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. 2021 Feb 1;153(2):e202012646.
doi: 10.1085/jgp.202012646.

Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation sites

Maxime Lorenzini  1 , Sophie Burel  1 , Adrien Lesage  1 , Emily Wagner  2 , Camille Charrière  1 , Pierre-Marie Chevillard  1 , Bérangère Evrard  1 , Dan Maloney  3 , Kiersten M Ruff  2 , Rohit V Pappu  2 , Stefan Wagner  4 , Jeanne M Nerbonne  5   6 , Jonathan R Silva  2 , R Reid Townsend  6   7 , Lars S Maier  4 , Céline Marionneau  1
Affiliations

Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation sites

Maxime Lorenzini et al. J Gen Physiol. .

Abstract

Phosphorylation of the voltage-gated Na+ (NaV) channel NaV1.5 regulates cardiac excitability, yet the phosphorylation sites regulating its function and the underlying mechanisms remain largely unknown. Using a systematic, quantitative phosphoproteomic approach, we analyzed NaV1.5 channel complexes purified from nonfailing and failing mouse left ventricles, and we identified 42 phosphorylation sites on NaV1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic NaV1.5 mutants revealed the roles of three phosphosites in regulating NaV1.5 channel expression and gating. The phosphorylated serines S664 and S667 regulate the voltage dependence of channel activation in a cumulative manner, whereas the nearby S671, the phosphorylation of which is increased in failing hearts, regulates cell surface NaV1.5 expression and peak Na+ current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, our results demonstrate that ventricular NaV1.5 is highly phosphorylated and that the phosphorylation-dependent regulation of NaV1.5 channels is highly complex, site specific, and dynamic.

