50 research outputs found
The antiarrhythmic compound efsevin directly modulates voltageâdependent anion channel 2 by binding to its inner wall and enhancing mitochondrial Ca2+ uptake
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 voltageâdependent anion channel 2 (VDAC2) in the outer mitochondrial membrane. Here, we investigated the molecular mechanism of the efsevinâVDAC2 interaction.
Experimental Approach
To evaluate the functional interaction of efsevin and VDAC2, we measured currents through recombinant VDAC2 in planar lipid bilayers. Using molecular ligandâprotein docking and mutational analysis, we identified the efsevin binding site on VDAC2. Finally, physiological consequences of the efsevinâinduced modulation of VDAC2 were analysed in HLâ1 cardiomyocytes.
Key Results
In lipid bilayers, efsevin reduced VDAC2 conductance and shifted the channel's open probability towards less anionâselective closed states. Efsevin binds to a binding pocket formed by the inner channel wall and the poreâlining Nâterminal αâhelix. Exchange of amino acids N207, K236 and N238 within this pocket for alanines abolished the channel's efsevinâresponsiveness. Upon heterologous expression in HLâ1 cardiomyocytes, both channels, wildâtype VDAC2 and the efsevinâinsensitive VDAC2AAA restored mitochondrial Ca2+ uptake, but only wildâtype 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 SRâmitochondria Ca2+ transfer. This study sheds new light on the function of VDAC2 and provides a basis for structureâaided chemical optimization of efsevin
Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function
Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand
Ceramides bind VDAC2 to trigger mitochondrial apoptosis
Ceramides draw wide attention as tumor suppressor lipids that act directly on mitochondria to trigger apoptotic cell death. However, molecular details of the underlying mechanism are largely unknown. Using a photoactivatable ceramide probe, we here identify the voltage-dependent anion channels VDAC1 and VDAC2 as mitochondrial ceramide binding proteins. Coarse-grain molecular dynamics simulations reveal that both channels harbor a ceramide binding site on one side of the barrel wall. This site includes a membrane-buried glutamate that mediates direct contact with the ceramide head group. Substitution or chemical modification of this residue abolishes photolabeling of both channels with the ceramide probe. Unlike VDAC1 removal, loss of VDAC2 or replacing its membrane-facing glutamate with glutamine renders human colon cancer cells largely resistant to ceramide-induced apoptosis. Collectively, our data support a role of VDAC2 as direct effector of ceramide-mediated cell death, providing a molecular framework for how ceramides exert their anti-neoplastic activity
The Value of Information in Markets with Heterogeneously Informed Traders - A Simulation and an Experimental Approach
Identification and functional characterization of the malignant hyperthermia mutation T1354S in the α1S subunit of the skeletal muscle voltage-gated calcium channel.
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High resolution structure and double electron-electron resonance of the zebrafish voltage-dependent anion channel 2 reveal an oligomeric population.
In recent years, there has been a vast increase in structural and functional understanding of VDAC1, but VDAC2 and -3 have been understudied despite having many unique phenotypes. One reason for the paucity of structural and biochemical characterization of the VDAC2 and -3 isoforms stems from the inability of obtaining purified, functional protein. Here we demonstrate the expression, isolation, and basic characterization of zebrafish VDAC2 (zfVDAC2). Further, we resolved the structure of zfVDAC2 at 2.8 Ă
resolution, revealing a crystallographic dimer. The dimer orientation was confirmed in solution by double electron-electron resonance spectroscopy and by cross-linking experiments disclosing a dimer population of âŒ20% in lauryldimethine amine oxide detergent micelles, whereas in lipidic bicelles a higher population of dimeric and higher order oligomers species were observed. The present study allows for a more accurate structural comparison between VDAC2 and its better-studied counterpart VDAC1
Recommended from our members
High resolution structure and double electron-electron resonance of the zebrafish voltage-dependent anion channel 2 reveal an oligomeric population.
In recent years, there has been a vast increase in structural and functional understanding of VDAC1, but VDAC2 and -3 have been understudied despite having many unique phenotypes. One reason for the paucity of structural and biochemical characterization of the VDAC2 and -3 isoforms stems from the inability of obtaining purified, functional protein. Here we demonstrate the expression, isolation, and basic characterization of zebrafish VDAC2 (zfVDAC2). Further, we resolved the structure of zfVDAC2 at 2.8 Ă
resolution, revealing a crystallographic dimer. The dimer orientation was confirmed in solution by double electron-electron resonance spectroscopy and by cross-linking experiments disclosing a dimer population of âŒ20% in lauryldimethine amine oxide detergent micelles, whereas in lipidic bicelles a higher population of dimeric and higher order oligomers species were observed. The present study allows for a more accurate structural comparison between VDAC2 and its better-studied counterpart VDAC1
TRPV4 channels are essential for alveolar epithelial barrier function as protection from lung edema.
Ischemia/reperfusion-induced edema (IRE), one of the most significant causes of mortality after lung transplantation, can be mimicked ex vivo in isolated perfused mouse lungs (IPL). Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel studied in endothelium; however, its role in the lung epithelium remains elusive. Here, we show enhanced IRE in TRPV4-deficient (TRPV4(-/-)) IPL compared with that of WT controls, indicating a protective role of TRPV4 in maintenance of the alveolar epithelial barrier. By immunohistochemistry, mRNA profiling, and electrophysiological characterization, we detected TRPV4 in bronchial epithelium, alveolar epithelial type I (ATI), and alveolar epithelial type II (ATII) cells. Genetic ablation of TRPV4 resulted in reduced expression of the water-conducting aqua porin-5 (AQP-5) channel in ATI cells. Migration of TRPV4(-)(/-) ATI cells was reduced, and cell barrier function was impaired. Analysis of isolated primary TRPV4(-/-) ATII cells revealed a reduced expression of surfactant protein C, and the TRPV4 activator GSK1016790A induced increases in current densities only in WT ATII cells. Moreover, TRPV4(-/-) lungs of adult mice developed significantly larger mean chord lengths and altered lung function compared with WT lungs. Therefore, our data illustrate essential functions of TRPV4 channels in alveolar epithelial cells and in protection from edema formation