Calmodulin limits pathogenic Na+ channel persistent current

Abstract

Increased "persistent" current, caused by delayed inactivation, through voltage-gated Na⁺ (Na_V) channels leads to cardiac arrhythmias or epilepsy. The underlying molecular contributors to these inactivation defects are poorly understood. Here, we show that calmodulin (CaM) binding to multiple sites within Na_V channel intracellular C-terminal domains (CTDs) limits persistent Na⁺ current and accelerates inactivation across the Na_V family. Arrhythmia or epilepsy mutations located in Na_V1.5 or Na_V1.2 channel CTDs, respectively, reduce CaM binding either directly or by interfering with CTD-CTD interchannel interactions. Boosting the availability of CaM, thus shifting its binding equilibrium, restores wild-type (WT)-like inactivation in mutant Na_V1.5 and Na_V1.2 channels and likewise diminishes the comparatively large persistent Na⁺ current through WT Na_V1.6, whose CTD displays relatively low CaM affinity. In cerebellar Purkinje neurons, in which Na_V1.6 promotes a large physiological persistent Na⁺ current, increased CaM diminishes the persistent Na⁺ current, suggesting that the endogenous, comparatively weak affinity of Na_V1.6 for apoCaM is important for physiological persistent current.

Figure 1.

LQT3 mutations in the IQ domain increase the persistent Na⁺ current amplitude. (A) Exemplar traces of the pseudo-WT Na_V1.5^TTX-S and Q1909R (magnified region on the right). Y axis of scale bars represents the percentage of persistent current amplitude normalized to the peak Na⁺ current. The inset shows I-V plots of peak current amplitude for Na_V1.5^TTX-S and Q1909R, demonstrating no significant difference in peak current amplitude between Na_V1.5^TTX-S and Q1909R. (B) Magnified region of exemplar traces showing persistent Na⁺ current for the LQT3 mutations in the CaM C-lobe–binding site and the L1917K. (C) Quantification of persistent Na⁺ current (as % peak of current). The white number in each bar indicates the number of cells, n. Data are presented as mean ± SEM. **, P < 0.01.

Figure 2.

LQT3 mutations in the IQ domain decrease apoCaM binding affinity and show reduced persistent Na⁺ current after CaM overexpression. (A and B) Exemplar ITC traces for apoCaM and a WT Na_V1.5 CTD (A) or a Q1909R Na_V1.5 CTD (B). (C) Western blot of CaM from lysates of HEK293T cells expressing Na_V1.5^TTX-S, Na_V β1, and empty vector (−) or CaM (+). (D) Exemplar traces of Na_V1.5^TTX-S with an additional Q1909R mutation coexpressed with CaM (red) or empty vector (black), showing rescue of the persistent Na⁺ current. (E) Exemplar traces recorded from cells expressing the pseudo-WT Na_V1.5^TTX-S coexpressed with CaM (red) or empty vector (black), showing no effect on the persistent Na⁺ current. (F) Quantification of persistent Na⁺ current amplitude as a percentage of peak current after overexpression of CaM (red) compared with Con (black; data from Fig. 1 C). Data are presented as mean ± SEM. **, P < 0.01.

Figure 3.

Na_V1.5 CTD–CaM heterodimer interaction is disrupted by the Y1795C LQT3 mutation. (A and B) Proposed interaction between two Na_V1.5 CTD–CaM heterodimers and position of Y1795C in one of the Na_V1.5 CTDs. The positions of Lys1878 in one Na_V1.5 CTD globular domain (sky blue) and Lys1922 in the IQ domain of a second Na_V1.5 CTD (green), which are available for cross-linking by DSG, are indicated. (C) Coomassie-blue–stained gel of Na_V1.5 CTD and CaM after cross-linking with DSG or buffer control (Con). Molecular weight markers are indicated. (D and F) XMapper display of LC/MS data showing DSG cross-linked peptide 1 for the WT Na_V1.5 CTD and CaM (WT) and the Y1795C mutant Na_V1.5 CTD and CaM. The intensity score (color code) indicates the number of peptides identified for each pairwise interaction. The position of cross-linking between Lys1878 and Lys1922 is circled and indicated by an arrow. (E) LC/MS data showing the cross-linked peptide. (G) Exemplar traces showing increased persistent Na⁺ current for the Y1795C and rescue by CaM overexpression. (H) Quantification of persistent Na⁺ current for the pseudo-WT Na_V1.5^TTX-S and the Y1795C Na_V1.5 mutant with and without CaM overexpression. Data are presented as mean ± SEM. **, P < 0.01.

Figure 4.

CaM overexpression does not affect channel function for mutants that do not display abnormal interactions with CaM. (A) Exemplar traces showing no increased persistent Na⁺ current for the D1790G and no change after CaM overexpression. (B) Hyperpolarizing shift in the V_1/2 of steady-state inactivation for the D1790G mutant Na_V1.5 and absence of an effect by CaM overexpression. Data are presented as mean ± SEM. (C) Exemplar traces showing increased persistent Na⁺ current for the ΔKPQ mutant and no change after CaM overexpression. (D) Gel filtration profiles of the III-IV linker fusion protein (green), Ca²⁺/CaM (red), and the mixture of the III-IV linker protein and Ca²⁺/CaM (black). (E) Gel filtration profiles of the III-IV linker fusion protein (green), apoCaM (red, treated with EGTA), and the mixture of the III-IV linker protein and apoCaM (black). (F) Gel filtration profiles of the III-IV linker fusion protein (green), the Na_V1.5 CTD (amino acids 1773–1940) complexed with apoCaM (red), and a mixture of the III-IV linker protein and the Na_V1.5 CTD–CaM after the addition of 5 mM Ca²⁺ (black). Insets show Coomassie blue–stained polyacrylamide gels; lane numbers correspond to the fractions labeled in the chromatograms. Molecular weight markers are indicated.

Figure 5.

ApoCaM regulates persistent Na⁺ current in neuronal Na_V channels. (A and B) Exemplar traces for Na_V1.2^TTX-R (WT) and the Na_V1.2 mutant R1918H showing increased persistent Na⁺ current for the R1918H mutant (but not for WT) and rescue by CaM overexpression for the R1918H mutant. (C) Summary data showing rescue by CaM for the Na_V1.2 mutants H1853R and R1918H. (D) Summary data showing increased τ of inactivation and rescue by CaM for the Na_V1.2 mutants H1853R and R1918H. (E) Exemplar traces for Na_V1.6^TTX-R (WT) expressed in HEK293T cells showing reduced persistent Na⁺ current by CaM overexpression. (F) Exemplar traces for Na_V1.6^TTX-R expressed in cultured cerebellar Purkinje neurons and reduced persistent Na⁺ current by CaM overexpression. (G) Exemplar traces of total Na_V Na⁺ current in cultured cerebellar Purkinje neuron showing reduction in persistent Na⁺ current after CaM overexpression. The inset shows GFP-expressing cultured cerebellar Purkinje neuron. Bar, 20 µm. (H) Summary data for Na_V1.6 expressed in HEK293T cells or in cultured cerebellar Purkinje neurons or total Na_V Na⁺ current in cultured cerebellar Purkinje neurons. Data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01.