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Figures

Figure S1.
Figure S1.
IP yields and relative quantification of NaV1.5 peptide abundances from sham and TAC mouse left ventricles (LVs). (A) Representative Western blots of total lysates and mαNaVPAN-IPs from sham and TAC LVs probed with the anti-NaV1.5 rabbit polyclonal (RbαNaV1.5) and anti-GAPDH mouse mAbs. (B) Normalization factors used in MS1 and MS2 analyses to correct for technical variabilities in NaV1.5 protein abundance in mαNaVPAN-IPs from sham and TAC LVs. (C) Distribution of TAC/sham log2-normalized ratios of NaV1.5 peptide spectrum matches. Both biochemical (A) and MS (B and C) analyses of NaV1.5 IP yields and peptide relative abundance demonstrate low technical variability.
Figure 1.
Figure 1.
Localization and quantification of 42 MS-identified NaV1.5 phosphorylation sites in mαNaVPAN-IPs from sham and TAC mouse left ventricles (LVs). (A) Schematic representation of phosphorylation sites on the NaV1.5 protein (UniProt reference sequence K3W4N7). Two phosphorylation site locations are possible at amino acids S1056-T1058. (B) The areas of extracted MS1 ion chromatograms, corresponding to MS2 spectra assigning phosphorylated (in red) and nonphosphorylated (in white) NaV1.5 peptides at indicated phosphorylation site(s), in mαNaVPAN-IPs from sham and TAC LVs are indicated. No red color is visible for the phosphorylated peptide at position T1809, because this phosphopeptide area is very small (area = 80,291 arbitrary unit) relative to the areas of the nonphosphorylated peptides (areas = 80,060,220 arbitrary unit). (C) The areas of extracted MS1 ion chromatograms, corresponding to MS2 spectra assigning phosphorylated peptides at indicated phosphorylation site(s), in mαNaVPAN-IPs from sham and TAC LVs are indicated. The brackets indicate the subgroups of phosphorylation sites analyzed in B. Independent quantification of S459 and S460 phosphorylated peptides was not possible, because localization of the phosphorylation site in most of the phosphorylated peptides could not be discriminated. Similar to B, no red bar is visible for the phosphorylated peptide at position T1809, because this phosphopeptide area is very small (area = 80,291 arbitrary unit), relative to the areas of the other phosphorylated peptides. (D) Distributions and mean ± SEM relative abundances of individual NaV1.5 phosphopeptides allowing assignments of indicated phosphorylation site(s), as well as of corresponding nonphosphorylated (NP) peptides, in TAC LV (n = 5, in black) versus sham LV (n = 4, in white) mαNaVPAN-IPs were obtained using TMT reporter ion intensities. The relative abundances of NaV1.5 phosphopeptides exhibiting phosphorylation(s) on serine 671 (S671; n = 12 peptides) alone or in combination with serine 664 (S664 + S671; n = 9 peptides) or serine 667 (S667 + S671; n = 7 peptides) are increased (**, P < 0.01; ***, P < 0.001; Mann-Whitney test) in TAC LV versus sham LV mαNaVPAN-IPs. (E) Experimental workflow used in the study. Once immunoprecipitated using the mαNaVPAN antibodies, the NaV channel complexes from sham and TAC mouse LVs were labeled individually with different TMT10 tags and combined in the same TMT set for multiplexed LC-MS/MS analysis. NaV1.5 phosphorylation sites were identified, quantified, and analyzed by clusters in whole-cell voltage-clamp recordings in HEK-293 cells.
Figure 2.
Figure 2.
NaV1.5 amino acid sequence coverage and localization of 42 NaV1.5 phosphorylation sites in mαNaVPAN-IPs from sham and TAC mouse left ventricles. Covered sequence and MS-identified phosphorylation sites are highlighted in yellow and red, respectively; transmembrane segments (S1-S6) in each domain (I–IV) are in bold and underlined in black; and loops I, II, and III correspond to interdomains I–II, II–III, and III–IV, respectively. The identification of peptides differing by the presence or absence of a glutamine (Q) at position 1080 ascertains the expression of two NaV1.5 variants: the Q1080 variant corresponds to UniProt reference sequence K3W4N7, and the Q1080del variant corresponds to UniProt reference sequence Q9JJV9. Two phosphorylation site locations are possible at amino acids S1056-T1058 (in green). C-TERM, C terminus; N-TERM, N terminus.
Figure S2.
Figure S2.
Conservation of phosphorylation sites in mouse and human NaV1.5. The mouse (reference sequence NP_001240789.1) and human (NP_000326.2) NaV1.5 sequences are aligned, and phosphorylation sites identified on the mouse sequence and conserved in human are highlighted in red. Two phosphorylation site locations are possible at amino acids S1056-T1058 (in green). 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–II, II–III, and III–IV, respectively. The seven phosphorylation clusters analyzed electrophysiologically are boxed in red. C-TERM, C terminus; N-TERM, N terminus.
Figure 3.
Figure 3.
