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. 2017 Oct 20;292(42):17431-17448.
doi: 10.1074/jbc.M117.787788. Epub 2017 Sep 7.

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

Sophie Burel  1 , Fabien C Coyan  1 , Maxime Lorenzini  1 , Matthew R Meyer  2 , Cheryl F Lichti  3 , Joan H Brown  4 , Gildas Loussouarn  1 , Flavien Charpentier  1 , Jeanne M Nerbonne  5   6 , R Reid Townsend  6   7 , Lars S Maier  8 , Céline Marionneau  9
Affiliations

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

Sophie Burel et al. J Biol Chem. .

Abstract

Voltage-gated Na+ (NaV) channels are key regulators of myocardial excitability, and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-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 NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na+ current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts.

Keywords: Ca2+/calmodulin-dependent protein kinase II (CaMKII); FGF13; Nav1.5; calmodulin (CaM); channel inactivation; heart; phosphoproteomics; phosphorylation; sodium channel.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Immunoprecipitation of NaV channel complexes from adult WT and CaMKIIδc-Tg mouse ventricles. A, representative NaV α Western blots of total lysates and immunoprecipitated proteins from adult WT and CaMKIIδc-Tg mouse ventricles with the anti-NaVPAN monoclonal antibody (mαNaVPAN-IPs) probed with the anti-NaV1.5 rabbit polyclonal (RbαNaV1.5) and the mαNaVPAN antibodies, respectively. B, mean ± S.E. relative NaV α protein abundance in WT (n = 4) and CaMKIIδc-Tg (n = 4) IPs. *, p < 0.05, Mann-Whitney test. C, SYPRO Ruby-stained gel of mαNaVPAN-IPs from WT and CaMKIIδc-Tg mouse ventricles. Relative abundance of proteins running at the molecular weight of NaV α subunits is higher in CaMKIIδc-Tg IPs than in WT IPs.
Figure 2.
Figure 2.
MS protein identification in immunoprecipitated NaV channel complexes from WT and CaMKIIδc-Tg mouse ventricles. A, NaV α subunits identified using the LTQ-Orbitrap XL, LTQ-Orbitrap Elite, and TripleTOF 5600 Plus mass spectrometers. The average numbers of exclusive unique peptides and total spectra for each NaV α subunit and the percent amino acid sequence coverages obtained for NaV1.5, including or excluding transmembrane domains (TD), are presented. In addition to NaV1.5, which is the most abundant protein in the mαNaVPAN-IPs, NaV1.4 is also detected, and the greater sensitivity of the Orbitrap Elite and TripleTOF mass spectrometers allowed the identification of NaV1.7, NaV1.8, and NaV1.3. B, amino acid sequence coverage obtained for the (mouse) NaV1.5 protein (NP_001240789). Detected peptides are highlighted in yellow; identified phosphorylation sites are highlighted in blue (sites already identified in our previous MS analyses) and red (newly identified sites in the present study); transmembrane segments (S1–S6) in each domain (I–IV) are in bold and underlined in black; loops I, II, and III correspond to interdomains I and II, II and III, and III and IV, respectively; and binding sites for iFGF and calmodulin (IQ-motif) are boxed in green and orange, respectively. C, relative abundances of NaV α subunits and previously characterized NaV1.5 channel-associated/regulatory proteins in the CaMKIIδc-Tg IPs (n = 4) versus the WT IPs (n = 4) were calculated from the entire (Orbitrap XL) MS1 peptide data set using the DAnTE statistical software (**, p < 0.01; ***, p < 0.001).
Figure 3.
Figure 3.
