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Langmuir 2007, 23, 3898-3905
Voltage-Dependent Anion Channel Transports Calcium Ions through Biomimetic Membranes Aure´lien Deniaud,† Claire Rossi,‡ Alexandre Berquand,§ Johanne Homand,‡ Sylvie Campagna,⊥ Wolfgang Knoll,| Catherine Brenner,† and Joe¨l Chopineau*,⊥ CNRS UMR 8159, Laboratoire de Ge´ ne´ tique et Biologie Cellulaire, UniVersite´ de Versailles/St Quentin, 45 AVenue des Etats-Unis, 78035 Versailles, France, UMR 6022 CNRS, UniVersite´ de Technologie de Compie` gne, BP 20529, 60205 Compie` gne cedex, France, UMR-CNRS 168 and LRC-CEA 34V, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France, Max-Planck Institut fu¨r Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany, and CNRS UMR 5048, Centre de Biochimie Structurale, INSERM U554, and UniVersite´ Montpellier 1 et 2, F34090 Montpellier, France ReceiVed October 23, 2006. In Final Form: January 9, 2007 The mitochondrial outer membrane channel (VDAC), a central player in mitochondria and cell death, was reconstituted in polymer-supported phospholipid bilayers. Highly purified VDAC was first reconstituted in vesicles; channel properties and NADH-ferricyanide reductase activity were ascertained before deposition onto solid substrates. 1-Palmitoyl-2oleoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-Nhydroxysuccinimide mixed vesicles containing VDAC were linked onto amine-grafted surfaces (glass and gold) and disrupted to form a VDAC-containing polymer-tethered planar bilayer. Surface plasmon spectroscopy, fluorescence microscopy, and atomic force microscopy measurements ascertained the membrane thickness, fluidity, and continuity. VDAC reconstituted in bilayers efficiently transported calcium ions and was modulable by two channel blockers, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and L-glutamate. The novel setup may allow the study of the assembly of a polyprotein complex centered on VDAC and its role in mitochondrial biology, calcium fluxes, and apoptosis.
Introduction The complexity of biological membranes and their interactions with intra- and extracellular partners and networks make their direct investigation a challenge. For such studies, phospholipid bilayers deposited on solid substrate have been used as experimental systems to incorporate proteins under native conditions.1-5 These biomimetic systems allowed functional investigations of membrane-spanning proteins and of polyprotein complexes with surface-sensitive techniques such as surface plasmon resonance (SPR),6,7 electrical impedance,8 quartz crystal microbalance,9 fluorescence microscopy,10 and atomic force microscopy (AFM).11-13 The formation of protein-free solidsupported membranes can be achieved by direct vesicle fusion, the Langmuir-Blodgett method, Langmuir-Schaffer transfers, self-assembly of various building blocks such as thiol on gold and silane on quartz, grafting of polymers, and ligand receptor * To whom correspondence should be addressed. E-mail:
[email protected]. † Universite ´ de Versailles/St Quentin. ‡ Universite ´ de Technologie de Compie`gne. § UMR-CNRS 168 and LRC-CEA 34V. | Max-Planck Institut fu ¨ r Polymerforschung. ⊥ Centre de Biochimie Structurale, INSERM U554, and Universite ´ Montpellier 1 et 2. (1) Brian, A. A.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 6159-6163. (2) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105-113. (3) Sackmann, E. Science 1996, 271, 43-48. (4) Sackmann, E.; Tanaka, M. Trends Biotechnol. 2000, 18, 58-64. (5) Sinner, E. K.; Knoll, W. Curr. Opin. Chem. Biol. 2001, 5, 705-711. (6) Duschl, C.; Knoll, W. J. Phys. Chem. 1988, 88, 4062-4069. (7) Heyse, S.; Ernst, O. P.; Dienes, Z.; Hofmann, K. P.; Vogel, H. Biochemistry 1998, 37, 507-522. (8) Gritsch, S.; Nollert, P.; Jahnig, P.; Sackmann, E. Langmuir 1998, 14, 31183125. (9) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397-1402. (10) Groves, J. T.; Ulman, N.; Boxer, S. G. Science 1997, 275, 651-653. (11) Dufrene, Y. F.; Lee, G. U. Biochim. Biophys. Acta 2000, 1509, 14-41. (12) Jass, J.; Tja¨rnhage, T.; Puu, G. Biophys. J. 2000, 79, 3153-3163. (13) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806-1815.
