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. 2020 Aug 14;295(33):11845-11865.
doi: 10.1074/jbc.RA120.014062. Epub 2020 Jul 1.

CaMKII enhances voltage-gated sodium channel Nav1.6 activity and neuronal excitability

Agnes S Zybura  1 , Anthony J Baucum 2nd  1   2 , Anthony M Rush  3 , Theodore R Cummins  1   2 , Andy Hudmon  4   5
Affiliations

CaMKII enhances voltage-gated sodium channel Nav1.6 activity and neuronal excitability

Agnes S Zybura et al. J Biol Chem. .

Abstract

Nav1.6 is the primary voltage-gated sodium channel isoform expressed in mature axon initial segments and nodes, making it critical for initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may have profound effects on input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism regulating ion channel function. Because Nav1.6 and the multifunctional Ca2+/CaM-dependent protein kinase II (CaMKII) are independently linked to excitability disorders, we sought to investigate modulation of Nav1.6 function by CaMKII signaling. We show that inhibition of CaMKII, a Ser/Thr protein kinase associated with excitability, synaptic plasticity, and excitability disorders, with the CaMKII-specific peptide inhibitor CN21 reduces transient and persistent currents in Nav1.6-expressing Purkinje neurons by 87%. Using whole-cell voltage clamp of Nav1.6, we show that CaMKII inhibition in ND7/23 and HEK293 cells significantly reduces transient and persistent currents by 72% and produces a 5.8-mV depolarizing shift in the voltage dependence of activation. Immobilized peptide arrays and nanoflow LC-electrospray ionization/MS of Nav1.6 reveal potential sites of CaMKII phosphorylation, specifically Ser-561 and Ser-641/Thr-642 within the first intracellular loop of the channel. Using site-directed mutagenesis to test multiple potential sites of phosphorylation, we show that Ala substitutions of Ser-561 and Ser-641/Thr-642 recapitulate the depolarizing shift in activation and reduction in current density. Computational simulations to model effects of CaMKII inhibition on Nav1.6 function demonstrate dramatic reductions in spontaneous and evoked action potentials in a Purkinje cell model, suggesting that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate neuronal excitability.

Keywords: Ca2+/calmodulin-dependent protein kinase II (CaMKII); CaMKII; Nav1.6; electrophysiology; phosphoproteomics; phosphorylation; post-translational modification (PTM); sodium channel.

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Conflict of interest statement

