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Sci Rep. 2017 Nov 9;7(1):15118. doi: 10.1038/s41598-017-15087-0.

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

Author information

1
School of Human Sciences, University of Western Australia, Crawley, Western Australia, Australia.
2
Harry Perkins Institute of Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia, Australia.
3
School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia, Australia.
4
ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, Western Australia, Australia.
5
School of Human Sciences, University of Western Australia, Crawley, Western Australia, Australia. livia.hool@uwa.edu.au.
6
Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia. livia.hool@uwa.edu.au.

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.

PMID:
29123182
PMCID:
PMC5680263
DOI:
10.1038/s41598-017-15087-0
[Indexed for MEDLINE]
Free PMC Article

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