PhosphoNav

Voltage-gated Na+ (Nav) channels are key determinants of myocardial excitability and defects in Nav channel functioning or regulation, associated with inherited and acquired cardiac disease, increase the risk of life-threatening arrhythmias. In heart failure, the inactivation gating properties of the main cardiac Nav channels, the Nav1.5 channels, are altered, resulting in decreased channel availability and increased late Na+ current. Among the major determinants suggested to cause these defects are the activation of specific kinases, primarily the Ca2+/Calmodulin Kinase II (CaMKII), and the direct phosphorylation of the Nav1.5 channel pore-forming subunit. Mass spectrometry (MS)-based phosphoproteomic analyses in the laboratory were undertaken to identify in situ the native phosphorylation sites on the Nav1.5 and associated/regulatory proteins purified from mouse cardiac ventricles in both basal (wild-type) and failing (CaMKIIdc-overexpressing) conditions. These experiments led to the identification of several native phosphorylation sites: nineteen were identified in Nav1.5 and one in Fibroblast Growth Factor 13 (FGF13), a key Nav channel associated/regulatory protein involved in channel inactivation. Of these newly-identified phosphorylation sites, interestingly, three in the C-terminus of Nav1.5 abut the binding sites for FGF13 and Ca2+/calmodulin, another key regulator of Nav channel inactivation, and conversely, the FGF13 phosphorylation site is in the Nav channel-binding site. We hypothesize that phosphorylation at one or several of these four sites regulates Nav1.5 channel inactivation gating by altering interaction with FGF13 and/or Ca2+/calmodulin. Specifically, we will test the hypotheses that (1) the regulation of cardiac Nav1.5 channel inactivation by FGF13 and Ca2+/calmodulin in basal physiological conditions depends on the phosphorylation of Nav1.5 at serine-1884 and of FGF13 at serine-218; and (2) the phosphorylation of Nav1.5 by CaMKII at serines-1933 and -1984 in heart failure adversely affects the interaction of Ca2+/calmodulin and FGF13 with the channel and the associated regulation of channel inactivation. By specifying mechanisms whereby phosphorylation regulates cardiac Nav1.5 channel function, our results will link cell signaling to membrane excitability and foster a better understanding of the role of Nav1.5 channels in health and disease, which is needed for improved prevention and treatment of arrhythmias.