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Probing the Orientation of Reconstituted Maltoporin Channels at the Single-protein Level

2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês

10.1074/jbc.m305434200

1083-351X

Christophe Danelon, Thérèse Brando, Mathias Winterhalter,

Lipid Membrane Structure and Behavior

Recently we have shown that maltoporin channels reconstituted into black lipid membranes have pronounced asymmetric properties in both ion conduction and sugar binding. This asymmetry revealed also that maltoporin insertion is directional. However, the orientation in the lipid bilayer remained an open question. To elucidate the orientation, we performed point mutations at each side of the channel and analyzed the ion current fluctuation caused by an asymmetric maltohexaose addition. In a second series we used a chemically modified maltohexaose sugar molecule with inhibited entry possibility from the periplasmic side. In contrast to the natural outer cell wall of bacteria, we found that the maltoporin inserts in artificial lipid bilayer in such a way that the long extracellular loops are exposed to the same side of the membrane than protein addition. Based on this orientation, the directional properties of sugar binding were correlated to physiological conditions. We found that nature has optimized maltoporin channels by lowering the activation barriers at each extremity of the pore to trap sugar molecules from the external medium and eject them most efficiently to the periplasmic side. Recently we have shown that maltoporin channels reconstituted into black lipid membranes have pronounced asymmetric properties in both ion conduction and sugar binding. This asymmetry revealed also that maltoporin insertion is directional. However, the orientation in the lipid bilayer remained an open question. To elucidate the orientation, we performed point mutations at each side of the channel and analyzed the ion current fluctuation caused by an asymmetric maltohexaose addition. In a second series we used a chemically modified maltohexaose sugar molecule with inhibited entry possibility from the periplasmic side. In contrast to the natural outer cell wall of bacteria, we found that the maltoporin inserts in artificial lipid bilayer in such a way that the long extracellular loops are exposed to the same side of the membrane than protein addition. Based on this orientation, the directional properties of sugar binding were correlated to physiological conditions. We found that nature has optimized maltoporin channels by lowering the activation barriers at each extremity of the pore to trap sugar molecules from the external medium and eject them most efficiently to the periplasmic side. The outer membrane of Gram-negative bacteria consists of an asymmetrical bilayer with phospholipids in the inner leaflet and lipopolysaccharides in the outer leaflet. The lipopolysaccharide headgroups are cross-linked by divalent cations, thereby providing an impermeable network for hydrophilic solutes and protecting the cell from damaging agents such as bile salts, lipases, and proteases. Uptake of nutrients or secretion of proteins through this barrier is accomplished by several channel forming proteins called porins (1Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Crossref PubMed Google Scholar). With a lack of other carbohydrate sources, the entry of maltodextrins has been optimized by the bacteria through the specific channel maltoporin, also called LamB (2Luckey M. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 167-171Crossref PubMed Scopus (179) Google Scholar, 3Nikaido H. Mol. Microbiol. 1992; 6: 435-442Crossref PubMed Scopus (258) Google Scholar, 4Boos W. Shuman H. Microbiol. Mol. Biol. Rev. 1998; 62: 204-229Crossref PubMed Google Scholar). The high resolution x-ray structure of maltoporin has been solved to 3.1 Å (5Schirmer T. Keller T.A. Wang Y.F. Rosenbusch J.P. Science. 1995; 267: 512-514Crossref PubMed Scopus (530) Google Scholar). The protein forms a homotrimer presenting three water-filled channels. A monomer consists of an 18-stranded antiparallel β-barrel embedded in the membrane, with large extracellular loops and short turns at the periplasmic side. The third loop, L3, folds inside the pore, contributing to a considerable constriction at the middle of the channel formed by the residue Tyr118. Interestingly, a line of six aromatic residues composed by Trp74* of an adjacent subunit, Tyr41, Tyr6, Trp420, Trp358, and Phe227, called the "greasy slide," extends from the entrance vestibule to the periplasmic outlet (see Fig. 1A) and follows the left-handed helical shape of the longer maltodextrins, conferring a screw-like character to the translocation process. Crystal structures of maltodextrins bound to maltoporin reveal that the sugar molecule is oriented with its nonreducing end pointing toward the periplasmic exit of the pore (6Dutzler R. Wang Y.F. Rizkallah P.J. Rosenbusch J.P. Schirmer T. Structure. 