recognition. To reduce the risk of friction and denaturation of membrane proteins, the membrane can be supported on a soft polymer such as poly(ethylene glycol) or poly(ethylene imine).3,14 Membrane proteins can then be reconstituted by addition of either a micellar protein suspension or proteoliposomes that are fused with an appropriate surface or interface. Small peptides (e.g., valinomycin or gramicidin) have been incorporated into supported membranes.15-17 Redox enzymes were recently immobilized in tethered lipid bilayer architectures as a crude membrane extract18,19 or in a purified form.20 However, the pore-forming toxin R-hemolysin was only partially reconstituted into supported bilayer membranes.21 Thus, functional assembly of membrane proteins into supported membranes is not yet a straightforward process.22 The voltage-dependent anion channel (VDAC) plays a central role in mitochondrial biology and apoptosis;23-26 see ref 23 for a critical review. VDAC is responsible for outer membrane permeability to hydrophilic molecules and ions such as calcium (14) Tanaka, M.; Sackmann, E. Nature 2005, 437, 656-663. (15) Cornell, B. A.; Braach-Maksvytis, V. L.; King, L. G.; Osman, P. D.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580-583. (16) Schiller, S. M.; Naumann, R.; Lovejoy, K.; Kunz, H.; Knoll, W. Angew. Chem., Int. Ed. 2003, 42, 208-211. (17) Romer, W.; Steinem, C. Biophys. J. 2004, 86, 955-965. (18) Jeuken, L. J. C.; Connell, S. D.; Nurnabi, M.; O’Reilly, J.; Henderson, P. J. F.; Evans, S. D.; Bushby, R. J. Langmuir 2005, 21, 1481-1488. (19) Elie-Caille, C.; Fliniaux, O.; Pantigny, J.; Maziere, J.-C.; Bourdillon, C. Langmuir 2005, 21, 4661-4668. (20) Jeuken, L. J. C.; Connell, S. D.; Henderson, P. J. F.; Gennis, R. B.; Evans, S. D.; Bushby, R. J. J. Am. Chem. Soc. 2006, 128, 1711-1716. (21) Glazier, S. A.; Vanderah, D. J.; Plant, A. L.; Bayley, H.; Valincius, G.; Kasianowicz, J. J. Langmuir 2000, 16, 10428-10435. (22) Michalke, A.; Schurholz, T.; Galla, H.-J.; Steinem, C. Langmuir 2001, 17, 2251-2257. (23) Colombini, M. Ann. N.Y. Acad. Sci. 1980, 341, 552-563. (24) Benz, R. Biochim. Biophys. Acta 1994, 1197, 167-196. (25) Colombini, M.; Blachly-Dyson, E.; Forte, M. Ion Channels 1996, 4, 169202. (26) Rostovtseva, T. K.; Tan, W.; Colombini, M. J. Bioenerg. Biomembr. 2005, 37, 129-142.
10.1021/la063105+ CCC: $37.00 © 2007 American Chemical Society Published on Web 02/22/2007
Calcium Ion Transport through Biomimetic Membranes
(Ca2+).27 VDAC participates in the formation of a polyprotein complex, called the permeability transition pore.28-31 It is also involved in the release of the apoptogenic cytochrome c into the cytosol under the control of Bax/Bcl-2 family members.32,33 The goal of this work was the functional reconstitution of VDAC into a tethered biomimetic membrane. Highly purified VDAC was reconstituted in liposomes and for the first time in a polymer-supported membrane. The whole procedure was followed step by step using SPR spectroscopy, fluorescence recovery after photobleaching (FRAP) experiments, and functionality measurements. Materials and Methods Materials. 