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Mass spectrometry-based identification of native cardiac Nav1.5 channel α subunit phosphorylation sites.

Authors
  • Marionneau, Céline
  • Lichti, Cheryl F
  • Lindenbaum, Pierre
  • Charpentier, Flavien
  • Nerbonne, Jeanne M
  • Townsend, R Reid
  • Mérot, Jean
Type
Published Article
Journal
Journal of Proteome Research
Publisher
American Chemical Society
Publication Date
Dec 07, 2012
Volume
11
Issue
12
Pages
5994–6007
Identifiers
DOI: 10.1021/pr300702c
PMID: 23092124
Source
Medline
License
Unknown

Abstract

Cardiac voltage-gated Na+ (Nav) channels are key determinants of action potential waveforms, refractoriness and propagation, and Nav1.5 is the main Nav pore-forming (α) subunit in the mammalian heart. Although direct phosphorylation of the Nav1.5 protein has been suggested to modulate various aspects of Nav channel physiology and pathophysiology, native Nav1.5 phosphorylation sites have not been identified. In the experiments here, a mass spectrometry (MS)-based proteomic approach was developed to identify native Nav1.5 phosphorylation sites directly. Using an anti-NavPAN antibody, Nav channel complexes were immunoprecipitated from adult mouse cardiac ventricles. The MS analyses revealed that this antibody immunoprecipitates several Nav α subunits in addition to Nav1.5, as well as several previously identified Nav channel associated/regulatory proteins. Label-free comparative and data-driven phosphoproteomic analyses of purified cardiac Nav1.5 protein identified 11 phosphorylation sites, 8 of which are novel. All the phosphorylation sites identified except one in the N-terminus are in the first intracellular linker loop, suggesting critical roles for this region in phosphorylation-dependent cardiac Nav channel regulation. Interestingly, commonly used prediction algorithms did not reliably predict these newly identified in situ phosphorylation sites. Taken together, the results presented provide the first in situ map of basal phosphorylation sites on the mouse cardiac Nav1.5 α subunit.

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