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• W2022101585 abstract "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. 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. It is a well established fact that the major form of apoptosis proceeds through the mitochondrial pathway, wherein mitochondria play a controlling role (1Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 3Waterhouse N.J. Ricci J.E. Green D.R. Biochimie (Paris). 2002; 84: 113-121Crossref PubMed Scopus (110) Google Scholar). Numerous proapoptotic molecules and pathological stimuli converge on mitochondria to induce the permeabilization of the mitochondria outer membrane (MOM), 2The abbreviations used are: MOM, mitochondria outer membrane; CL, cardiolipin; BzATP, benzoylbenzoyl-ATP; VDAC, voltage-dependent anion channel; PC, phosphatidylcholine; PE, phosphatidylethanolamine; DOPC, dioleoylphosphatidylcholine; DOPE, dioleoylphosphatidylethanolamine; nS, nanosiemens. 2The abbreviations used are: MOM, mitochondria outer membrane; CL, cardiolipin; BzATP, benzoylbenzoyl-ATP; VDAC, voltage-dependent anion channel; PC, phosphatidylcholine; PE, phosphatidylethanolamine; DOPC, dioleoylphosphatidylcholine; DOPE, dioleoylphosphatidylethanolamine; nS, nanosiemens. which leads to cytochrome c release from the intermembrane space, consequent caspase activation, and irreversible apoptotic cell death. Although it was shown that many proteins could inhibit or prevent MOM permeabilization by acting on mitochondrial membranes, the mechanism of MOM permeabilization during apoptosis remains controversial (4Green D.R. Kroemer G. Science. 2004; 305: 626-629Crossref PubMed Scopus (2751) Google Scholar). The local regulation and execution of MOM permeabilization, predominantly orchestrated by proteins from the Bcl-2 family, involves mitochondrial lipids. For example, a reversible conformational change of the proapoptotic Bcl-2 family protein Bax, which occurs prior to Bax oligomerization, was obtained upon interaction of monomeric Bax with the membrane surface (5Yethon J.A. Epand R.F. Leber B. Epand R.M. Andrews D.W. J. Biol. Chem. 2003; 278: 48935-48941Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Phospholipids were shown to significantly affect the activity of another proapoptotic BH3 domain-only protein Bid in cell-free assays (6Esposti M.D. Erler J.T. Hickman J.A. Dive C. Mol. Cell Biol. 2001; 21: 7268-7276Crossref PubMed Scopus (124) Google Scholar). A caspase-8-cleaved Bid (tBid) was found to display lipid transfer activity (6Esposti M.D. Erler J.T. Hickman J.A. Dive C. Mol. Cell Biol. 2001; 21: 7268-7276Crossref PubMed Scopus (124) Google Scholar) and promote negative membrane curvature and, as a result, destabilize bilayer membranes (7Epand R.F. Martinou J.C. Fornallaz-Mulhauser M. Hughes D.W. Epand R.M. J. Biol. Chem. 2002; 277: 32632-32639Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). It was proposed (8Basanez G. Sharpe J.C. Galanis J. Brandt T.B. Hardwick J.M. Zimmerberg J. J. Biol. Chem. 2002; 277: 49360-49365Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) that Bax-type apoptotic proteins could form lipidic pore-type nonbilayer structures in the membrane. It was also shown that cardiolipin (CL), a lipid characteristic of mitochondria membrane, increases binding of tBid to pure lipid vesicles as well as to MOM (9Lutter M. Fang M. Luo X. Nishijimat M. Xie X.S. Wang X. Nat. Cell Biol. 2000; 2: 754-756Crossref PubMed Scopus (404) Google Scholar) and promotes formation of large pores by tBid and monomeric Bax (10Kuwana T. Mackey M.R. Perkins G. Ellisman M.H. Latterich M. Schneiter R. Freen D.R. Newmeyer D. Cell. 2002; 111: 331-342Abstract Full Text Full Text PDF PubMed Scopus (1203) Google Scholar). CL is a unique phospholipid with four acyl chains. It is found in high concentrations in the inner membrane, where CL is the only major (up to 20 weight % of the