2-Mercaptoethylamine (cysteamine hydrochloride) (g99%), (aminopropyl)dimethylethoxysilane (ADMS; 99%), 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-NBD (7-nitro2,1,3-benzoxadiazol-4-yl) (DPPE-NBD), 1,2-distearoyl-sn-glycero3-phosphoethanolamine (DSPE), Triton X-100, DIDS (4,4′diisothiocyanatostilbene-2,2′-disulfonic acid) disodium salt, L-glutamate, potassium ferricyanide (K3Fe(CN)6), β-NADH, and reactive red agarose were purchased from Sigma-Aldrich (St QuentinFallavier, France). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was from Avanti Polar Lipids (Alabaster, AL). 1,2Distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)N-hydroxysuccinimide (DSPE-PEG3400-NHS) was from Shearwater Polymers (Huntsville, AL). Fluo-4 dextran (MW 10000) potassium salt was from Interchim (Montluc¸ on, France). All other chemicals used in this work were of analytical grade. Hydroxyapatite and BioBeads SM-2 absorbent (20-50 mesh) were from Bio-Rad (Hercules, CA), Celite was from Prolabo (Paris, France). Glass microscope slides were from Dutscher (Brumath, France), chromium and gold were from Sigma-Aldrich, and silicon wafers were from CrysTec GmbH (Berlin, Germany). The two-component epoxy glue for optical purposes (EPO-TEK 353ND-4) was purchased from Polytec GmbH (Waldbronn, Germany). All buffers prepared from Milli-Q water (resistivity higher than 18.2 MΩ‚cm) were filtered and thoroughly degassed. The glass material used in this work was carefully cleaned with a 2% Hellmanex (Hellmanex, France) solution and thoroughly rinsed with Milli-Q water. Porin Purification and Characterization. VDAC was purified according to a procedure from De Pinto et al.34 and modified by Gincel et al.35 In brief, mitochondria (5 mg/mL protein) were lysed by osmotic shock in 10 mM Tris-HCl buffer (pH 7) (buffer A), 45 min at 4 °C. After centrifugation (50000g, 60 min) the pellet containing mitochondrial membranes was solubilized in buffer A containing 3% Triton X-100 to obtain a final protein concentration of 5 mg/mL. This protein mixture was incubated for 45 min at 4 °C. After centrifugation (50000g, 60 min) the supernatant was loaded onto a dry hydroxyapatite/Celite (2:1) column (1.3 × 10 cm). Then porin was collected in the flow through. The VDAC-containing fractions were diluted three times with 10 mM Tris-HCl (pH 7), mixed with reactive red agarose resin, and incubated for 2 h at 4 °C. The beads were collected on a column and extensively washed with buffer B (Tris, 10 mM, pH 7, 0.3% TX-100), and finally the protein was eluted with 0.5 M NaCl. The VDAC purified fraction (27) Gincel, D.; Zaid, H.; Shoshan-Barmatz, V. Biochem. J. 2001, 358, 147155. (28) Haworth, R. A.; Hunter, D. R. Arch. Biochem. Biophys. 1979, 195, 460467. (29) Beutner, G.; Ruck, A.; Riede, B.; Welte, W.; Brdiczka, D. FEBS Lett. 1996, 396, 189-195. (30) Zoratti, M.; Szabo, I. Biochim. Biophys. Acta 1995, 1241, 139-176. (31) Marzo, I.; Brenner, C.; Zamzami, N.; Susin, S. A.; Beutner, G.; Brdiczka, D.; Remy, R.; Xie, Z. H.; Reed, J. C.; Kroemer, G. J. Exp. Med. 1998, 187, 1261-1271. (32) Zamzami, N.; Kroemer, G. Nat. ReV. Mol. Cell. Biol. 2001, 2, 67-71. (33) Vyssokikh, M.; Zorova, L.; Zorov, D.; Heimlich, G.; Jurgensmeier, J.; Schreiner, D.; Brdiczka, D. Biochim. Biophys. Acta 2004, 1644, 27-36. (34) De Pinto, V.; Prezioso, G.; Palmieri, F. Biochim. Biophys. Acta 1987, 905, 499-502. (35) Gincel, D.; Silberberg, S. D.; Shoshan-Barmatz, V. J. Bioenerg. Biomembr. 2000, 32, 571-583.