1996; 4: 127-134Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 7Meyer J.E.W. Hofnung M. Schulz G.E. J. Mol. Biol. 1997; 266: 761-775Crossref PubMed Scopus (110) Google Scholar). The complex also reveals three glucosyl-binding subsites where the apolar pyranose rings are in van der Waals' interaction with the residues Tyr41, Tyr6, and Trp420 at the central part of the greasy slide, whereas the moieties at either end of the sugar curl away from the residues at the periphery (Trp74* at the vestibule and Trp358 and Phe227 at the periplasmic end). Simultaneously, sugar-hydroxyl groups are engaged in hydrogen bonds with two "polar tracks." All of these interactions are supposed to provide a specific sugar translocation pathway. Specific binding of maltodextrins increases the local concentration inside the channel and thus facilitates translocation at low substrates concentrations. Maltoporin has been extensively studied using the black lipid membrane technique. The maltoporin proteins are incorporated into planar lipid bilayers and characterized by their conductance (8Schindler G. Rosenbusch J.P. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3751-3755Crossref PubMed Scopus (251) Google Scholar, 9Benz R. Schmid A. Vos-Sheperkeuter G.H. J. Membr. Biol. 1987; 100: 21-29Crossref PubMed Scopus (147) Google Scholar, 10Winterhalter M. Colloids Surf. 1999; 149: 547-551Crossref Scopus (22) Google Scholar, 11Van Gelder P. Dumas F. Winterhalter M. Biophys. Chem. 2000; 85: 153-167Crossref PubMed Scopus (74) Google Scholar). Based on the observation that sugar addition reduces channel conductance in a concentration-dependent manner, conductance measurements can be used as a probe to reveal the thermodynamic parameter of the sugar binding. Information about the rate constants of sugar translocation can be obtained using the spectral analysis of the ion current fluctuations (10Winterhalter M. Colloids Surf. 1999; 149: 547-551Crossref Scopus (22) Google Scholar, 11Van Gelder P. Dumas F. Winterhalter M. Biophys. Chem. 2000; 85: 153-167Crossref PubMed Scopus (74) Google Scholar, 12Nekolla S. Andersen C. Benz R. Biophys. J. 1994; 66: 1388-1397Abstract Full Text PDF PubMed Scopus (88) Google Scholar, 13Andersen C. Jordy M. Benz R. J. Gen. Physiol. 1995; 105: 385-401Crossref PubMed Scopus (75) Google Scholar). In previous studies conducted on a large ensemble of porins, site-directed mutagenesis was employed to probe the functional role of the greasy slide (14Hilty C. Winterhalter M. Phys. Rev. Lett. 2001; 86: 5624-5627Crossref PubMed Scopus (41) Google Scholar, 15Van Gelder P. Dumas F. Bartoldus I. Saint N. Prilipov A. Winterhalter M. Wang Y. Philippsen A. Rosenbusch J.P. Schirmer T. J. Bacteriol. 2002; 184: 2994-2999Crossref PubMed Scopus (35) Google Scholar, 16Orlik F. Andersen C. Benz R. Biophys. J. 2002; 83: 309-321Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). The mutations affecting the aromatic residues at the channel center considerably reduces the stability constants as suggested by the crystallographic data. The effect of the mutation is less pronounced for residues located at either end of the slide (W74*A, W358A and F227A) but demonstrates that they are also involved in the facilitated substrate transport through maltoporin channels. Recent conductance measurements performed on single maltoporin trimers show that the temporary binding of a maltodextrin molecule inside the channel can be observed as ion current fluctuation as penetrating sugar molecules constrain the passage of ions (17Bezrukov S.M. Kullman L. Winterhalter M. FEBS Lett. 2000; 476: 224-228Crossref PubMed Scopus (70) Google Scholar, 18Kullman L. Winterhalter M. Bezrukov S.M. Biophys. J. 2002; 82: 803-812Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 19Schwarz G. Danelon C. Winterhalter M. Biophys. J. 2003; 84: 2990-2998Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Reconstitution experiments at the single-channel level allowed unambiguous exploration of the directional properties of maltoporin channels. These studies clearly showed that the channel is highly asymmetric with respect