Abstract 13360: Mass Spectrometry-Based Identification of Native Cardiac Nav1.5 Channel Complexes and Phosphorylation Sites
Cardiac voltage-gated Na+ (Nav) channels are key determinants of action potential waveforms, refractoriness and propagation, and defects in Nav channel functioning or regulation, associated with inherited and acquired cardiac disease, are linked to increased risk of life threatening arrhythmias. Although previous studies have suggested roles for specific proteins and kinases in mediating Nav channel modulation, the in situ regulatory components of cardiac Nav channels remain largely unknown. A mass spectrometry (MS)-based proteomic approach was developed here to identify native cardiac Nav channel complexes and in situ phosphorylation sites on the Nav1.5 pore-forming (α) subunit. Using anti-NavPAN specific antibodies (directed against all Nav α subunits), Nav channel complexes were immunoprecipitated from adult wild-type mouse heart and channel components were identified by MS. In addition to Nav1.5, which was the most abundant protein identified, these analyses identified three other Nav α subunits, Nav1.4, Nav1.3 and Nav1.9, as well as some of the previously identified Nav channel associated/regulatory proteins, such as the Calmodulin, the CamKII δ subunit and Fibroblast Growth Factor 13. Interestingly, these analyses also revealed two novel membrane targeting proteins, the Eps15 interacting protein 2 (Epsin2) and the Protein kinase C and casein kinase II substrate protein 3 (Pacsin3) in native cardiac Nav channel complexes. Additional biochemical experiments confirmed that Nav1.5 co-immunoprecipitates with both Epsin2 and Pacsin3 when co-expressed in heterologous cells. In addition to interacting proteins, phosphoproteomic analyses of purified cardiac Nav1.5 protein identified 11 serine/threonine phosphorylation sites, 9 of which are novel. With the exception of one residue located in the cytoplasmic N-terminus, all the phosphorylation sites identified are in the first intracellular linker loop, suggesting a critical role for this region in phosphorylation-dependent regulation of Nav1.5 channel expression and functioning. In addition to identifying two novel cardiac Nav1.5 associated proteins, the unbiased MS-based analyses used here provide the first in situ phosphorylation map of cardiac Nav1.5-encoded channels.
- © 2012 by American Heart Association, Inc.