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. 2018 Sep 5:9:1230.
doi: 10.3389/fphys.2018.01230. eCollection 2018.

Key Role of the Membrane Trafficking of Nav1.5 Channel Protein in Antidepressant-Induced Brugada Syndrome

Xi Chen  1   2 , Chao Zhu  2 , Hao Zhou  2 , Yu Zhang  2 , Zhongqi Cai  2 , Honglin Wu  3 , Xiaomeng Ren  2 , Lei Gao  2 , Jiancheng Zhang  3 , Yang Li  2
Affiliations

Key Role of the Membrane Trafficking of Nav1.5 Channel Protein in Antidepressant-Induced Brugada Syndrome

Xi Chen et al. Front Physiol. .

Abstract

Anti-depressant treatment has been found to be associated with the development of Brugada syndrome (BrS) through poorly defined mechanisms. Herein, this study aimed to explore the molecular basis for amitriptyline-induced BrS. The effects of long-term treatments of amitriptyline on Nav1.5 were investigated using neonatal rat ventricular myocytes. The electrophysiological properties, expression and distribution of Nav1.5 were studied using the patch clamp, Western blot and confocal laser microscopy assays. Interactions between Nav1.5 and its interacting proteins, including ankyrin-G and dystrophin, were evaluated by co-immunoprecipitation. A larger decrease in the peak INa occurred after long-term treatments to amitriptyline (56.64%) than after acute exposure to amitriptyline (28%). Slow recovery from inactivation of Nav1.5 was observed after acute or long-term treatments to amitriptyline. The expression of Nav1.5 on the cell membrane showed a larger decrease by long-term treatments to amitriptyline than by acute exposure to amitriptyline. After long-term treatments to amitriptyline, we observed reduced Nav1.5 proteins on the cell membrane and the disrupted co-localization of Nav1.5 and ankyrin-G or dystrophin. Co-immunoprecipitation experiments further testified that the combination of Nav1.5 and ankyrin-G or dystrophin was severely weakened after long-term treatments to amitriptyline, implying the failed interaction between Nav1.5 and ankyrin-G or dystrophin. Our data suggest that the long-term effect of amitriptyline serves as an important contribution to BrS induced by amitriptyline. The mechanisms of BrS induced by amitriptyline were related to Nav1.5 trafficking and could be explained by the disrupted interaction of ankyrin-G, dystrophin and Nav1.5.

Keywords: Brugada syndrome; Nav1.5; antidepressant; interacting proteins; long-term effect; trafficking.

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Figures

FIGURE 1
FIGURE 1
Effect of amitriptyline on Nav1.5 currents. (A) Protocols for INa eliciting. (B) Representative INa in control cells and in cells with acute treatments of amitriptyline. (C) I-V curves for control cells and cells with acute treatments of amitriptyline. (D) Analysis of peak INa densities for the acute effect of amitriptyline. (E) Representative INa in control cells and cells with long-term treatments of amitriptyline. (F) I-V curves for control cells and cells with long-term treatments of amitriptyline. (G) Analysis of peak INa densities for the long-term effect of amitriptyline. ∗∗∗P < 0.001.
FIGURE 2
FIGURE 2
Effect of amitriptyline on the gating properties of Nav1.5 currents. (A) Protocols for steady-state activation (SSA) recording. (B) SSA curves for the acute effect of amitriptyline. (C) SSA cures for the long-term effect of amitriptyline. (D) Protocols for steady-state inactivation (SSI) recording. (E) SSI curves for the acute effect of amitriptyline. (F) SSI curves for the long-term effect of amitriptyline. (G) Protocols for recovery from inactivation (RFI) recording. (H) RFI curves for the acute effect of amitriptyline. (I) RFI curves for the long-term effect of amitriptyline.
FIGURE 3
FIGURE 3
Effect of amitriptyline on intermediate-state inactivation (ISI) and closed-state inactivation (CSI) of Nav1.5 currents. (A) Protocols for ISI recording. (B) Protocols for CSI recording. (C) ISI curves for the acute effect of amitriptyline. (D) ISI cures for the long-term effect of amitriptyline. (E) CSI curves for the acute effect of amitriptyline. (F) CSI cures for the long-term effect of amitriptyline.
FIGURE 4
FIGURE 4
Long-term effect of amitriptyline on protein expression and distribution of Nav1.5. (A) Protein expression of Nav1.5 in control cells, cells with acute treatments of amitriptyline and cells with long-term treatments with amitriptyline. (B) Representative confocal images of Nav1.5 (red) and ankyrin-G (green) without or with long-term treatments with amitriptyline. (C) Representative confocal images of Nav1.5 (red) and dystrophin (green) without or with long-term treatments with amitriptyline. (D) Representative confocal images of Nav1.5 and ankyrin-G with nocodazole treatments. (E) Representative confocal images of Nav1.5 and dystrophin with nocodazole treatments. The blue signal indicates the nucleus. Scale bar: 10 μm. ∗P < 0.05.
FIGURE 5
FIGURE 5
Long-term effect of amitriptyline on the Nav1.5-ankyrin-G interaction and Nav1.5-dystrophin interaction. (A) With the anti-ankyrin-G antibody, Nav1.5 was detectable without long-term treatments with amitriptyline but was not clearly observed after long-term treatments with amitriptyline. (B) With anti-dystrophin antibody, Nav1.5 was detectable without long-term treatments with amitriptyline but was not clearly observed after the long-term treatments with amitriptyline. Input: Western blotting of total protein lysates.
FIGURE 6
FIGURE 6
Effect of other antidepressants on Nav1.5 currents. (A) Representative INa current traces recorded from cells under control conditions and acute clomipramine treatments of (B,C) I-V curves and analysis of the peak INa densities for the acute effect of clomipramine. (D) Representative INa current traces recorded from cells with long-term treatments of clomipramine. (E,F) I-V curves and peak INa densities of cells with long-term treatmentsn of clomipramine. (G–I) Representative INa in cells with acute desipramine treatments. (J–L) Representative INa in cells with long-term treatments of desipramine. (M–R) Representative INa in cells with acute and long-term treatments of nortriptyline. ∗P < 0.05, ∗∗P < 0.01.
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