to the sugar entrance side and applied voltages and revealed that the maltoporin insertion in artificial membranes is always unidirectional (17Bezrukov S.M. Kullman L. Winterhalter M. FEBS Lett. 2000; 476: 224-228Crossref PubMed Scopus (70) Google Scholar, 18Kullman L. Winterhalter M. Bezrukov S.M. Biophys. J. 2002; 82: 803-812Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 19Schwarz G. Danelon C. Winterhalter M. Biophys. J. 2003; 84: 2990-2998Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). However, the real orientation of maltoporin remained an open question. In multichannel reconstitution experiments, the direction of maltoporin incorporation has been a matter of debate. Based on the binding asymmetry of a pseudooligosaccharide modified at its nonreducing end (20Brunkhorst C. Andersen C. Schneider C. J. Bacteriol. 1999; 181: 2612-2619Crossref PubMed Google Scholar), on mutants deleted in the large extracellular loops, and on asymmetrical pH-induced closure of the channel proteins, Benz and co-workers (21Andersen C. Schiffler B. Charbit A. Benz R. J. Biol. Chem. 2002; 277: 41318-41325Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 22Andersen C. Bachmeyer C. Täuber H. Benz R. Wang J. Michel V. Newton S.M.C. Hofnung M. Charbit A. Mol. Microbiol. 1999; 32: 851-867Crossref PubMed Scopus (25) Google Scholar) propose that maltoporin preferentially inserts with the short periplasmic turns moving through the membrane. In contrast, Van Gelder et al. (23Van Gelder P. Dumas F. Rosenbusch J.P. Winterhalter M. Eur. J. Biochem. 2000; 267: 1-7Crossref PubMed Google Scholar) used bacteriophage-λ and observed the opposite orientation. In the present study, we address the question of maltoporin orientation at the single channel level. First we investigate the influence of the mutations W74A at the extracellular vestibule and F227A at the periplasmic exit on the kinetics of maltohexaose binding. A second method to probe the orientation is based on the obvious asymmetric binding process of maltodextrin to maltoporin requiring an orientated penetration. We synthesized an ANDS 1The abbreviations used are: ANDS, 3-amino-naphtalene-2,7-disulfonic acid; M6-ANDS, ANDS-maltoheptaose derivative.1The abbreviations used are: ANDS, 3-amino-naphtalene-2,7-disulfonic acid; M6-ANDS, ANDS-maltoheptaose derivative.-maltoheptaose derivative (noted M6-ANDS in the following), a maltohexaose analogue with a bulky ANDS cap at its reducing end (see Fig. 1B). This modification prevents sugar molecules from penetrating the channel with the reducing end. The knowledge of maltoporin orientation associated with the functional role of the outermost residues of the greasy slide allowed us for the first time to provide a quantitative description of carbohydrates transport involving an asymmetric energy profile for a sugar molecule permeating the channel. Preparation and Characterization of M6-ANDS—Maltoheptaose was purchased from Senn Chemicals (Basel, Switzerland), and ANDS was from Interchim (Paris, France). 60 mg of maltoheptaose (around 50 μmol) were labeled as a result of reductive amination by the adding of 250 μl of 0.2 m ANDS in 15% acetic acid and the same volume of NaBH3CN 1 m in tetrahydrofuran. The reaction was incubated for 3 h at 55 °C. The choice of ANDS-maltodextrin cap to probe maltoporin orientation was motivated by the fact that synthesis and characterization of ANDS-oligosaccharide derivatives have already been described (24Chiesa C. Horvàth C. J. Chromatogr. 1993; 645: 337-352Crossref PubMed Scopus (164) Google Scholar). Maltoheptaose ANDS-derivative, called here M6-ANDS (see Fig. 1B), was first analyzed by capillary electrophoresis, and the major peak of M6-ANDS was identified. Characterization of this peak was performed by capillary electrophoresis coupled with electrospray ionization mass spectrometry under the conditions described below. The negative mass spectrum obtained was dominated by peaks at m/z 1438 and 718.9 assigned to single and doubly charged deprotonated molecular ions (M – H)– and (M – 2H)2– of M6-ANDS. Capillary electrophoresis-mass spectroscopy analyses were carried out on a CE system P/ACE™ MDQ (Beckman Coulter, Inc.) with a 75-μm × 80-cm fused silica capillary. The outlet of the capillary was integrated into the ESI spray needle that was directly coupled to an ion trap mass spectrometry system (LCQ™ DUO; Thermofinnigan, Inc.). The separations were monitored with a Beckman laser-induced fluorescence detection system using a 4 mW argon ion laser with an excitation wavelength of 488 nm and an emission filter of 520 nm. During analysis, the temperature was maintained constant (25 °C) along the capillary, and a voltage of 4 kV was applied at the outlet end of this capillary. The sheath liquid (water/isopropanol, 20/80, v/v) at the rate of 5 μl/min and the sheath gas (nitrogen, 20 units) were infused coaxially to the CE capillary. For measurements, the negative mode was used, and all of the data were collected on X Calibur software (see Ref. 25Monsarrat B. Brando T. Coudouret P. Nigou J. Puzo G. Glycobiology. 