Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites

Abstract

The cardiac Na(+) channel Na(V)1.5 current (I(Na)) is critical to cardiac excitability, and altered I(Na) gating has been implicated in genetic and acquired arrhythmias. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is up-regulated in heart failure and has been shown to cause I(Na) gating changes that mimic those induced by a point mutation in humans that is associated with combined long QT and Brugada syndromes. We sought to identify the site(s) on Na(V)1.5 that mediate(s) the CaMKII-induced alterations in I(Na) gating. We analyzed both CaMKII binding and CaMKII-dependent phosphorylation of the intracellularly accessible regions of Na(V)1.5 using a series of GST fusion constructs, immobilized peptide arrays, and soluble peptides. A stable interaction between δ(C)-CaMKII and the intracellular loop between domains 1 and 2 of Na(V)1.5 was observed. This region was also phosphorylated by δ(C)-CaMKII, specifically at the Ser-516 and Thr-594 sites. Wild-type (WT) and phosphomutant hNa(V)1.5 were co-expressed with GFP-δ(C)-CaMKII in HEK293 cells, and I(Na) was recorded. As observed in myocytes, CaMKII shifted WT I(Na) availability to a more negative membrane potential and enhanced accumulation of I(Na) into an intermediate inactivated state, but these effects were abolished by mutating either of these sites to non-phosphorylatable Ala residues. Mutation of these sites to phosphomimetic Glu residues negatively shifted I(Na) availability without the need for CaMKII. CaMKII-dependent phosphorylation of Na(V)1.5 at multiple sites (including Thr-594 and Ser-516) appears to be required to evoke loss-of-function changes in gating that could contribute to acquired Brugada syndrome-like effects in heart failure.

FIGURE 1.

CaMKII phosphorylates L1 of Na_V1.5. A, human Na_V1.5 has five major intracellular regions (N and C termini plus loops 1–3 (L1–L3). Potential CaMKII phosphorylation sites in the L1 are highlighted. Single-letter amino acid codes are shown. B, hNa_V1.5 immunoprecipitated from HEK293 cells was exposed to activated α-CaMKII with [γ-³²P]ATP. Western blot (left) indicates hNa_V1.5 in stably expressing HEK293 cells, and the autoradiogram (right) indicates phosphorylation at Na_V1.5 molecular weight. C and D, bacterially expressed GST fusion proteins of intracellular hNa_V1.5 regions were exposed to activated δ_C-CaMKII with [γ-³²P]ATP. C, autoradiograph indicates phosphorylation of the L1 after δ_C-CaMKII phosphorylation. D, average ³²P incorporation (n = 3, ±S.D. (error bars)) of GST fusion proteins detected following Cerenkov counting.

FIGURE 2.

Autophosphorylated CaMKII tethers to L1 of Na_V1.5. A, HEK293 cells expressing hNaV1.5 and GFP-δ_C-CaMKII were lysed, pulled down with monoclonal GFP antibody, and subsequently blotted for GFP-δ_C-CaMKII (right) and hNa_V1.5 (left). Full-length hNa_V1.5 co-immunoprecipitated (IP) with GFP-δ_C-CaMKII. B, Western blot detection of δ_C-CaMKII bound to GST-tagged fragments of the major intracellular loops of hNa_V1.5 under naive conditions (top), in the presence of Ca²⁺/CaM (middle), and when δ_C-CaMKII was preautophosphorylated (bottom). C, image of 1 μg of Ca²⁺/CaM/autophosphorylated DyLight₈₀₀-labeled δ_C-CaMKII bound to GST-L1 and not GST alone (right lanes). Naive δ_C-CaMKII (left lanes) did not bind GST-L1. D, average phosphorylation (n = 3, ± S.D. (error bars)) of syntide-2 with GST-L1 pull-down fragments bound to autophosphorylated δ_C-CaMKII (right bars) and not naive CaMKII (left bars). Data are represented as specific activity with 1 μg of multimeric human δ_C-CaMKII. *, significant difference compared with GST alone (p < 0.05; t test).

FIGURE 3.

