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

H. Cserne Szappanos; P. Muralidharan; E. Ingley; J. Petereit; H.A. Millar; L.C. Hool

doi:10.1038/s41598-017-15087-0

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

Journal article

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Peer reviewed

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

H. Cserne Szappanos, P. Muralidharan, E. Ingley, J. Petereit, H.A. Millar and L.C. Hool

Scientific Reports, Vol.7(1), art. no. 15118

2017

DOI: https://doi.org/10.1038/s41598-017-15087-0

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Abstract

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 Ca2+ channel (Cav1.2) is absolutely required. Here we identify the functionally relevant site for PKA phosphorylation on the human cardiac L-type Ca2+ 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

Details

Title: Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel
Authors/Creators: H. Cserne Szappanos (Author/Creator)
P. Muralidharan (Author/Creator) - The University of Western Australia
E. Ingley (Author/Creator) - Harry Perkins Institute of Medical Research
J. Petereit (Author/Creator) - ARC Centre of Excellence in Plant Energy Biology
H.A. Millar (Author/Creator)
L.C. Hool (Author/Creator) - The University of Western Australia
Publication Details: Scientific Reports, Vol.7(1), art. no. 15118
Publisher: Springer Nature
Identifiers: 991005545180107891
Copyright: © 2017 The Author(s)
Murdoch Affiliation: School of Veterinary and Life Sciences
Language: English
Resource Type: Journal article

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Collaboration types: Domestic collaboration
Citation topics: 1 Clinical & Life Sciences; 1.79 Molecular & Cell Biology - Physiology; 1.79.239 Ion Channelopathies
Web Of Science research areas: Biochemistry & Molecular Biology
ESI research areas: Biology & Biochemistry