Mass Spectrometry-Based Identification of Native Cardiac Nav1.5 Channel α Subunit Phosphorylation Sites

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.