Voltage Gating of VDAC Is Regulated by Nonlamellar Lipids of Mitochondrial Membranes

Laboratory of Physical and Structural Biology and National Center for Medical Rehabilitation Research, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 01/2007; 281(49):37496-506. DOI: 10.1074/jbc.M602548200
Source: PubMed


Evidence is accumulating that lipids play important roles in permeabilization of the mitochondria outer membrane (MOM) at the early stage of apoptosis. Lamellar phosphatidylcholine (PC) and nonlamellar phosphatidylethanolamine (PE) lipids are the major membrane components of the MOM. Cardiolipin (CL), the characteristic lipid from the mitochondrial inner membrane, is another nonlamellar lipid recently shown to play a role in MOM permeabilization. We investigate the effect of these three key lipids on the gating properties of the voltage-dependent anion channel (VDAC), the major channel in MOM. We find that PE induces voltage asymmetry in VDAC current-voltage characteristics by promoting channel closure at cis negative applied potentials. Significant asymmetry is also induced by CL. The observed differences in VDAC behavior in PC and PE membranes cannot be explained by differences in the insertion orientation of VDAC in these membranes. Rather, it is clear that the two nonlamellar lipids affect VDAC gating. Using gramicidin A channels as a tool to probe bilayer mechanics, we show that VDAC channels are much more sensitive to the presence of CL than could be expected from the experiments with gramicidin channels. We suggest that this is due to the preferential insertion of VDAC into CL-rich domains. We propose that the specific lipid composition of the mitochondria outer membrane and/or of contact sites might influence MOM permeability by regulating VDAC gating.

1 Follower
8 Reads
  • Source
    • "Although the response of multichannel VDAC conductance to decreasing voltage (channel opening) has been extensively studied (Colombini 1989; Mangan and Colombini 1987; Rostovtseva et al. 2006; Schein et al. 1976; Teijido et al. 2014; Zizi et al. 1995, 1998), as far as we are aware full hysteresis curves have never been published. We therefore studied the conductance of multichannel membranes in response to a voltage ramp (piecewise linear voltage vs. time). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Hysteresis in the conductance of voltage-sensitive ion channels is observed when the transmembrane voltage is periodically varied with time. Although this phenomenon has been used in studies of gating of the voltage-dependent anion channel, VDAC, from the outer mitochondrial membrane for nearly four decades, full hysteresis curves have never been reported, because the focus was solely on the channel opening branches of the hysteresis loops. We studied the hysteretic response of a multichannel VDAC system to a triangular voltage ramp the frequency of which was varied over three orders of magnitude, from 0.5 mHz to 0.2 Hz. We found that in this wide frequency range the area encircled by the hysteresis curves changes by less than a factor of three, suggesting broad distribution of the characteristic times and strongly non-equilibrium behavior. At the same time, quasi-equilibrium two-state behavior is observed for hysteresis branches corresponding to VDAC opening. This enables calculation of the usual equilibrium gating parameters, gating charge and voltage of equipartitioning, which were found to be almost insensitive to the ramp frequency. To rationalize this peculiarity, we hypothesize that during voltage-induced closure and opening the system explores different regions of the complex free energy landscape, and, in the opening branch, follows quasi-equilibrium paths.
    Biophysics of Structure and Mechanism 06/2015; 44(6). DOI:10.1007/s00249-015-1049-2 · 2.22 Impact Factor
  • Source
    • "We asked whether the electrophysiological characteristics of this stable protein nanopore are impacted by the nature of the lipid environment within the membrane. It is already established that the membrane composition [29], surface tension [30], surface charge [31] [32] [33] [34], the nature of polar headgroup [35] [36] [37] [38], the degree of acyl chain unsaturation [39], the hydrophobic thickness [37] [40] [41] and the bilayer curvature [42] [43] can play a critical role in promoting the activity, stability, folding, gating and ligand-binding dynamics of the transmembrane proteins. In this "
    [Show abstract] [Hide abstract]
    ABSTRACT: Using rational membrane protein design, we were recently able to obtain a β-barrel protein nanopore that was robust under an unusually broad range of experimental circumstances. This protein nanopore was based upon the native scaffold of the bacterial ferric hydroxamate uptake component A (FhuA) of E. coli. In this work, we expanded the examinations of the open-state current of this engineered protein nanopore, also called FhuA ΔC/Δ4L, employing an array of lipid bilayer systems that contained charged and uncharged as well as conical and cylindrical lipids. Remarkably, systematical single-channel analysis of FhuA ΔC/Δ4L indicated that most of its biophysical features, such as the unitary conductance and the stability of the open-state current, were not altered under the conditions tested in this work. However, electrical recordings at high transmembrane potentials revealed that the presence of conical phospholipids within the bilayer catalyzes the first, stepwise current transition of the FhuA ΔC/Δ4L protein nanopore to a lower-conductance open state. This study reinforces the stability of the open-state current of the engineered FhuA ΔC/Δ4L protein nanopore under various experimental conditions, paving the way for further critical developments in biosensing and molecular biomedical diagnosis.
    Biochimica et Biophysica Acta 12/2012; 1828(3). DOI:10.1016/j.bbamem.2012.12.003 · 4.66 Impact Factor
  • Source
    • "After VDAC channels were inserted and their parameters were monitored, tubulin was added to one or both sides of the membrane under constant stirring for 2 min. Current recordings were performed as described previously [49] using an Axopatch 200B amplifier (Axon Instruments, Inc., Foster City, CA) in the voltage clamp mode. Single-channel data were filtered by a low-pass 8-pole Butterworth filter (Model 9002, Frequency Devices, Inc., Haverhill, MA) at 15 kHz and directly saved into the computer memory with a sampling frequency of 50 kHz. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Tubulin was recently found to be a uniquely potent regulator of the voltage-dependent anion channel (VDAC), the most abundant channel of the mitochondrial outer membrane, which constitutes a major pathway for ATP/ADP and other metabolites across this membrane. Dimeric tubulin induces reversible blockage of VDAC reconstituted into a planar lipid membrane and dramatically reduces respiration of isolated mitochondria. Here we show that VDAC phosphorylation is an important determinant of its interaction with dimeric tubulin. We demonstrate that in vitro phosphorylation of VDAC by either glycogen synthase kinase-3β (GSK3β) or cAMP-dependent protein kinase A (PKA), increases the on-rate of tubulin binding to the reconstituted channel by orders of magnitude, but only for tubulin at the cis side of the membrane. This and the fact the basic properties of VDAC, such as single-channel conductance and selectivity, remained unaltered by phosphorylation allowed us to suggest the phosphorylation regions positioned on the cytosolic loops of VDAC and establish channel orientation in our reconstitution experiments. Experiments on human hepatoma cells HepG2 support our conjecture that VDAC permeability for the mitochondrial respiratory substrates is regulated by dimeric tubulin and channel phosphorylation. Treatment of HepG2 cells with colchicine prevents microtubule polymerization, thus increasing dimeric tubulin availability in the cytosol. Accordingly, this leads to a decrease of mitochondrial potential measured by assessing mitochondrial tetramethylrhodamine methyester uptake with confocal microscopy. Inhibition of PKA activity blocks and reverses mitochondrial depolarization induced by colchicine. Our findings suggest a novel functional link between serine/threonine kinase signaling pathways, mitochondrial respiration, and the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation.
    PLoS ONE 10/2011; 6(10):e25539. DOI:10.1371/journal.pone.0025539 · 3.23 Impact Factor
Show more