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

Figures

Figure 1.
Figure 1.
CaMKII inhibition reduces sodium currents in acutely dissociated Purkinje neurons. Shown are a representative family of transient (A) and persistent (B) sodium current traces recorded from acutely dissociated Purkinje neurons with no peptide (left, black), tatCN21 (middle, red), and tatCN21Ala (right, gray). Boldface traces represent sodium current at −25 mV. C, transient current density values (−25 mV). F(2, 326) = 44.5, p < 0.0001 by two-way ANOVA performed across voltages ranging from −100 to +15 mV. D, persistent current density values (−25 mV). F(2, 72) = 13.45, p < 0.0001 by two-way ANOVA performed across voltages ranging from −65 to −15 mV. *, p < 0.05; **, p < 0.01; ****, p < 0.0001. n = 3–7. Two-way ANOVA ± S.E. (error bars), Tukey's post hoc test. pF, picofarads.
Figure 2.
Figure 2.
CaMKII inhibition reduces Nav1.6 transient current density. A, representative family of transient current traces generated by TTX-R hNav1.6 transiently expressed in ND7/23 cells recorded with no peptide (right), CN21 (middle), and CN21Ala (right) in the patch pipette. Currents were elicited with 50-ms step depolarizations ranging from −80 to +60 mV (inset). Peak current traces (0 mV) are in boldface. B, transient current density-voltage curve (left) for Nav1.6 with no peptide (black circles), CN21 (red triangles), and CN21Ala (gray squares) in the pipette. Current density values were calculated by normalizing the current amplitude at each voltage by the cell capacitance. Peak current density values are plotted on the right. With CN21 treatment, Nav1.6 transient currents are significantly attenuated compared with no peptide and CN21Ala controls (F(2, 841) = 367.7, p < 0.0001. No peptide and CN21Ala are not significantly different (p = 0.4800). ****, p < 0.0001. n = 11–12. Two-way ANOVA ± S.E. (error bars), Tukey's post hoc test.
Figure 3.
Figure 3.
CaMKII inhibition reduces Nav1.6 persistent current density. A, representative family of persistent current traces generated by TTX-R hNav1.6 transiently expressed in ND7/23 cells with no peptide (black), CN21 (red), or CN21Ala (gray) in the patch pipette. Currents were elicited with 50-ms step depolarizations ranging from −80 to +60 mV (inset) and were measured at 45 ms into the current trace. B, percentage persistent current-voltage curve (left) for Nav1.6 with no peptide (black circles), CN21 (red triangles), and CN21Ala (gray squares) in the pipette (F(2, 483) = 45.91, p < 0.0001 with two-way ANOVA). Peak persistent current density values are plotted on the right and were calculated by normalizing the current amplitude at each voltage by the cell capacitance. There is a significant reduction of Nav1.6 persistent sodium currents with CaMKII inhibition by CN21 compared with no peptide and CN21Ala controls (F(2, 267) = 42.07, p < 0.0001). No peptide and CN21Ala persistent currents are not significantly different (p = 0.9855). *, p < 0.05; **, p < 0.01. n = 9–10. Two-way ANOVA ± S.E. (error bars), Tukey's post hoc test.
Figure 4.
Figure 4.
CaMKII inhibition produces a depolarizing shift in Nav1.6 voltage-dependence of activation. A, voltage dependence of steady-state activation and inactivation (channel availability) curves fit with a Boltzmann function. Steady-state inactivation was measured by holding cells for 500 ms at a range of prepulse voltages from −130 to +40 mV, followed by a 20-ms test pulse to 0 mV to measure channel availability (inset). B, activation (top) and availability (bottom) midpoints. CN21 treatment produced a significant depolarizing shift in the activation midpoint (F(2, 26) = 6.220, p = 0.0062); however, it had no significant effect on the midpoint of inactivation (F(2, 29) = 2.229, p = 0.126). *, p < 0.05; **, p < 0.01; n = 6–12. One-way ANOVA ± S.E. (error bars), Tukey's post hoc test.
Figure 5.
Figure 5.
Topology of voltage-gated sodium channel SCN8A. Potential CaMKII phosphorylation sites are shown in L1 and labeled as follows: Ser-541 (pink), Ser-561 (green), Ser-641 (dark purple), and Thr-642 (light purple).
Figure 6.
Figure 6.
MS/MS spectra for CaMKII phosphorylation sites on Nav1.6 identified by nanoflow LC-ESI/MS. A, representative tandem mass spectrum of human Nav1.6 peptide containing pSer-561. The spectrum of a singly phosphorylated Nav1.6 peptide at m/z 695.32 was fragmented to produce a tandem mass spectrum with y-ion and b-ion series that correspond to the sequence HNSKSSIFSFR (amino acids 559–569) and confirm the phosphosite at Ser-561. B, representative tandem mass spectrum for a phosphorylated Nav1.6 peptide at m/z 778.50 containing adjacent sites Ser-641 and Thr-642 that could not be confidently differentiated by MS/MS analysis. The peptide was fragmented to produce the tandem mass spectrum with a-ion and b-ion series that correspond to the sequence NSTVDcNGVVSLIGGPGSHIGGR (amino acids 640–662).
Figure 7.
Figure 7.