Langmuir, Vol. 23, No. 7, 2007 3899 was dialyzed against pH 7.5 phosphate buffer, 10 mM, 100 mM NaCl, 0.3% TX-100. For gel electrophoresis and immunoblot analyses, proteins were solubilized in sample buffer (300 mM Tris, pH 6.8, 50% glycerol, 12.5% SDS, 50 mM dithiothreitol, 0.05% bromophenol blue, 1:5 buffer/protein, v/v), boiled for 10 min, and separated by SDSPAGE (10%).36 Gels were silver-stained or transferred onto a 0.4 µm PVDF membrane (Hercules, CA) for VDAC immunorevelation by an anti-VDAC polyclonal serum (Eurogentec, Seraing, Belgium) using ECL (enhanced chemoluminescence) Plus according to the manufacturer’s instructions (GE Healthcare, Saclay, France). VDAC channel activity was measured from conductance experiments; virtually solvent-free planar lipid bilayers were formed by the Montal and Mueller technique.37 An azolectin membrane was formed over a 100-150 µm hole in a Teflon film (10 µm thick) that had been treated with a 1:40 mixture (v/v) of hexadecane/hexane, separating two glass half-cells. After 2 h, lipid monolayers had spread over the top of the electrolyte solution (1 M KCl, 10 mM HEPES, pH 7.4) in both compartments. Calcium chloride (1 mM) was added before measurements in both sides. Bilayers were formed by lowering and raising the level of electrolyte in one or both sides and monitoring the capacitance responses. Voltage was applied through a Ag/AgCl electrode on the “cis” side (the “trans” side corresponds to ground). The protein was added to the cis compartment, typically at 10-11 to 10-10 M. In single-channel recordings, currents were amplified and potentials applied simultaneously with a patch-clamp amplifier (RK 300, Bio-Logic). Single-channel currents were monitored using an oscilloscope (TDS 3012, Tektronix, Beaverton, OR), filtered at 300 Hz, and stored on a CD recorder (DRA 200, Bio-Logic) for offline analysis. Data were analyzed with Windac32 (http:// www.shareit.com) and Biotools (Bio-Logic). All experiments were performed at room temperature. The NADH-ferricyanide reductase activity of VDAC was measured according to Baker et al.38 using a microplate reader (Tecan Genios, Trappes, France). Reduction of ferricyanide to ferrocyanide was monitored in a total volume of 200 µL of a reaction mixture containing 10 mM sodium phosphate buffer, pH 7.5, 0.3% TritonX100, 100 mM NaCl, 250 µM NADH. The reaction was performed at room temperature and was initiated by the addition of 250 µM potassium ferricyanide. Absorbance was measured at 340 nm during 15 min. The enzymatic initial rates were calculated from the linear part of the curve using a molar extinction coefficient of 6220 M-1 cm-1 for NADH. Preparation of the Proteoliposomes. A dried lipid film of POPC/ DSPE-PEG3400-NHS (5%, w/w) was formed from a lipid chloroform solution by removing the organic solvent under a nitrogen stream followed by 1/2 h of drying under vacuum. The dried lipid film was suspended in 10 mM sodium phosphate buffer (pH 7.5) containing 100 mM NaCl (liposome buffer). This lipid solution was mixed with porin and TX-100 to obtain a TX-100:lipid weight ratio of 2:1 in a total volume of 200 µL. After 1 h, 6 mg of Bio-Beads was added four times at 30 min intervals at room temperature.39 After elimination of Bio-Beads, the lipid suspension was extruded 19 times through 50 nm size calibrated polycarbonate membranes using a syringe-type extruder (Liposofast, Avestin Inc., Ottawa, Canada). The hydrodynamic mean diameters of the vesicles were determined by quasielastic light scattering (Zetasizer 1000/3000, Malvern Instruments, U.K.). For FRAP measurements, the fluorescent probe (DPPE-NBD) was added at a 2% molar ratio to the lipid/ chloroform solution. Formation and Characterization of Membranes. Glass microscope coverslips (12 mm diameter) were cleaned and silanized by (aminopropyl)dimethylethoxysilane. Gold-coated glass slides obtained by thermal evaporation of gold were functionalized with 2-mercaptoethylamine as previously described.40 Both amine-grafted (36) Laemmli, U. K. Nature 1970, 227, 680-685. (37) Montal, M.; Mueller, P. Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 35613566. (38) Baker, M. A.; Lane, D. J.; Ly, J. D.; De Pinto, V.; Lawen, A. J. Biol. Chem. 2004, 279, 4811-4819. (39) Rigaud, J. L.; Levy, D. Methods Enzymol. 2003, 372, 65-86.