total lipids) negatively charged phospholipid (11De Kroon A.I.P.M. Dolis D. Mayer A. Lill R. De Kruijff B. Biochim. Biophys. Acta. 1997; 1325: 108-116Crossref PubMed Scopus (193) Google Scholar, 12Ardail D. Privat J.P. Egret-Charlier M. Levrat C. Lerme F. Louisot P. J. Biol. Chem. 1990; 265: 18797-18802Abstract Full Text PDF PubMed Google Scholar). In the outer membrane, CL is present in much lower concentrations, but its content is higher in the contact sites, the points of close proximity between the inner and outer mitochondrial membranes (12Ardail D. Privat J.P. Egret-Charlier M. Levrat C. Lerme F. Louisot P. J. Biol. Chem. 1990; 265: 18797-18802Abstract Full Text PDF PubMed Google Scholar, 13Simbeni R. Pon L. Zinser E. Paltauf F. Daum G. J. Biol. Chem. 1991; 266: 10047-10049Abstract Full Text PDF PubMed Google Scholar). CL is strongly bound to various enzymes and protein complexes involved in transport processes across mitochondrial inner membrane (14Powell G.L. Knowles P.F. Marsh D. Biochemistry. 1987; 26: 8138-8145Crossref PubMed Scopus (67) Google Scholar, 15Hoch F.L. Biochim. Biophys. Acta. 1992; 1113: 71-133Crossref PubMed Scopus (539) Google Scholar). For example, CL is required for the cytochrome c oxidase activity (16Fry M. Green D.E. Biochim. Biophys. Res. Commun. 1980; 93: 1238-1460Crossref PubMed Scopus (176) Google Scholar, 17Vik S.B. Georgevich G. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 1456-1460Crossref PubMed Scopus (183) Google Scholar). It was demonstrated that tBid interacts with CL on functional mitochondria (18Kim T.H. Zhao Y. Ding W.X. Shin J.N. He X. Seo Y.W. Chen J. Rabinowich H. Amoscato A.A. Yin X.M. Mol. Biol. Cell. 2004; 15: 3061-3072Crossref PubMed Scopus (149) Google Scholar). This interaction occurs mainly in the contact sites and might contribute to mitochondrial cristae reorganization and cytochrome c release. Recent experiments with CL-deficient yeast mitochondria provide further evidence of the requirement of this lipid for tBid binding to mitochondria (19Gonzalvez F. Pariselli F. Dupaigne P. Budihardjo I. Lutter M. Antonsson B. Diolez P. Manon S. Martinou J.C. Goubern M. Wang X. Bernard S. Petit P.X. Cell Death Differ. 2005; 12: 614-626Crossref PubMed Scopus (132) Google Scholar, 20Gonzalvez F. Bessoule J.J. Rocchiccioli F. Manon S. Petit P.X. Cell Death Differ. 2005; 12: 659-667Crossref PubMed Scopus (59) Google Scholar). Moreover, the reported inhibition of state-3 respiration and ATP synthesis by tBid also requires the presence of CL (19Gonzalvez F. Pariselli F. Dupaigne P. Budihardjo I. Lutter M. Antonsson B. Diolez P. Manon S. Martinou J.C. Goubern M. Wang X. Bernard S. Petit P.X. Cell Death Differ. 2005; 12: 614-626Crossref PubMed Scopus (132) Google Scholar, 20Gonzalvez F. Bessoule J.J. Rocchiccioli F. Manon S. Petit P.X. Cell Death Differ. 2005; 12: 659-667Crossref PubMed Scopus (59) Google Scholar). These observations, despite sometimes contrasting results (21Iverson S.L. Enoksson M. Gogvadze V. Ott M. Orrenius S. J. Biol. Chem. 2004; 279: 1100-1107Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 22Polčic P. Su X. Fowlkes J. Blachly-Dyson E. Dowhan W. Forte M. Cell Death Differ. 2005; 12: 310-312Crossref PubMed Scopus (20) Google Scholar), have increased current awareness of the role of CL in the mechanism of MOM permeabilization by proapoptotic Bcl-2 proteins. The major channel in MOM is the voltage-dependent anion channel (VDAC). VDAC is known to be primarily responsible for metabolite flux across the MOM (23Colombini M. Curr. Top. Membr. 1994; 42: 73-101Crossref Scopus (73) Google Scholar, 24Hodge T. Colombini M. J. Membr. Biol. 1997; 157: 271-279Crossref PubMed Scopus (218) Google Scholar). One of the characteristic properties of the VDAC channel reconstituted into planar lipid membranes is its voltage gating (25Colombini M. Blachly-Dyson E. Forte M. Narahashi T. Ion Channels. 