Immobilized peptide arrays of potential CaMKII phosphorylation sites in Nav1.6. A, schematic of the SPOTs immobilized peptide tiling assay performed in B and C. B, phosphorylation intensity of peptides tiled from the intracellular regions of human Nav1.6 after αCaMKII phosphorylation with [γ-32P]ATP. Corresponding intracellular regions are labeled and single-letter amino acid codes/phosphorylation motifs for each putative phosphorylation site are shown. Inset, representative phosphor image of immobilized tiled peptide spots (darkness intensity indicates 32P incorporation in that peptide). Phosphosites were identified if two or more successive peptides were phosphorylated at the indicated threshold (dotted line) of known phosphorylated substrates AC2 and GluN2B (positive controls). C, average phosphorylation of immobilized peptides with WT and Ala point mutations introduced at phosphosites identified by peptide array in B and nanoflow LC-ESI/MS analysis. Double Ala mutations were introduced at the adjacent sites Ser-641/Thr-642 to identify preferential contribution of CaMKII phosphorylation. The N terminus of EAAT1 served as the negative control; AC2 and GluN2B phosphorylation served as positive controls.
Figure 8.
Figure 8.
CaMKII modulates Nav1.6 sodium currents at Ser-641/Thr-642. Shown are transient current density-voltage curves (A) and corresponding bar graphs (B) of peak current densities for Nav1.6 phospho-null mutations with no peptide (left), CN21 (middle), and CN21Ala (right) in the pipette. The dotted line serves as a visual control representing Nav1.6 WT current density with no peptide. S541A and S641A increase Nav1.6 current density, whereas T642A and S641A/T642A decrease current density compared with WT Nav1.6 in the absence of CaMKII inhibition, while S561A displayed no change (F(5, 1419) = 66.84, p < 0.0001 by two-way ANOVA). When treated with CN21, only S561A and S641A/T642A maintain a similar current density to WT Nav1.6 with CN21 treatment (F(5, 1101) = 91.15, p < 0.0001 by two-way ANOVA). Similar to the no peptide condition, S541A and S641A increased Nav1.6 current density compared with WT treated with CN21Ala whereas S641A/T642A displayed a significant decrease, and S561A displayed no change (F(5, 956) = 59.17, p < 0.0001). No peptide: S561A versus WT, p = 0.9029. CN21: S561A versus WT, p = 0.4133 and S641A/T642A versus WT, p = 0.4861. CN21Ala: S561A versus WT, p > 0.9999 and T642A versus WT, p = 0.3856. *, p < 0.05; **, p < 0.01; #, p < 0.001; Ψ, p < 0.0001. n = 4–15. Two-way ANOVA ± S.E. (error bars), Dunnett's post hoc test versus WT within treatment.
Figure 9.
Figure 9.
Effects of CaMKII phospho-null mutations in Nav1.6 on voltage-dependence of activation and channel availability. A, voltage dependence of steady-state activation and inactivation (channel availability) curves fit with a Boltzmann function comparing WT and phospho-null mutants with no peptide (left), CN21 (middle), or CN21Ala (right) in the patch pipette. Steady-state inactivation was measured by holding cells for 500 ms at a range of prepulse voltages from −130 to +40 mV, followed by a 20-ms test pulse to 0 mV to measure channel availability. B, voltage dependence of steady-state activation midpoints. Compared with WT, S561A is the only mutant that displays a significant depolarizing shift in activation midpoint independent of CaMKII inhibition. No peptide: F(5, 42) = 3.114, p = 0.0177. CN21: F(5, 35) = 0.6870, p = 0.6365. CN21Ala: F(5, 31) = 2.865, p = 0.0307. C, voltage dependence of steady-state inactivation midpoints. No peptide: F(5, 41) = 2.392, p = 0.0541. CN21: F(5, 37) = 2.109, p = 0.0861. CN21Ala: F(5, 34) = 2.760, p = 0.0338. *, p < 0.05; **, p < 0.01. n = 4–12. One-way ANOVA ± S.E. (error bars), Dunnett's post hoc test (versus WT). Conductance: no peptide versus WT: S541A, p = 0.0749; S561A, p = 0.0304; S641A, p = 0.0893; T642A, p = 0.1760; S641A/T642A, p = 0.9999. CN21 versus WT: S541A, p = 0.9868; S561A, p = 0.8290; S641A, p > 0.9999; T642A, p = 0.8514; S641A/T642A, p = 0.7207. CN21Ala versus WT: S541A, p = 0.2407; S561A, p = 0.0050; S641A, p = 0.1456; T642A, p = 0.6502; S641A/T642A, p = 0.1812. Availability: no peptide versus WT: S541A, p = 0.8900; S561A, p = 0.8494; S641A, p = 0.9530; T642A, p = 0.9620; S641A/T642A, p = 0.0889. CN21 versus WT: S541A, p = 0.9847; S561A, p = 0.9824; S641A, p = 0.9998; T642A, p = 0.7540; S641A/T642A, p = 0.0265. CN21Ala versus WT: S541A, p = 0.5310; S561A, p = 0.1049; S641A, p = 0.4346; T642A, p = 0.8963; S641A/T642A, p = 0.6977.
Figure 10.
Figure 10.
Voltage-clamp simulations of modeled Nav1.6 channels with and without CN21-mediated functional alterations. A, voltage dependence of activation; B, normalized current density-voltage curves for modeled (left) and experimental (right) channels. The model exemplifies the relative functional alterations (shift in activation and reduction in current density) that were observed with CN21 treatment in our experimental data.
Figure 11.
Figure 11.
Current-clamp simulations of modeled Nav1.6 channels on action potential firing in simulated Purkinje neurons. Shown is quantification of spontaneous (A) and evoked action potential frequency (B) from modeled Purkinje neurons with either no peptide control (black), CN21 (pink), Ser-561 (teal), or Ser-641/Thr-642 (purple) modeled Nav1.6 currents. Neither the CN21, Ser-561, nor Ser-641/Thr-642 modeled currents fire spontaneous action potentials. Representative traces of evoked action potentials at 40 pA are shown in C.
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