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Figure 1. VDAC purification and characterization. (A) Purification. Mitochondrial membranes (Mb) were isolated and treated with 3% TX-100. Solubilized proteins were separated onto a hydroxyapatite/Celite column, and hydroxyapatite-eluted proteins (HA) were loaded onto a reactive red agarose column to yield an eluate composed of 95% VDAC (VDAC). A 2 µg portion of proteins was separated in SDS-PAGE for silver staining and for immunodetection. (B) Single-channel traces of VDAC in 1 M KCl, 100 mM Hepes, pH 7.4, 1 mM CaCl2 with respect to the applied voltages (mV). A 5 ng portion of protein was added on the cis side. The dashed lines represent zero current. (C) Enzymatic activity. Various purification fractions were analyzed for NADH-ferricyanide reductase activity in the presence or absence of 25 µM DIDS or 5 mM L-Glu. substrates were stored under nitrogen before being used. Formation of the supported bilayers on gold surfaces was followed using an SPR apparatus.41 Reflectivity was recorded as a function of the incident angle and the optical thickness determined.42 Fluorescence microscopy was performed as previously described.40 A vesicle suspension was added over the freshly silanized glass surface. Bilayer formation was observed after rinsing with buffer. The lateral diffusion coefficient and the mobile fraction of the fluorescent probe were determined as previously described.43 AFM measurements were carried out using a dimension 3100 Nanoscope IIIa (Digital Instruments, Santa Barbara, CA). All samples were observed in buffer at room temperature. Experiments were performed in contact mode using DNP-20 and NP-STT20 cantilevers (tip curvature radius about 20 nm and spring constant 0.12 N‚m-1) from Digital Instruments. The loading force was set as low as possible. The scan rate was between 1.5 and 3 Hz. Calcium Measurements. The Ca2+ uptake into proteoliposomes or under the tethered bilayer was measured using a spectrofluorimeter microplate reader (Tecan Genios) using white 96-well (200 µL) plates for proteoliposomes or white 24-well (500 µL) plates for tethered bilayer experiments. For the measurements of Ca2+ uptake into proteoliposomes, the Ca2+ probe Fluo4 dextran (250 µg/mL) (λexc ) 485 nm, λem ) 535 nm) was added to the mixed lipid/ detergent micelle mixture, and the external free probe was removed by ultracentrifugation at 100000g, 60 min, 4 °C (Beckman ultracentrifuge, rotor SW60). For tethered bilayer experiments, the probe (250 µg/mL) was added to the lipid suspension before extrusion, and the measurement cell was washed after bilayer formation. Experiments were performed at room temperature. After fluorescence stabilization, two pulses of Ca2+ (CaCl2) were applied at a 5 min interval. Fluorescence was measured every 30 s during 5 min, and (40) Rossi, C.; Homand, J.; Bauche, C.; Hamdi, H.; Ladant, D.; Chopineau, J. Biochemistry 2003, 42, 15273-15283. (41) Lingler, S.; Rubinstein, I.; Knoll, W.; Offenha¨usser, A. Langmuir 1997, 13, 7085-7091. (42) Bunjes, N.; Schmidt, E. K.; Jonczyk, A.; Rippmann, F.; Beyer, D.; Ringsdorf, H.; Gra¨ber, P.; Knoll, W.; Naumann, R. Langmuir 1997, 13, 61886194. (43) Berquand, A.; Mazeran, P. E.; Pantigny, J.; Proux-Delrouyre, V.; Laval, J. M.; Bourdillon, C. Langmuir 2003, 19, 1700-1707.
2% TX-100 was added to disrupt the bilayer.44 The maximal fluorescence obtained was measured after 15 min assuming that all probe molecules were in contact with Ca2+. Inhibitor was added for 5 min before the first Ca2+ pulse. The fluorescence increase (%) for each Ca2+ addition or after TX-100 treatment was calculated as the ratio of the increase in fluorescence for one addition to the total increase in fluorescence (two Ca2+ pulses plus addition of TX-100). The percentage of inhibition was the ratio of the percentage of one pulse of Ca2+ without inhibitor to the percentage of the same pulse of Ca2+ in the presence of inhibitor. The fluorescence response was corrected for the effect of the inhibitor on the bilayer without VDAC.
Results and Discussion Preparation and Characterization of VDAC Proteoliposomes. VDAC was purified to homogeneity (Figure 1) and reconstituted into proteoliposomes (Figure 2). Mouse liver mitochondrial membranes were extracted by 3% TX-100, a surfactant frequently used to solubilize native mitochondrial proteins. Then, VDAC was purified using a two-step chromatography method. After the first separation, the flow through contained about 80% VDAC. The fractions were mixed with red agarose resin and eluted with 0.5 M NaCl in the presence of 0.3% TX-100. Enrichment in VDAC up to 95% was finally obtained, as indicated by immunoblotting and silver staining (Figure 1A). The channel activity of VDAC was checked by channel conductance experiments carried out using black lipid membranes (Figure 1B). We observed, in the presence of 1 mM CaCl2, the classical hallmarks of the VDAC: a practically permanent full open state at low potentials (