4. Plenum Press, New York1996: 169-202Google Scholar). VDAC channels can exist in two functional states that differ in their ability to pass nonelectrolytes and to conduct ions (24Hodge T. Colombini M. J. Membr. Biol. 1997; 157: 271-279Crossref PubMed Scopus (218) Google Scholar, 26Schein S.J. Colombini M. Finkelstein A. J. Membr. Biol. 1976; 30: 99-120Crossref PubMed Scopus (411) Google Scholar, 27Colombini M. J. Membr. Biol. 1989; 111: 103-111Crossref PubMed Scopus (252) Google Scholar). Elevated voltages favor “closed” states, the states of smaller conductance. These states are characterized by weak cationic selectivity, as compared with weak anionic selectivity in the open state, and are virtually impermeable for negatively charged metabolites, such as ATP (28Rostovtseva T. Colombini M. J. Biol. Chem. 1996; 271: 28006-28008Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Therefore, VDAC closure greatly diminishes metabolite flux across MOM. However, closed states are able to transport small ions, such as K+ and Cl–. The gating mechanism for VDAC is still under discussion, but most of the experimental and theoretical evidence supports the model proposed by Colombini and co-workers (25Colombini M. Blachly-Dyson E. Forte M. Narahashi T. Ion Channels. 4. Plenum Press, New York1996: 169-202Google Scholar, 29Song J. Midson C. Blachly-Dyson E. Forte M. Colombini M. J. Biol. Chem. 1998; 273: 24406-24413Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), where the existence of a mobile domain in the wall of the channel, called the voltage sensor, is postulated. VDAC responds to the electric field applied to the membrane by moving the sensor to the surface of the membrane, which results in a pore of diminished diameter and inverted selectivity (30Peng S. Blachly-Dyson E. Forte M. Colombini M. Biophys. J. 1992; 62: 123-135Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 32Song J.M. Midson C. Blachly-Dyson E. Forte M. Colombini M. Biophys. J. 1998; 74: 2926-2944Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In the present study, we hypothesize that the effect of membrane lipid composition on VDAC voltage gating is related to the elastic stress of lipid packing. The physical mechanisms by which membrane proteins respond to the elastic stress are attracting significant interest (33Suchyna T.M. Tape S.E. Koeppe II, R.E. Andersen O.S. Sachs F. Gottlieb P.A. Nature. 2004; 430: 235-240Crossref PubMed Scopus (241) Google Scholar, 39Lundbaek J.A. J. Phys. Condens. Matter. 2006; 18: S1305-S1344Crossref PubMed Scopus (67) Google Scholar). The general idea is that protein conformational transitions must be coupled to some mechanical displacements that change the elastic stress of surrounding lipids and vice versa. In other words, if a membrane channel switches between two different conformation states, such as open and closed, one of these states might be more energetically favorable due to the protein interaction with the surrounding hydrophobic lipid phase. In order to test this hypothesis, we investigated the effect of three key mitochondria lipids: phosphatidylcholine (PC), lamellar lipid with small spontaneous curvature, and two nonlamellar lipids with high negative spontaneous curvature, phosphatidylethanolamine (PE) and CL. The rationale for our study is that when a nonlamellar lipid, like PE, which spontaneously forms inverted hexagonal phase, is forced into the planar bilayer structure, this results in a significant stress of lipid packing in the region of the acyl chains (34van den Killian J.A. Brink-van der Laan E. de Kruijff B. Biochim. Biophys. Acta. 2004; 1666: 275-288Crossref PubMed Scopus (324) Google Scholar, 37Booth P.J. Curr. Opin. Struct. Biol. 2005; 15: 435-440Crossref PubMed Scopus (54) Google Scholar, 39Lundbaek J.A. J. Phys. Condens. Matter. 