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 efsevininduced 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 Ca 2+ 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 Ca 2+ transfer. This study sheds new light on the function of VDAC2 and provides a basis for structure-aided chemical optimization of efsevin.Abbreviations: CPVT, catecholaminergic polymorphic ventricular tachycardia; DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine; hVDAC2, human voltage-dependent anion channel 2; IRES, internal ribosome entry site; MCU, mitochondrial calcium uniporter; MOI, multiplicity of infection; mVDAC2, mouse voltage-dependent anion channel 2; OMM, outer mitochondrial membrane; P O , open probability; RuR, ruthenium red; shRNA, short hairpin RNA; SR, sarcoplasmic reticulum; VDAC, voltage-dependent anion channel; zVDAC2, zebrafish voltage-dependent anion channel 2.

Targeting the Multiple Physiologic Roles of VDAC With Steroids and Hydrophobic Drugs

“…Authors concluded that this mechanism could explain the efsevin-induced facilitated transfer of calcium from the sarcoplasmic reticulum into mitochondria in zebra fish. Interestingly, in these studies, efsevin has not been observed to be involved in apoptosis (Schweitzer et al, 2017;Wilting et al, 2020) despite the accepted role of VDAC2 in apoptosis through recruitment of Bax or Bak (Cheng et al, 2003;Chin et al, 2018).…”

“…Efsevin is thought to enhance mitochondrial uptake of calcium through VDAC2, thereby preventing calcium accumulation in the cytosol (Schweitzer et al, 2017). Lately, using an in vitro system of reconstituted zfVDAC2 channels, Wilting et al (2020) found that efsevin promotes channel closure and shifts its selectivity toward less anionic thus, according to the previous studies (Rostovtseva et al, 2005;Tan and Colombini, 2007), ultimately causing an increase of VDAC2 permeability for calcium. Authors concluded that this mechanism could explain the efsevin-induced facilitated transfer of calcium from the sarcoplasmic reticulum into mitochondria in zebra fish.…”

“…An additional characterization using photoaffinity labeling with mass spectrometry identified a cholesterol binding pocket in the lipid-facing outer surface of the mVDAC1 β-barrel coordinated by the membrane-facing residue E73 (Budelier et al, 2017). Cholesterol is, maybe, the clearest example of a hydrophobic (Budelier et al, 2017) MD Simulations (Weiser et al, 2014b) No (Queralt-Martin et al, 2019) No (Queralt-Martin et al, 2019) Yes (van Blitterswijk et al, 1987;Weinrich et al, 2009) Allopregnanolone Photoaffinity labeling (Cheng et al, 2019) No (Cheng et al, 2019) No (Cheng et al, 2019) Yes (Alakoskela et al, 2007;Cheng et al, 2019) Olesoxime Photoaffinity labeling (Bordet et al, 2007) Yes (Rovini et al, 2019) No (Rovini et al, 2019) No (Rovini et al, 2019) Propofol Photoaffinity labeling (Weiser et al, 2013;Weiser et al, 2014a) Yes (Weiser et al, 2014a) No (Weiser et al, 2014a) No (Bahri et al, 2007;Weiser et al, 2014a) Itraconazole Photoaffinity labeling and affinity pulldown (Head et al, 2015) TBD TBD TBD Efsevin Affinity pulldown (Shimizu et al, 2015) Yes (Wilting et al, 2020) No (Wilting et al, 2020) TBD Erastin Binds to VDAC2; Affinity pulldown (Yagoda et al, 2007) No (Maldonado et al, 2013) No (Maldonado et al, 2013) Increases VDAC2 permeability to NADH (Bauer et al, 2011) Yes (Jacobs, personal communication)…”

VDAC2-mediated regulation of calcium homeostasis in cardiomyocytes (a review)

“…VDAC2 образован бочкообразной структурой, состоящей из 19 антипараллельных β-листов и N-концевой α-спирали, выстилающей внутреннюю стенку канала [25]. Сайт связывания с эфсевином расположен в так называемой канавке между внутренней стенкой канала и α-спиралью [26]. VDAC подвергается зависящему от напряжения стробированию между анион-селективным состоянием высокой проводимости, называемым классическим открытым состоянием, и несколькими катион-селективными состояниями низкой проводимости, именуемыми закрытыми состо-яниями [27,28], т. е. закрытые состояния VDAC более катион-селективные по сравнению с открытым состоянием [29].…”

“…Кроме того, связывание эфсевина с другими мишенями, включая изоформы VDAC1 и VDAC3, никогда не тестировалось. Таким образом, его неизвестная селектив-ность, низкая стабильность эфсевина указывают на необходимость химической оптимизации соединения перед проведением дальнейших доклинических и клинических исследований [26].…”