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

Céline Marionneau, Cheryl F. Lichti, Pierre Lindenbaum, Flavien Charpentier, Jeanne M. Nerbonne, R. Reid Townsend, Jean Mérot

Research output: Contribution to journalArticle

37 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)5994-6007
Number of pages14
JournalJournal of Proteome Research
Volume11
Issue number12
DOIs
StatePublished - Dec 7 2012

Keywords

  • Nav1.5 channels
  • heart
  • label-free comparative and data-driven LC-MS/MS analyses
  • mass spectrometric identifications
  • native phosphorylations

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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    Marionneau, C., Lichti, C. F., Lindenbaum, P., Charpentier, F., Nerbonne, J. M., Townsend, R. R., & Mérot, J. (2012). Mass spectrometry-based identification of native cardiac Nav1.5 channel α subunit phosphorylation sites. Journal of Proteome Research, 11(12), 5994-6007. https://doi.org/10.1021/pr300702c