2006; 18: S1305-S1344Crossref PubMed Scopus (67) Google Scholar, 42Gullingsrud J. Schulten K. Biophys. J. 2004; 86: 3496-3509Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Estimates show that the difference in the corresponding lateral pressure between PE and PC membranes can reach several hundred atmospheres. It is plausible then that such change in the pressure may change VDAC conformational equilibrium. Here we compare the voltage gating of VDAC channels reconstituted into planar membranes formed from two lipids, PC and PE, whose spontaneous curvature, packing density, and hydrophobic thickness are well known (40Keller S.L. Bezrukov S.M. Gruner S.M. Tate M.W. Vodyanoy I. Parsegian V.A. Biophys. J. 1993; 65: 23-27Abstract Full Text PDF PubMed Scopus (227) Google Scholar, 43Gawrisch K. Parsegian V.A. Hajduk D.A. Tate M.W. Gruner S.M. Fuller N.L. Rand R.P. Biochemistry. 1992; 31: 2856-2864Crossref PubMed Scopus (137) Google Scholar, 46Petrache H.I. Tristram-Nagle S. Gawrisch K. Harries D. Parsegian V.A. Biophys. J. 2004; 86: 1574-1586Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). We find that the relative probability of the open and closed states changes with lipid substitution. The VDAC gating obtained in the multichannel membranes made of PE, a nonlamellar lipid with high negative spontaneous curvature and high packing stress, is more asymmetric in voltage than in the membranes formed from PC, a lamellar lipid with relaxed bilayer structure. We also demonstrate that CL, which induces negative curvature (47Verkleij A.J. de Kruijff B. Leunissen-Bijvelt J. Van Echteld C.J. Hille J. Rijnbout H. Biochim. Biophys. Acta. 1982; 693: 1-12Crossref PubMed Scopus (87) Google Scholar, 48Rand R.P. Sengupta S. Biochim. Biophys. Acta. 1972; 255: 484-492Crossref PubMed Scopus (263) Google Scholar), promotes similar voltage-gating asymmetry. As an internal control of the membrane mechanical properties, we use gramicidin A channels, whose association-dissociation kinetics is known to depend on several bilayer parameters, such as thickness, compression-expansion modulus, bending rigidity, interfacial tension (39Lundbaek J.A. J. Phys. Condens. Matter. 2006; 18: S1305-S1344Crossref PubMed Scopus (67) Google Scholar), and dipole potential (49Rokitskaya T.I. Antonenko Y.N. Kotova E.A. Biophys. J. 1997; 73: 850-854Abstract Full Text PDF PubMed Scopus (93) Google Scholar), but also on the lipid packing stress. Comparison of VDAC and gramicidin channel behavior in the membranes of the same lipid composition allows us to suggest that VDAC preferably inserts into the CL-rich domains. VDAC from Neurospora crassa mitochondrial outer membranes, isolated and purified according to standard methods (50Mannella C.A. J. Cell Biol. 1982; 94: 680-687Crossref PubMed Scopus (128) Google Scholar, 51Freitag H. Benz R. Neupert W. Methods Enzymol. 1983; 97: 286-294Crossref PubMed Scopus (29) Google Scholar), was a generous gift of Marco Colombini (University of Maryland, College Park). Dioleoylphosphatidylcholine (DOPC), Dioleoylphosphatidylethanolamine (DOPE), and CL were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). BzATP was purchased from Sigma. The mixtures of DOPC and DOPE or CL were prepared from aliquots of two lipid solutions in chloroform, followed by drying lipid mixtures with nitrogen and then redissolving them in hexane or pentane to a total lipid concentration of 5 mg/ml. Bilayer membranes were formed from monolayers across 70–90-μm diameter orifices in a 15-μm-thick Teflon partition that separated two chambers (52Rostovtseva T.K. Bezrukov S.M. Biophys. J. 1998; 74: 2365-2373Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The experimental Teflon chamber was sonicated for 15 min in a chloroform/methanol (2:1) mixture, and a new partition was used each time when lipid composition was changed in order to avoid any traces of a “foreign” lipid. The membrane potential was maintained using Ag/AgCl electrodes with 3 m KCl and 