CaMKII phosphorylates Ser-516 and Ser-593/Thr-594 in L1. A, phosphor image of immobilized tiled peptide array of L1 after δ_C-CaMKII phosphorylation with [γ-³²P]ATP (darkness indicates ³²P incorporation on that peptide). Single-letter amino acid codes are shown. B, phosphorylation intensity of each peptide spot in A, detected using MultiGauge version 3.0. C, soluble peptide (50 μm) phosphorylation by monomeric δ_C-CaMKII (n = 3, ±S.D. (error bars); *, p < 0.05 versus control peptide). 15-mers were centered near the indicated sites. D, time course of monomeric δ_C-CaMKII phosphorylation of 500 μm control peptide, original unbiased Ser-571 peptide, the biased 571* peptide (see “Experimental Procedures” for sequence), and 50 μm AC2, a known CaMKII substrate. E, average phosphorylation (n = 3, ±S.D.) of soluble peptides in D at the 1 min time point. F, average phosphorylation (n = 3, ±S.D.) of immobilized peptides with wild-type control, as well as Ser-593/Thr-594 single point Ala mutations and a double-point Ala mutation. S593A/T594A and T594A exhibit decreased phosphorylation compared with wild-type control (n = 3, ±S.D.; *, p < 0.05 versus control Ser-493/Thr-494; one-way ANOVA, post hoc Dunnett's test).

FIGURE 4.

CaMKII phosphorylation of Nav1.5 L1-GST fragments. A, Coomassie stain (top) and phosphor image (bottom) of GST-tagged wild-type L1 and various L1 mutants. B, average phosphorylation (n = 3, ±S.D. (error bars)) of L1 constructs shown in A. S516A, T594A, and S516A/T594A are significantly decreased compared with wild-type GST-L1, as indicated (*, p < 0.05, one-way ANOVA, post hoc Dunnett's test). C, phosphorylation time course of GST-L1 wild-type and mutant fragments. D, stoichiometry of phosphorylation of GST-L1 wild-type and mutant fragments following 180-min phosphorylation by monomeric δ_C-CaMKII (time of saturation in C). Ala and Glu substitutions at Ser-516 and Thr-594 result in significantly decreased phosphorylation compared with wild-type and Ser-571 mutants (n = 3, ±S.D.; *, p < 0.05, one-way ANOVA, post hoc Dunnett's test).

FIGURE 5.

CaMKII shifts I_Na availability but not activation. I_Na was measured in HEK cells expressing WT, S516A, S593A/T594A, T594A, S571A, or S593A mutant hNa_V1.5 plus GFP-δ_C-CaMKII (pipette containing 1 μm Ca²⁺ plus 1 μm CaM with or without AIP). A, representative raw I_Na and mean current-voltage relationships. Protocols for I-V (top) and steady state inactivation (SSI; bottom) for D and E are shown. B and C, voltage dependence and V₅₀ of activation were not different among groups or with/without AIP. D and E, I_Na availability was shifted to more negative V_m by δ_C-CaMKII in WT and S593A control, which was prevented by AIP. S516A, S593A/T594A, T594A, and S571A did not show this effect (n as indicated, ±S.E. (error bars); *, p < 0.05 by t test versus with AIP and two-way ANOVA but no difference among the indicated groups).

FIGURE 6.

CaMKII enhances I_Na entry into intermediate inactivation. A, representative raw WT I_Na trace of enhanced entry into intermediate inactivation and two-pulse voltage protocol. B, time dependence of accumulation into intermediate inactivation during pulses to −20 mV was significantly greater in WT under CaMKII-activating conditions and 516E/594E phosphomimetic (+AIP) versus WT + AIP and phosphomutants. C, average accumulation of intermediate inactivation at 2.5 s from the bounding box in B (n as indicated, ±S.E. (error bars); *, p < 0.05 by ANOVA or t test versus with AIP; #, p < 0.05 by t test versus with AIP; p = not significant versus WT-AIP).

FIGURE 7.

S516E and T594E phosphomimetics shift I_Na availability independent of CaMKII. A, V₅₀ of activation was not different among phosphomimetics or compared with WT. B, T594E and S516E I_Na V₅₀ of availability was shifted to negative V_m in the presence of AIP and without δ_C-CaMKII co-expression (indicated by striping). V₅₀ of availability for S571E and S516E/T594E was not significantly different from WT with AIP (*, p < 0.05 by ANOVA versus WT with AIP; bracket indicates no difference by t test for S516E and T594E versus WT with δ_C-CaMKII). C, expanded version of B, for comparing the effect of AIP omission (versus with AIP) in the pipette for different Na_V1.5. For the T594E phosphomimetic, availability was not altered when endogenous CaMKII was allowed to function (versus with AIP). For S516E, endogenous CaMKII partly reversed the effect of the phosphomimetic. S571E, which did not mimic the CaMKII-induced shift in V₅₀ when AIP was present (in contrast to S516E and T594E) could still shift V₅₀ negative by endogenous CaMKII activity. (n as indicated, ±S.E. (error bars); AIP and exogenous CaMKII present only where indicated). #, p < 0.05 by t test versus with AIP for the same Na_V1.5 type.