C-terminal phosphorylation of NaV1.5 impairs FGF13-dependent regulation of channel inactivation

Abstract

Voltage-gated Na (Na) channels are key regulators of myocardial excitability, and Ca/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in Na1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of Na1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify the phosphorylation sites in the Na1.5 proteins purified from adult WT and failing CaMKIIδ-overexpressing (CaMKIIδ-Tg) mouse ventricles. Of 19 native Na1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδ-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of Na1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases Na1.5 channel availability and decreases late Na current, two effects that were abrogated with Na1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to Na1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with Na1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of Na1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδ-dependent arrhythmogenic disorders in failing hearts.

Conduction in the right and left ventricle is differentially regulated by protein kinases and phosphatases: implications for arrhythmogenesis

“…This finding would be in line with those cellular studies that showed that both CaMKII and PKA activities upregulate I Na (2,44,52,83,85,89). Studies from different groups revealed that both PKA and CaMKII phosphorylate the cardiac ␣-subunit (Nav1.5) at multiple residues (1,5,12,25,34,55,92), the majority located on the intracellular/cytosolic loop connecting domains 1 and 2 (1st intracellular linker loop) (12,30,50,51). It remains undetermined whether there are targets in Nav1.5 shared by the two kinases.…”

“…Given that the effects of the CaMKII blocker KN93 and PKA blocker H89 on conduction delay distribution are very similar (see Fig. 6, C and D) and given that previous studies indicated that CaMKII and PKA target distinct Nav1.5 phosphorylation sites (1,5,12,25,34,55,92), we hypothesized that regulation of conduction by the two kinases might be synergistic. Hence, we tested the effect of combined administration of KN93 and H89.…”

“…Thus I Na regulation by kinases/phosphatases directly impacts cellular excitability. It is well established that Nav1.5, the I Na pore-forming ␣-subunit of the main cardiac sodium channel, contains various phosphorylation sites that are mostly located in the first intracellular linker loop of the channel (12,30,50,51), some of which have been identified as targets for various protein kinases including CaMKII (5,12,25,34), PKA (1,55,92), PKC (27), and others (20,50). While an effective/ optimal phosphorylation level is likely achieved by the combined action of protein kinases and phosphatases (14,87), which phosphatases are responsible for this regulation and how dephosphorylation specificity for Nav1.5 is achieved remain largely unknown.…”

Phosphorylation of cardiac voltage‐gated sodium channel: Potential players with multiple dimensions

“…Among these, phosphorylation of serine residues at position 1938 and 1989 was increased by CaMKIIδ C overexpression. Both serine residues (1938 and 1989) are conserved in the human Na V 1.5 channel and when the orthologous serine residues at position 1933 and 1984 in human Na V 1.5 (hH1c) are mutated, interaction of FGF‐13 with Na V 1.5 channels is disrupted; this alters fast inactivation, increases late I Na and decreases channel availability . In another recent study, mass‐spectrometry based analysis of immunopurified and CaMKIIδ C ‐mediated in vitro phosphorylation of human Na V 1.5 expressed in HEK‐293 cells revealed 31 serine and three threonine phosphorylated residues .…”

“Heart Oddity”: Intrinsically Reduced Excitability in the Right Ventricle Requires Compensation by Regionally Specific Stress Kinase Function

“…Interestingly, the conductivity of channels involved in the generation and transmission of the ventricular impulse (notably the cardiac Na + channel, Nav1.5) may be modulated by phosphorylation, and thus are amenable to regulation by protein kinases responding to various neural and hormonal signals, in particular transmitted through activation of G-protein-coupled receptors (GPCRs) (Sato et al, 2015). Prominent in these signaling pathways are the calcium/calmodulin-dependent protein kinase II (CaMKII) (Hund et al, 2010;Ashpole et al, 2012;Glynn et al, 2015;Burel et al, 2017) and the cAMP-activated protein kinase A (PKA) (Murphy et al, 1996;Zhou et al, 2002;Aiba et al, 2014), collectively known as "stress" kinases for their involvement in the "fight or flight" physiological response (Wehrens et al, 2004;Wu et al, 2016). This mini-review will focus on very recent (and still limited) information regarding how the electrical wave propagation through ventricular chambers is regulated by stress kinases.…”