15% (w/v) agarose bridges. Aqueous solutions of 250 mm or 1 m KCl and 1 mm CaCl2 were buffered by 5 mm HEPES at pH 7.0 or 7.6. VDAC channel insertion was achieved by adding 0.01–0.1 μl of a 1% Triton X-100 solution of purified VDAC to the 1-ml aqueous phase in the cis compartment while stirring. Potential is defined as positive when it is greater at the side of the VDAC addition (cis-side). For more details see Refs. 53Bezrukov S.M. Vodyanoy I. Biophys. J. 1993; 64: 16-25Abstract Full Text PDF PubMed Scopus (176) Google Scholar and 54Rostovtseva T.K. Antonsson B. Suzuki M. Youle R.J. Colombini M. Bezrukov S.M. J. Biol. Chem. 2004; 279: 13575-13583Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar. The voltage-dependent properties of a VDAC-containing membrane were assessed following the protocol devised by Colombini and colleagues (26Schein S.J. Colombini M. Finkelstein A. J. Membr. Biol. 1976; 30: 99-120Crossref PubMed Scopus (411) Google Scholar, 27Colombini M. J. Membr. Biol. 1989; 111: 103-111Crossref PubMed Scopus (252) Google Scholar, 55Zizi M. Byrd C. Boxus R. Colombini M. Biophys. J. 1998; 75: 704-713Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) in which gating is inferred from the channel response to a slowly changing periodic transmembrane voltage. In our experiments, we used a symmetrical 5-mHz triangular voltage wave with ±60 mV amplitude from a function waveform generator (model 33120A; Hewlett Packard). Data were acquired with the help of a Digidata 1322A board (Axon Instruments, Inc.) at a sampling frequency of 1 Hz and analyzed using the pClamp 9 software (Axon Instruments). Voltage-dependent channels respond to the transmembrane voltage (V) by changing their conformational equilibrium. For a two-state channel, the equilibrium probabilities of the open and closed states, Popen(V) and Pclosed(V), obey the Boltzmann distribution (56Ehrenstein G. Lecar H. Nossal R. J. Gen. Physiol. 1970; 55: 119-133Crossref PubMed Scopus (155) Google Scholar), Pclosed(V)/Popen(V)=exp((V0-V)ne/kT)(Eq. 1) where n is the net effective gating charge, which is a measure of the voltage dependence steepness, and V0 is the voltage at which half the channels are open; e, k, and T have their usual meanings of the elementary charge, Boltzmann constant, and absolute temperature. In the case of VDAC two distinctly different gating processes take place at negative and positive potentials (25Colombini M. Blachly-Dyson E. Forte M. Narahashi T. Ion Channels. 4. Plenum Press, New York1996: 169-202Google Scholar). We use Equation 1 for the gating at negative applied potentials and define Popen(V) as the ratio, Popen(V)=(G(V)-Gmin)/(Gmax-Gmin)(Eq. 2) where Gmax and Gmin are the maximum and the minimum conductances, corresponding to almost all channels open (small voltages) and almost all channels closed (high voltages), respectively. The 5-mHz voltage wave is usually slow enough to obtain quasi-equilibrium G(V) distributions, which allow comparison of VDAC gating under different experimental conditions (for more detailed discussion of this protocol, see Refs. 27Colombini M. J. Membr. Biol. 1989; 111: 103-111Crossref PubMed Scopus (252) Google Scholar, 55Zizi M. Byrd C. Boxus R. Colombini M. Biophys. J. 1998; 75: 704-713Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 57Zizi M. Thomas L. Blachly-Dyson E. Forte M. Colombini M. J. Membr. Biol. 1995; 144: 121-129Crossref PubMed Scopus (50) Google Scholar, and 58Doring C. Colombini M. J. Membr. Biol. 1985; 83: 81-86Crossref PubMed Scopus (46) Google Scholar). To visualize the quality of the fitting and to find parameters n and V0, we use the logarithmic version of Equation 1, ln((1-Popen(V))/Popen(V))=(V0-V)ne/kT(Eq. 3) where n is determined from the slope of the dependence, and V0 is determined from the intersection with zero ordinate. Gramicidin A (a generous gift of O. S. Andersen, Cornell University Medical