The antiarrhythmic compound efsevin directly modulates voltage-dependent anion channel 2 by binding to its inner wall and enhancing mitochondrial Ca2+ uptake

Abstract

Background and purpose: The synthetic compound efsevin was recently identified to suppress arrhythmogenesis in models of cardiac arrhythmia, making it a promising candidate for antiarrhythmic therapy. Its activity was shown to be dependent on the voltage-dependent anion channel 2 (VDAC2) in the outer mitochondrial membrane. Here, we investigated the molecular mechanism of the efsevin-VDAC2 interaction.

Experimental approach: To evaluate the functional interaction of efsevin and VDAC2, we measured currents through recombinant VDAC2 in planar lipid bilayers. Using molecular ligand-protein docking and mutational analysis, we identified the efsevin binding site on VDAC2. Finally, physiological consequences of the efsevin-induced modulation of VDAC2 were analysed in HL-1 cardiomyocytes.

Key results: In lipid bilayers, efsevin reduced VDAC2 conductance and shifted the channel's open probability towards less anion-selective closed states. Efsevin binds to a binding pocket formed by the inner channel wall and the pore-lining N-terminal α-helix. Exchange of amino acids N207, K236 and N238 within this pocket for alanines abolished the channel's efsevin-responsiveness. Upon heterologous expression in HL-1 cardiomyocytes, both channels, wild-type VDAC2 and the efsevin-insensitive VDAC2^AAA restored mitochondrial Ca²⁺ uptake, but only wild-type VDAC2 was sensitive to efsevin.

Conclusion and implications: In summary, our data indicate a direct interaction of efsevin with VDAC2 inside the channel pore that leads to modified gating and results in enhanced SR-mitochondria Ca²⁺ transfer. This study sheds new light on the function of VDAC2 and provides a basis for structure-aided chemical optimization of efsevin.

FIGURE 1

Effects of efsevin on zVDAC2 currents in planar lipid bilayers. (a) Typical current recordings from zVDAC2 inserted into painted planar DPhPC lipid bilayers in 1‐M KCl in response to 10 s test pulses from 0 to 10, 40, and 50 mV, respectively, under control conditions (left) and after addition of 8‐μM efsevin (right) to the same channel. Gating between three major states open (o), closed1 (c1) and closed2 (c2) and few subconductance states can be observed (pulse protocol in shown in grey). (b) Current–voltage relationship of average zVDAC2 currents before (n = 9 individual channels, black circles) and after addition of efsevin (n = 6 individual channels, red triangles, Unpaired Student's t‐test). (c) Conductance–voltage relationship of zVDAC2 before (n = 9 individual channels, black circles) and after addition of efsevin (n = 6 individual channels, red triangles, Unpaired Student's t‐test)

FIGURE 2

Effects of efsevin on zVDAC2 conductance and open probability (P_O). (a) Representative recordings of zVDAC2 in a painted lipid bilayer under control conditions (upper trace) and after addition of 8‐μM efsevin (lower trace) at potentials where typical gating behaviour between the three states is observed, that is, very low/high potentials under control conditions and moderate potentials after addition of efsevin. Currents through the open and two distinct closed states of the channel are indicated by dashed lines. (b) Addition of 8‐μM efsevin does not change the conductance of the three distinct conductance states of zVDAC2 (n = 12 individual channels for control and 6 individual channels after addition of efsevin). (c) Analysis of the open probability (P_O) of zVDAC2 shows a significant shift of the channel towards the closed states across all voltages after addition of efsevin (n = 12 for control and n = 6 for efsevin, Mann–Whitney U test). (d) Representative conductance histograms from recordings of zVDAC2 with and without efsevin at 30 mV and 60 mV show a shift of the channel from the classical open state to the closed states. (e,f) Current–voltage plots for the open and the efsevin‐induced closed state 1 obtained from zVDAC2 in a folded lipid bilayer using a 0.2‐M to 1‐M KCl gradient reveal a shift in the reversal potential (V _rev). (f) Quantitative analysis of selectivity measurements reveals a significant reduction in anion selectivity for the efsevin‐induced closed state 1 (n = 6 for control, n = 5 for efsevin, Unpaired Student's t‐ test)

FIGURE 3

Predicted binding site of efsevin on zVDAC2 obtained by molecular docking. (a) Side view of the channel (pdb: 4bum, light brown) displays a binding pocket (red) for efsevin (light blue) located in the interspace between the inner channel wall and the N‐terminal pore‐lining α‐helix. (b) Analysis of 25 different molecular dockings (rows) reveals interactions between efsevin and 18 amino acid rests of zVDAC2 (columns) through hydrogen bonds (blue) and hydrophobic interactions (yellow). Residues with the most interactions (N207 in β‐sheet 14, K236, and N238 in β‐sheet 16 of the barrel) are boxed in red. (c) Detailed view of one representative docking with efsevin binding to the channel through hydrogen bonds between the most prominent amino acids highlighted in (b) and efsevin. (d) Surface representation of residues forming hydrophobic interactions in the conformation shown in (c) reveals a cavity that accommodates the p‐tolyl group of efsevin on the hinge of the flexible N‐terminal α‐helix. Nitrogen is shown in blue, oxygen in red, and sulfur in yellow

FIGURE 4

Effects of efsevin on conductance, and open probability (P_O) of zVDAC2^AAA in lipid bilayers. (a) Conductance–voltage relation of zVDAC2^AAA in painted bilayer recordings reveals a comparable shape before and after addition of 8‐μM efsevin. (b) No differences in single channel conductance for open and closed states of zVDAC2^AAA were observed after addition of efsevin. (c) Finally, in zVDAC2^AAA, efsevin was unable to induce the reduction of P_O observed for wild‐type zVDAC2 (n = 4 individual channels for control and 3 individual channels after addition of efsevin)

FIGURE 5

Sarcoplasmic reticulum (SR)‐mitochondria Ca²⁺ transfer upon heterologous expression of zVDAC2 and zVDAC2^AAA in HL‐1 cardiomyocytes. (a) Representative recordings of mitochondrial Ca²⁺ upon application of 10‐mM caffeine to induce SR calcium release from permeabilized HL‐1 cardiomyocytes. Traces from control conditions (black), recordings in the presence of 10‐μM ruthenium red to block mitochondrial Ca²⁺ uptake (RuR, grey) and in the presence of 10‐μM efsevin (red) are shown for native HL‐1 cells (native), cells transduced with shRNA targeting the endogenous mouse mVDAC2 (shmVDAC2), and cells overexpressing zVDAC2 and zVDAC2^AAA, respectively. (b) Statistical analysis of SR‐mitochondria Ca²⁺ transfer experiments. While native HL‐1 cardiomyocytes showed an efsevin‐sensitive uptake of Ca²⁺ into mitochondria (n = 21 for control, n = 7 for RuR, and n = 18 for efsevin), this uptake was abolished upon knock‐down of the endogenous mVDAC2 (shmVDAC2, n = 24 for control, n = 8 for RuR, n = 15 for efsevin). Subsequent heterologous expression of zVDAC2 (shmVDAC2, n = 21 for control, n = 7 for RuR, n = 15 for efsevin) and zVDAC2^AAA (shmVDAC2^AAA, n = 18 for control, n = 6 for RuR, n = 18 for efsevin) revealed restoration of SR‐mitochondria Ca²⁺ transfer. However, only zVDAC2 but not zVDAC2^AAA was sensitive to efsevin (Kruskal–Wallis test with Dunn's post hoc test)