Localization of MS-identified in situ phosphorylation sites on the mouse ventricular NaV1.5 α subunit protein. Among the 19 phosphorylation sites identified, 10 (in blue) had already been identified in our previous MS analyses and 9 (in red) are novel. Four and two phosphorylation site locations are possible at amino acids 36–42 and 524–525, respectively. The three newly identified C-terminal phosphoserines at positions 1888, 1937, and 1938 (pSer-1888, pSer-1937, and pSer-1938) are in close proximity to the binding sites for iFGF and calmodulin (IQ-motif).
Figure 4.
Figure 4.
Quantification analysis of NaV1.5 phosphorylation sites in the CaMKIIδc-Tg versus the WT mαNaVPAN-IPs. A, relative abundances of 32 NaV1.5 phosphopeptides allowing assignments of the listed phosphorylation sites, in the CaMKIIδc-Tg (n = 4) versus the WT (n = 4) IPs, were calculated using label-free quantification of the (Orbitrap XL) MS1 data. The mean ± S.E. relative abundance of unphosphorylated NaV1.5 peptides in the CaMKIIδc-Tg versus the WT IPs was calculated from 86 unphosphorylated NaV1.5 peptides (minimum Scaffold peptide probability scores of 95%). Consistent with the biochemistry data (Fig. 1) and the DAnTE protein statistical analysis (Fig. 2C), the unphosphorylated NaV1.5 peptides are 3.6-fold more represented in the CaMKIIδc-Tg IPs than in the WT IPs (red dashed line). The relative abundances of individual NaV1.5 phosphopeptides are significantly (*, p < 0.05, Mann-Whitney test) different in the CaMKIIδc-Tg compared with the WT IPs. B, Tukey whisker analysis of NaV1.5 peptide relative abundance in CaMKIIδc-Tg versus WT IPs. Of the 118 (86 unphosphorylated and 32 phosphorylated) NaV1.5 peptides, only the phosphopeptides AT(pS)DNLPVR, RL(pS)(pS)GTEDGGDDR, and AL(pS)AVSVLTSALEELEESHRK (marked with † in A), exhibiting phosphorylation(s) on serines 1989 (pSer-1989), 483 and 484 (pSer-483 and pSer-484), and 664 (pSer-664), respectively, present fold change ratios (8.96-, 7.13-, and 0.58-fold) significantly different from the median ratio. C, mean ± S.E. intensities of phosphopeptides Q(−17.03)QAGSSGLSDEDAPER, assigning pSer-1937 and/or pSer-1938 (absence in WT IPs and presence in CaMKIIδc-Tg IPs), and AT(pS)DNLPVR, assigning pSer-1989 (8.96-fold change ratio), in WT (n = 4) and CaMKIIδc-Tg (n = 4) IPs. *, p < 0.05, Mann-Whitney test. D, conservation of the two C-terminal NaV1.5 serines 1938 and 1989 across orthologs.
Figure 5.
Figure 5.
Phosphorylation at serines 1933 and 1984 disrupts the FGF13-dependent increase in steady-state NaV1.5 channel availability. A, representative whole-cell voltage-gated Na+ currents recorded from transiently transfected HEK293 cells. Currents were obtained 48 h following transfection of HEK293 cells with NaV1.5-WT (black), NaV1.5-WT + FGF13 (green), NaV1.5-S1933E/S1984E + FGF13 (NaV1.5-EE + FGF13, red), NaV1.5-S1933A/S1984A + FGF13 (NaV1.5-AA + FGF13, blue), NaV1.5-S1933E/S1984E (NaV1.5-EE, purple), and NaV1.5-S1933A/S1984A (NaV1.5-AA, pink) using the protocols illustrated in each panel. B, mean ± S.E. peak Na+ current (INa) densities are plotted as a function of test potential. C, voltage dependence of current activation. Mean ± S.E. normalized conductances (GNa) are plotted as a function of test potential and fitted using a Boltzmann equation. D, voltage dependence of steady-state current inactivation. Mean ± S.E. normalized current amplitudes are plotted as a function of prepulse potential and fitted using a Boltzmann equation. FGF13 