College) was added from 0.1–1 nm ethanol stock solutions to both aqueous compartments at the amount sufficient to give a single-channel activity. Aqueous solutions of 1 m KCl were buffered by 5 mm HEPES at pH 7.4. In the experiments with gramicidin channels, data were filtered by a low pass 8-pole Butterworth filter at 5 kHz and saved into the computer memory with a sampling frequency of 10 kHz. Data were analyzed using pClamp 9 software. A digital 8-pole Bessel low pass filter set at 50 Hz was applied to all records, and then single channels were discriminated. Gramicidin lifetimes were calculated by fitting logarithmic single exponentials to logarithmically binned histograms of at least 250 single-channel events (59Sigworth F.J. Sine S.M. Biophys. J. 1987; 52: 1047-1054Abstract Full Text PDF PubMed Scopus (641) Google Scholar). Nine different logarithmic probability fits were generated using different fitting procedures, and the mean and S.E. values of the fitted time constants were used as mean and S.E. for the lifetime. All measurements were made at room temperature, T = 23 ± 1.0 °C. VDAC in PC, PE, and PC/CL Membranes—Voltage gating is one of the characteristic properties of a VDAC channel reconstituted into planar bilayer membranes. Once inserted, the channel remains in a high conducting or “open” state at low applied potentials (<30 mV by modulus). However, at relatively high potentials (>30 mV), the channel moves into one of the multiple low conducting or “closed” states (Fig. 1). Sometimes channel conductance fluctuates between one open and multiple closed states. At 0 mV, the channel reopens and closes again if high potential (50 mV, as in Fig. 1) is applied to the membrane. Channel closure occurs at both positive and negative potentials. The most convenient and reliable method to study VDAC channel gating was designed by Colombini and co-workers (26Schein S.J. Colombini M. Finkelstein A. J. Membr. Biol. 1976; 30: 99-120Crossref PubMed Scopus (411) Google Scholar, 27Colombini M. J. Membr. Biol. 1989; 111: 103-111Crossref PubMed Scopus (252) Google Scholar, 55Zizi M. Byrd C. Boxus R. Colombini M. Biophys. J. 1998; 75: 704-713Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In this method, the slowly changing periodic voltages applied to the membrane allow the collection of necessary statistics. The channel closure at different potentials is obtained from G/V plots at increasing (by modulus) transmembrane voltages. Fig. 2A illustrates current responses to a symmetrical triangular voltage wave of 5 mHz and 60-mV amplitude (lower trace) applied to a multichannel membranes formed from PC (upper trace) and PE (middle trace). It is seen that in what concerns voltage polarity, the current trace is symmetric in PC membrane and asymmetric in PE membrane. At negative applied voltages, the current amplitude in PE membrane is lower than at positive voltages, due to more pronounced VDAC closure at negative potentials. Currents in PE membrane also tend to demonstrate a more pronounced hysteretic behavior. Although the gating is a random phenomenon (note the variability in responses to the repeated identical sweeps of the applied voltage in Fig. 2A), with many channels and sufficient averaging, a clear bell-shaped voltage dependence of conductance is observed (Fig. 2B). To take into account the variable number of channels in each experiment, the G/V plots in Fig. 2B are expressed as a normalized conductance. Each data point is an average of a few independent experiments performed on individual multichannel membranes. Again, Fig. 2B shows that VDAC gating obtained on the membranes made from pure PE is more asymmetric than on the membranes formed from pure PC. Specifically, the channel closure at the cis negative voltages in PE membranes is more pronounced than in PC membranes. In order to quantify the observed asymmetry of the voltage dependences with