1999; 9: 1-8Crossref PubMed Scopus (49) Google Scholar for details). Planar Lipid Bilayer Experiments—Planar lipid bilayers have been prepared of diphytanoyl phosphatidylcholine (Avanti Polar Lipids Inc.) according to the technique of Montal and Mueller (26Montal M. Mueller P. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 3561-3566Crossref PubMed Scopus (1570) Google Scholar). They are formed across a 60-μm-diameter hole in a 25-μm-thick Teflon film (Goodfellow, Cambridge, UK) being sandwiched between two Delrine chambers, each containing 2 ml of an aqueous solution (1 m KCl, 1 mm CaCl2, 10 mm Tris buffered to pH 7.4). The whole set-up was shielded from external electromagnetic fields as well as from vibrations to minimize extra noise from other sources. The Delrine cell was enclosed in a double isolated Faraday cage connected with the signal ground and also with a homemade acoustically isolating closet placed on a piezo-electric vibration isolating table (model "Elite 3"; Newport Corp., Irvine, CA). The quality of the bilayer membranes was checked by capacitance and residual conductance measurements. The capacitance of the whole system proved to be about 150 picofarads. The residual membrane conductance (less than 7 picosiemens) was subtracted from the overall conductance. The apparatus had been connected with the external circuit through a pair of homemade Ag/AgCl electrodes encased in 200-μl pipette tips filled with 5% agarose soaked with 1 m KCl during the fabrication process. The electrode on the so called cis-side of the measuring cell was grounded, whereas the other (on trans) was connected with the head stage of an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) in the voltage clamp mode. The mutagenesis and purification of maltoporin have previously been described in detail (15Van Gelder P. Dumas F. Bartoldus I. Saint N. Prilipov A. Winterhalter M. Wang Y. Philippsen A. Rosenbusch J.P. Schirmer T. J. Bacteriol. 2002; 184: 2994-2999Crossref PubMed Scopus (35) Google Scholar). Small amounts of wild type maltoporin from a 0.1 μg/ml buffer solution with 1% octyl-POE detergent (Alexis, Lauchringen, Switzerland) were injected into the cis-side compartment. Incorporation of maltoporin into the bilayer was promoted by applying a transmembrane voltage of 100–200 mV to tentatively disturb the membrane and by stirring for a few seconds after addition. We have optimized the protocol to observe single porin insertion in less than 10 min and to inhibit further insertion. A single maltoporin molecule in the bilayer could be kept stable for several hours without any significant change of its physical properties. The concentration of maltohexaose (Senn Chemicals) was adjusted by adding appropriate small amounts of a concentrated stock solution. The exact sugar concentration was then determined by means of optical polarization measurements (Perkin-Elmer 241). After sugar addition the aqueous solution was homogenized by stirring during a few seconds. Then signals were recorded 20 min later. Titrations for the whole range of applied voltages were carried out with the same maltoporin molecule to avoid possible divergences between different individual single protein molecules. All of the measurements were performed at room temperature. The applied transmembrane voltage refers to the potential on the cis-side relative to the trans-side. The data were filtered with the low pass Bessel filter of the amplifier at 2–5 kHz and then monitored with a Lecroy (Geneva, Switzerland) LT342 digital storage oscilloscope. The entire experiment was recorded on video tape with a digital type recorder (DTR-1204; Biologic, Claix, France). The average power spectrum of the current noise was recorded using the fast Fourier transform module of the oscilloscope. To overcome the additional noise sources mentioned above, the background spectrum of the membrane without sugar was subtracted from each individual spectrum. The curve fitting was carried out using the Marquart-Levenberg method. The