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Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel

The “Fight or Flight” response is elicited by extrinsic stress and is necessary in many species for survival. The response involves activation of the β-adrenergic signalling pathway. Surprisingly the mechanisms have remained unresolved. Calcium influx through the cardiac L-type Ca(2+) channel (Ca(v)...

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Autores principales: Cserne Szappanos, Henrietta, Muralidharan, Padmapriya, Ingley, Evan, Petereit, Jakob, Millar, A. Harvey, Hool, Livia C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680263/
https://www.ncbi.nlm.nih.gov/pubmed/29123182
http://dx.doi.org/10.1038/s41598-017-15087-0
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author Cserne Szappanos, Henrietta
Muralidharan, Padmapriya
Ingley, Evan
Petereit, Jakob
Millar, A. Harvey
Hool, Livia C.
author_facet Cserne Szappanos, Henrietta
Muralidharan, Padmapriya
Ingley, Evan
Petereit, Jakob
Millar, A. Harvey
Hool, Livia C.
author_sort Cserne Szappanos, Henrietta
collection PubMed
description The “Fight or Flight” response is elicited by extrinsic stress and is necessary in many species for survival. The response involves activation of the β-adrenergic signalling pathway. Surprisingly the mechanisms have remained unresolved. Calcium influx through the cardiac L-type Ca(2+) channel (Ca(v)1.2) is absolutely required. Here we identify the functionally relevant site for PKA phosphorylation on the human cardiac L-type Ca(2+) channel pore forming α1 subunit using a novel approach. We used a cell free system where we could assess direct effects of PKA on human purified channel protein function reconstituted in proteoliposomes. In addition to assessing open probability of channel protein we used semi-quantitative fluorescent phosphoprotein detection and MS/MS mass spectrometry analysis to demonstrate the PKA specificity of the site. Robust increases in frequency of channel openings were recorded after phosphorylation of the long and short N terminal isoforms and the channel protein with C terminus truncated at aa1504. A protein kinase A anchoring protein (AKAP) was not required. We find the novel PKA phosphorylation site at Ser1458 is in close proximity to the Repeat IV S6 region and induces a conformational change in the channel protein that is necessary and sufficient for increased calcium influx through the channel.
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spelling pubmed-56802632017-11-17 Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel Cserne Szappanos, Henrietta Muralidharan, Padmapriya Ingley, Evan Petereit, Jakob Millar, A. Harvey Hool, Livia C. Sci Rep Article The “Fight or Flight” response is elicited by extrinsic stress and is necessary in many species for survival. The response involves activation of the β-adrenergic signalling pathway. Surprisingly the mechanisms have remained unresolved. Calcium influx through the cardiac L-type Ca(2+) channel (Ca(v)1.2) is absolutely required. Here we identify the functionally relevant site for PKA phosphorylation on the human cardiac L-type Ca(2+) channel pore forming α1 subunit using a novel approach. We used a cell free system where we could assess direct effects of PKA on human purified channel protein function reconstituted in proteoliposomes. In addition to assessing open probability of channel protein we used semi-quantitative fluorescent phosphoprotein detection and MS/MS mass spectrometry analysis to demonstrate the PKA specificity of the site. Robust increases in frequency of channel openings were recorded after phosphorylation of the long and short N terminal isoforms and the channel protein with C terminus truncated at aa1504. A protein kinase A anchoring protein (AKAP) was not required. We find the novel PKA phosphorylation site at Ser1458 is in close proximity to the Repeat IV S6 region and induces a conformational change in the channel protein that is necessary and sufficient for increased calcium influx through the channel. Nature Publishing Group UK 2017-11-09 /pmc/articles/PMC5680263/ /pubmed/29123182 http://dx.doi.org/10.1038/s41598-017-15087-0 Text en © The Author(s) 2017 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Cserne Szappanos, Henrietta
Muralidharan, Padmapriya
Ingley, Evan
Petereit, Jakob
Millar, A. Harvey
Hool, Livia C.
Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel
title Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel
title_full Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel
title_fullStr Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel
title_full_unstemmed Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel
title_short Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel
title_sort identification of a novel camp dependent protein kinase a phosphorylation site on the human cardiac calcium channel
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680263/
https://www.ncbi.nlm.nih.gov/pubmed/29123182
http://dx.doi.org/10.1038/s41598-017-15087-0
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