respect to the sign of applied voltage, we introduced an asymmetry factor (Fig. 3A) as a ratio of conductances at positive and negative applied voltages. VDAC gating in PC membranes was symmetrical up to 40 mV, and slight asymmetry appeared at higher potentials with an asymmetry factor of 1.1. The asymmetry factor for PE membranes increased with voltage and reached a steady state level of 1.9 at potentials higher than 40 mV. In membranes made from the 1:1 (mol/mol) mixture of PC and PE, the asymmetry factor was between those obtained for pure PC and PE membranes, as could be expected. A comparison of asymmetries measured at 45 mV for different lipid compositions is given in Fig. 3B. In the membranes that contained more than 10 channels and up to 130 channels, the asymmetry factor was 1.1 ± 0.2 for PC and 1.9 ± 0.3 for PE membranes (Fig. 3B). CL is another lipid that is known to induce high negative spontaneous curvature. We studied voltage gating of VDAC in CL-containing PC membranes. Figs. 2B and 3, A and B, show that in the membranes made from the mixture of PC with 4 mol % of CL, the asymmetry of VDAC gating is similar to that observed in pure PE membranes. At voltages higher than 40 mV, the asymmetry factor reached a steady level of about 2. Thus, lipids with high spontaneous curvature, PE and CL, promote VDAC voltage-gating asymmetry. A possible explanation of the difference in the voltage-gating asymmetry between PE and PC multichannel membranes could be the difference in the degree of orientation of VDAC channel insertion. Indeed, suppose that insertion is random in PC bilayers and predominantly directional in PE bilayers. Then, if the gating asymmetry is an intrinsic property of VDAC channels, this could explain the observed difference. Zizi et al. (57Zizi M. Thomas L. Blachly-Dyson E. Forte M. Colombini M. J. Membr. Biol. 1995; 144: 121-129Crossref PubMed Scopus (50) Google Scholar) demonstrated that VDAC insertion into planar phospholipid membrane made of asolectin (mixture of soy bean lipids) is an oriented process in the sense that the second and following channels insert in the same direction as the first one. Interestingly, the orientation of the first channel was found to be random. The present data suggest that in pure PE membrane, most of the channels are inserted in the same orientation, not only in a particular experiment but in the orientation that persists from experiment to experiment. However, the symmetric gating in PC membranes could be explained by random insertion of VDAC channels into these membranes, wherein the channel asymmetry is compensated for, on average, by the oppositely orientated channels. To test the orientation of VDAC channel insertion in PC membranes, we used a photoaffinity analog of ATP, BzATP. The idea is based on our finding that the symmetric addition of BzATP to both sides of the membrane generates asymmetric noise in the current through the channel. A typical experiment is demonstrated in Fig. 4A. Trace a represents ion current through four VDAC channels in PC membrane before the BzATP addition. The current is symmetric, and the total conductance at +30 mV is 15.6 nS. This corresponds to a single-channel conductance of 3.9 nS, a typical value in 1 m KCl solutions (23Colombini M. Curr. Top. Membr. 1994; 42: 73-101Crossref Scopus (73) Google Scholar). The addition of BzATP to both sides of the membrane in equal conce" @default.
• W2022101585 created "2016-06-24" @default.
• W2022101585 creator A5002985843 @default.
• W2022101585 creator A5047582684 @default.
• W2022101585 creator A5055120180 @default.
• W2022101585 creator A5070614397 @default.
• W2022101585 date "2006-12-01" @default.
• W2022101585 modified "2023-10-16" @default.
• W2022101585 title "Voltage Gating of VDAC Is Regulated by Nonlamellar Lipids of Mitochondrial Membranes" @default.
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