significantly (p < 0.01 versus NaV1.5-WT, one-way ANOVA followed by the Dunnett's post hoc test) shifts the voltage dependence of NaV1.5 channel inactivation toward depolarized potentials, an effect reversed with the NaV1.5-EE phosphomutant (p < 0.01 versus NaV1.5-WT + FGF13, one-way ANOVA followed by the Dunnett's post hoc test). E, no significant changes in voltage dependence of steady-state current inactivation were observed between NaV1.5-WT, NaV1.5-EE, and NaV1.5-AA in the absence of FGF13. Detailed densities, properties, and statistics are provided in Table 2.
Figure 6.
Figure 6.
Phosphorylation at serines 1933 and 1984 disrupts the FGF13-dependent decrease in INaL. TTX-sensitive late Na+ current (INaL) recordings were obtained 48 h after transfection of HEK293 cells in three different data sets. The first data set (A and B) was obtained from cells expressing NaV1.5-WT (black), NaV1.5-WT + FGF13 (green), and NaV1.5-S1933E/1984E + FGF13 (NaV1.5-EE + FGF13, red); the second data set (C and D) from cells expressing NaV1.5-WT (black), NaV1.5-WT + FGF13 (green), and NaV1.5-S1933A/S1984A + FGF13 (NaV1.5-AA + FGF13, blue); and the third data set (E) from cells expressing NaV1.5-WT (black), NaV1.5-S1933E/S1984E (NaV1.5-EE, purple), and NaV1.5-S1933A/S1984A (NaV1.5-AA, pink). A and C, representative TTX-sensitive INaL recordings evoked by prolonged depolarizations (350 ms at −20 mV) from a holding potential of −120 mV. The scale bars indicate a current amplitude of 10 pA, which corresponds to a current density of 0.8 pA/pF and time (50 ms). B, D, and E, distributions and mean ± S.E. TTX-sensitive late Na+ current (INaL) densities. *, p < 0.05; ***, p < 0.001 versus NaV1.5-WT; #, p < 0.05 versus NaV1.5-WT + FGF13; ns, non-significant; Kruskal-Wallis one-way ANOVA followed by the Dunn's post hoc test. Detailed densities and statistics are provided in Table 3.
Figure 7.
Figure 7.
Phosphorylation at serines 1933 and 1984 decreases the interaction of FGF13 and consequently of CaM with NaV1.5. Forty eight hours following transfection of HEK293 cells with NaV1.5-WT, NaV1.5-S1933A/S1984A (NaV1.5-AA), NaV1.5-S1933E/S1984E (NaV1.5-EE), NaV1.5-S1933A (1933A), NaV1.5-S1933E (1933E), NaV1.5-S1984A (1984A), NaV1.5-S1984E (1984E), FGF13, and/or CaM, cell lysates were prepared and used for IPs with the mαNaVPAN antibody. A, D, and F, representative Western blots of the lysates (left panel) and the immunoprecipitates (right panel) with the monoclonal anti-NaVPAN, anti-FGF13, and/or anti-CaM antibodies. Relative mean ± S.E. FGF13 (B) and CaM (C) abundances in mαNaVPAN-IPs from cells expressing NaV1.5-WT (WT, n = 8), NaV1.5-AA (AA, n = 8), and NaV1.5-EE (EE, n = 8); NaV1.5-WT (WT, n = 5 and 4, respectively), 1933A (n = 6 and 4, respectively), and 1933E (n = 6 and 4, respectively); and NaV1.5-WT (WT, n = 6), 1984A (n = 6), and 1984E (n = 6). E, relative mean ± S.E. CaM abundances in mαNaVPAN-IPs from cells expressing NaV1.5-WT (n = 8) and NaV1.5-WT + FGF13 (n = 12), and FGF13 abundances in mαNaVPAN-IPs from cells expressing NaV1.5-WT + FGF13 (n = 4) and NaV1.5-WT + FGF13 + CaM (n = 4). **, p < 0.01; ***, p < 0.001 versus NaV1.5-WT, Kruskal-Wallis one-way ANOVA followed by the Dunn's post hoc test (B and C), or Mann-Whitney test (E). FGF13 and CaM abundances in each IP were first normalized to immunoprecipitated NaV1.5 and then expressed relative to FGF13 or CaM abundances in IPs from control conditions.
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