Cell-free electrophysiology of human VDACs incorporated into nanodiscs: An improved method

Abstract

Voltage-dependent anion-selective channel (VDAC) is one of the main proteins of the outer mitochondrial membrane of all eukaryotes, where it forms aqueous, voltage-sensitive, and ion-selective channels. Its electrophysiological properties have been thoroughly analyzed with the planar lipid bilayer technique. To date, however, available results are based on isolations of VDACs from tissue or from recombinant VDACs produced in bacterial systems. It is well known that the cytosolic overexpression of highly hydrophobic membrane proteins often results in the formation of inclusion bodies containing insoluble aggregates. Purification of properly folded proteins and restoration of their full biological activity requires several procedures that considerably lengthen experimental times. To overcome these restraints, we propose a one-step reaction that combines in vitro cell-free protein expression with nanodisc technology to obtain human VDAC isoforms directly integrated in a native-like lipid bilayer. Reconstitution assays into artificial membranes confirm the reliability of this new methodological approach and provide results comparable to those of VDACs prepared with traditional protein isolation and reconstitution protocols. The use of membrane-mimicking nanodisc systems represents a breakthrough in VDAC electrophysiology and may be adopted to further structural studies.

Voltage-Dependent Anion Selective Channel 3: Unraveling Structural and Functional Features of the Least Known Porin Isoform

“…Careful analyzes showed that, after treatment with reducing agents, VDAC3 occasionally reaches the characteristic conductance level of a fully open VDAC ( Okazaki et al, 2015 ; Reina et al, 2016 ). Very recently, the group of De Pinto further confirmed what was previously reported in Checchetto et al (2014) using nanodisc-stabilized human VDACs: accordingly, in the absence of any reductants, VDAC3 inserted into artificial membranes as small, non-gated channels ( Conti Nibali et al, 2021 ). When cysteines are found to be reduced in mass spectrometry analysis (following DTT and iodoacetamide treatment) it means that those cysteines were probably involved in disulfide bridges (otherwise DTT could not have reduced them back to SH).…”

Voltage Dependent Anion Channel 3 (VDAC3) protects mitochondria from oxidative stress

“…Subsequently, Checchetto et al described very small and ungated channels upon VDAC3 reconstitution under non-reducing conditions [ 22 ]. The addition of reductants to the refolding procedures [ 23 ] or pre-incubation with DTT before electrophysiological analysis [ 24 ] significantly increased the current through VDAC3 which behaves as canonical voltage-gated VDAC pores albeit with a much lower insertion rate than isoform 1 and 2. Swapping experiments had previously suggested the involvement of cysteine residues in VDAC3 pore-activity [ 25 ]: the replacement of the VDAC3 N-terminus, containing two cysteines, with the homologous sequence of VDAC1, which has none, allowed isoform 3 to completely rescue the porin-less yeast temperature-sensitive phenotype [ 25 ].…”

Electrophysiological properties and structural prediction of the SARS-CoV-2 viroprotein E

“…Electrophysiological experiments were carried out using the Planar Lipid Bilayer (PLB) technique according to ( Checchetto et al, 2014 ; Reina et al, 2016 ; Conti Nibali et al, 2021 ). In brief, 1% DiphPC (Avanti Polar Lipids, Alabaster, AL) in n-decane was used for painting bilayer membranes with an approximate 110–150 pF capacity on a 200 μm aperture of a Delrin cuvette (Warner Instruments).…”