1. Plasmids for production of recombinant truncated intracellular regions of hGIRK4 fused to GST were produced according to [25] using suitable primers are listed in supplementary Table S2. All constructs and mutations were verified by sequencing.…”

Section: Methodsmentioning

confidence: 99%

Molecular basis of the facilitation of the heterooligomeric GIRK1/GIRK4 complex by cAMP dependent protein kinase

Treiber

Rosker

Keren-Raifman

et al. 2013

Biochimica et Biophysica Acta (BBA) - Biomembranes

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G-protein activated inwardly rectifying K+ channels (GIRKs) of the heterotetrameric GIRK1/GIRK4 composition mediate IK + ACh in atrium and are regulated by cAMP dependent protein kinase (PKA). Phosphorylation of GIRK1/GIRK4 complexes promotes the activation of the channel by the G-protein Gβγ-dimer (“heterologous facilitation”). Previously we reported that 3 serines/threonines (S/Ts) within the GIRK1 subunit are phosphorylated by the catalytic subunit of PKA (PKA-cs) in-vitro and are responsible for the acute functional effects exerted by PKA on the homooligomeric GIRK1F137S (GIRK1⁎) channel. Here we report that homooligomeric GIRK4WT and GIRK4S143T (GIRK4⁎) channels are clearly regulated by PKA phosphorylation. Heterooligomeric channels of the GIRK1S385CS401CT407C/GIRK4WT composition, where the GIRK1 subunit is devoid of PKA mediated phosphorylation, exhibited reduced but still significant acute effects (reduction during agonist application was ≈ 49% compared to GIRK1WT/GIRK4WT). Site directed mutagenesis of truncated cytosolic regions of GIRK4 revealed four serines/threonines (S/Ts) that were heavily phosphorylated by PKA-cs in vitro. Two of them were found to be responsible for the acute effects exerted by PKA in vivo, since the effect of cAMP injection was reduced by ≈ 99% in homooligomeric GIRK4⁎T199CS412C channels. Coexpression of GIRK1WT/GIRK4T199CS412C reduced the acute effect by ≈ 65%. Only channels of the GIRK1S385CS401CT407C/GIRK4T199CS412C composition were practically devoid of PKA mediated effects (reduction by ≈ 97%), indicating that both subunits contribute to the heterologous facilitation of IK + ACh.

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Identification of the structural determinant responsible for the phosphorylation of G‐protein activated potassium channel 1 by cAMP‐dependent protein kinase

Cited by 9 publications

References 36 publications

The Roles of Gβγ and Gα in Gating and Regulation of GIRK Channels

The Roles of Gβγ and Gα in Gating and Regulation of GIRK Channels

Cloning and characterisation of GIRK1 variants resulting from alternative RNA editing of the KCNJ3 gene transcript in a human breast cancer cell line

Molecular basis of the facilitation of the heterooligomeric GIRK1/GIRK4 complex by cAMP dependent protein kinase

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