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Macromolecular Voltage-Gated Na+ Channel Complexes in the Regulation of Normal and Diseased Cardiac Excitability

Final Report Summary - NAVEX (Macromolecular Voltage-Gated Na+ Channel Complexes in the Regulation of Normal and Diseased Cardiac Excitability)

The general objective of the three years-NavEx project contract was to characterize the molecular and cellular mechanisms involved in controlling and/or modulating the expression and the functioning of the cardiac voltage-gated Na+ (Nav) channels in native cardiac cells.

To achieve these goals, a MS-based proteomic approach was developed and utilized to identify in situ the molecular components of native cardiac Nav channel complexes. Nav channel complexes were immunoprecipitated from adult mouse cardiac ventricles using an anti-NavPAN specific antibody. The MS analyses of cardiac Nav channel immunoprecipitates revealed that the anti-NavPAN antibody immunoprecipitates several Nav  subunits in addition to Nav1.5 from adult mouse ventricles, as well as several previously identified Nav channel associated/regulatory proteins. Additionally, and directly of interest here, these analyses also resulted in the identification of several novel putative associated/regulatory proteins of Nav channels, among which three have been selected for further analyses: the Eps15 interacting protein 2 (Epsin2), the Oxysterol-binding protein-Related Protein 11 (ORP11) and the plakoglobin. Additional biochemical experiments confirmed that Nav1.5 co-immunoprecipitates with each of the three proteins identified when co-expressed in HEK293 cells, which provides an independent validation of the association with Nav1.5. Preliminary analyses in primary cultured neonatal rat ventricular myocytes in which ORP11 or plakoglobin expression was eliminated transiently by knockdown, however, did not show any changes in Nav1.5 total or cell surface expression. Additional voltage-clamp analyses are therefore required to test whether biophysical properties of cardiac Nav1.5 channels are altered.

In addition to interacting proteins, phosphoproteomic analyses of purified cardiac Nav1.5 protein identified 11 serine/threonine phosphorylation sites, 8 of which are novel. With the exception of 1 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. The results of this phosphoproteomic study have been published in the Journal of Proteome Research (Marionneau et al, 2012). Biochemical experiments have since been undertaken and demonstrated that two (serine to glutamate) phosphomutants show increased cell surface expression as compared with wild-type channels, suggesting critical roles for these two novel serine phosphorylation sites in modulating the cell surface expression of Nav1.5 channels. Together, these analyses: (1) provided the first in situ phosphorylation map of cardiac Nav1.5 channels; (2) demonstrated that native cardiac Nav1.5 channels are highly phosphorylated; and (3) identified two serine phosphorylation sites as critical determinants of Nav1.5 channel cell surface expression in HEK293 cells.

Although many investigators have studied the biochemical and biophysical properties of the cardiac Nav1.5 channel, which is a proven therapeutic target for the development of anti-arrhythmic agents, many of the mechanisms that regulate the expression and the functioning of these channels in situ are not characterized. The long-term investigations of the research projects developed in NavEx recognize this gap, and the studies of the novel Nav1.5 associated/regulatory proteins and phosphorylation sites could have a real impact in various clinical settings, including the prevention, the diagnostic and the treatment of cardiac arrhythmias. Although complete validation of our proteomic results has not been obtained during this (3 year) funding period, NavEx has enabled to generate and validate sufficient results to put us in a strong position to compete for other national and European financial supports