The phosphorylation sites at positions S664-671 regulate the voltage dependence of current activation and peak INa density. (A) Representative whole-cell voltage-gated INa recorded 48 h following transfection of HEK-293 cells with NaV1.5-WT + NaVβ1 (black), NaV1.5-S664-671A + NaVβ1 (blue), NaV1.5-S664-671E + NaVβ1 (red), or NaV1.5-S664-671D + NaVβ1 (green) using the protocols illustrated in each panel. Scale bars are 1 nA and 2 ms. (B) Voltage dependences of current activation and steady-state inactivation. The voltage dependence of current activation is shifted toward depolarized potentials in cells expressing the NaV1.5-S664-671A or NaV1.5-S664-671E quadruple phosphomutants compared with cells expressing NaV1.5-WT or the NaV1.5-S664-671D quadruple phosphomimetic channels. (C) Mean ± SEM peak INa densities plotted as a function of test potential. The peak INa density is reduced in cells expressing the NaV1.5-S664-671E mutant compared with cells expressing NaV1.5-WT. (D–F) Mean ± SEM times to peak (D), fast (τfast; E), and slow (τslow; F) inactivation time constants plotted as a function of test potential. The times to peak, τfast, and τslow are higher in cells expressing the NaV1.5-S664-671A or NaV1.5-S664-671E quadruple phosphomutants than in cells expressing NaV1.5-WT or NaV1.5-S664-671D. Current densities, time- and voltage-dependent properties, and statistical comparisons across groups are provided in Fig. S3 and Table 3. GNa, sodium conductance.
Figure S3.
Figure S3.
Distributions and mean ± SEM membrane potentials for half-activation and half-inactivation, peak INa densities, and time constants of recovery from inactivation of WT and mutant NaV1.5 channels. Half-activation (A), peak INa densities (B), half-inactivation (C), and time constants of recovery from inactivation (D) of WT and mutant NaV1.5 channels. Currents were recorded as described in the legend to Fig. 3. The INa densities presented were determined from analyses of records obtained on depolarizations to −20 mV (HP = −120 mV). #, P < 0.05 versus NaV1.5-WT, one-way ANOVA followed by the Dunnett’s post-hoc test. ***, P < 0.001 versus NaV1.5-WT, Kruskal-Wallis followed by the Dunn’s post-hoc test. Current densities, time- and voltage-dependent properties, and statistical comparisons across groups are provided in Table 3.
Figure 4.
Figure 4.
The S664 and S667 phosphorylation sites regulate the voltage dependence of current activation, whereas the S671 phosphorylation site regulates the peak INa density. Currents were recorded as described in the legend to Fig. 3. (A–D) The voltage dependence of current activation is shifted toward more depolarized potentials in cells expressing NaV1.5-S664A (A), NaV1.5-S664E (A), NaV1.5-S667A (B), or NaV1.5-S667E (B) than in cells expressing NaV1.5-WT, whereas no significant differences are observed with the NaV1.5-T670 (C) or NaV1.5-S671 (D) phosphomutants. (E–H) The mean ± SEM peak INa densities are plotted as a function of test potential. The peak INa density is reduced in cells expressing NaV1.5-S671E (H), compared with cells expressing NaV1.5-WT, whereas no significant differences are observed with the other phosphomutants. Current densities, time- and voltage-dependent properties, and statistical comparisons across groups are provided in Table 4. GNa, sodium conductance.
Figure S4.
Figure S4.
Distributions and mean ± SEM. (A and B) TTX-sensitive INaL densitiesof quadruple S664-671 (A) and simple S671 (B) NaV1.5 phosphomutants. TTX-sensitive INaL were evoked during prolonged depolarizations (350 ms at −20 mV; HP = −120 mV) 48 h after transfection of HEK-293 cells with WT (black), phosphosilent (blue), and phosphomimetic (red) NaV1.5 channels and NaVβ1. No significant differences between mutant and WT channels were observed.
Figure 5.
Figure 5.
The S671 phosphorylation site regulates the cell surface expression of NaV1.5. (A) Representative Western blots of total (left panel) and cell surface (right panel) NaV1.5 from HEK-293 cells transiently transfected with NaV1.5-WT + NaVβ1, NaV1.5-S671A + NaVβ1, or NaV1.5-S671E + NaVβ1. Samples were probed in parallel with the anti–transferrin receptor (TransR) and anti-GAPDH antibodies. (B) Mean ± SEM total and cell surface NaV1.5 protein expression in transiently transfected HEK-293 cells (n = 12 in six different experiments). Expression of NaV1.5 in each sample was first normalized to the TransR protein in the same blot and then expressed relative to NaV1.5 protein expression (total or cell surface) in cells transfected with NaV1.5-WT + Navβ1. Relative (mean ± SEM) NaV1.5 cell surface expression is different (***, P < 0.001, one-way ANOVA followed by the Dunnett’s post-hoc test) in cells expressing NaV1.5-WT, NaV1.5-S671A, and NaV1.5-S671E channels.
Figure 6.
Figure 6.
Simulations of phosphorylation of segments of the first intracellular linker loop of NaV1.5. The sequential distance between a pair of residues is on the x axis, and the average spatial distance between a pair of residues separated by the specified sequential distance is on the y axis. <Rij> is the average simulated spatial distance between all residue pairs separated in the amino acid sequence by |j-i| residues. The WT sequences are plotted in black. Phosphorylation is simulated by single or multiple replacement of serines/threonines with glutamates (E), and resulting simulations are plotted in gradations of red. The FRC (in blue) and EV (in purple) limits are plotted for reference (see text). (A) Sequence 441-480 contains the phosphosites S457, S459, and S460. (B) Sequence 465-501 contains the phosphosites S483, S484, and T486. (C) Sequence 481-515 contains the phosphosites S497 and S499. (D) Sequence 651-684 contains the phosphosites S664, S667, T670, and S671.
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