total current recording was transferred to a personal computer via a GPIB card using the graphical program LabVIEW 4.01 from National Instruments. Statistical analysis of the blockade events was performed by means of the BioPatch Analysis (Science Instruments) and homemade software. Derivation of the Individual Rate Constants of Sugar Binding—The binding of carbohydrates to the maltoporin channels was assumed for a long time to be symmetric (9Benz R. Schmid A. Vos-Sheperkeuter G.H. J. Membr. Biol. 1987; 100: 21-29Crossref PubMed Scopus (147) Google Scholar, 12Nekolla S. Andersen C. Benz R. Biophys. J. 1994; 66: 1388-1397Abstract Full Text PDF PubMed Scopus (88) Google Scholar), so that once a sugar molecule is bound inside the pore it would have equal probability to translocate or to exit to the same side. Only recent measurements performed on individual maltoporin trimers demonstrated that the association rate is 3–5-fold different depending on the sugar entrance side (18Kullman L. Winterhalter M. Bezrukov S.M. Biophys. J. 2002; 82: 803-812Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 19Schwarz G. Danelon C. Winterhalter M. Biophys. J. 2003; 84: 2990-2998Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The reaction for maltodextrin (M) binding has been proposed to be of first order. The internal binding site, P, is accessible from either side of the membrane (cis as well as trans). A bound substrate molecule, PM, closes the channel to ion current. The related reaction scheme is written as follows, P+Mcis⇄koffciskoncisPM⇄kontranskofftransP+MtransReactionIREACTION 1 where koncis and kontrans refer to the association rate constants for a sugar molecule entering the channel from the cis- and the trans-sides, respectively; and koffcis and kofftrans stand for the dissociation rate constants of a sugar molecule exiting from the binding site, respectively, to the cis- and the trans-sides. The two respective pairs of on-off rate constants associated with the cis- and trans-sides are related to the single thermodynamic equilibrium constant, K, according to the detailed balance principle. K=koncis/koffcis=kontrans/kofftrans(Eq. 1) The on rate constants koncis and kontrans can individually be found out from the respective simple cases where sugar is only added to the cis-side or the trans-side of the channel. We cannot directly access the off rate constants koffcis and kofftrans from the current fluctuation measurements because they are not distinguishable. Nevertheless, they can easily be derived from the apparent koff=koffcis+kofftrans and Equation 1. The relaxation time constant of the binding process, τ, that modulates the ion current depends on the rate constants and sugar concentration, [M], as follows. τ-1=2πfc=koncis·[M]cis+kontrans·[M]trans+koffcis+kofftrans(Eq. 2) The parameter f c can be determined by fitting the power spectral density of the sugar-induced ion fluctuations with the Lorentzian form (12Nekolla S. Andersen C. Benz R. Biophys. J. 1994; 66: 1388-1397Abstract Full Text PDF PubMed Scopus (88) Google Scholar), S(f)=S0/(1+(f/fc)2)(Eq. 3) where S 0 is the plateau power density at frequencies f ≪ f c, and f c is the corner frequency at S(f) = S 0/2. Effect of the Mutations W74A and F227A on Ion Conduction—As seen in Fig. 1A, the residue Trp74 is located at the entrance, and Phe227 is located at the periplasmic exit, and both were changed into alanine. To study the influence of these mutations on the directional insertion of maltoporin channels, we first recorded the ion current through single mutant W74A at different applied voltages and calculated the corresponding conductance values. In a second series of measurements, we repeated the recording with the F227A mutant. The results of both recordings are shown in Fig. 2. We observe that the conductance of the wild type and mutated channels increases with the applied voltage and depends on the sign of the external potential. Repeated recordings on porin insertion revealed in all the cases (more than 200 attempts for the wild type maltoporin and more than 50 attempts for each mutated proteins) an orientation having the high conductance under smaller potentials on the side of channel addition, i.e. at negative voltages according to our sign convention. At ±190 mV, the asymmetry is 14% for both mutant channels. It is noteworthy that larger conductance at negative voltages has been obtained for all insertions independently on the sign of the potential applied during channel incorporation. However, it depends on the side of protein addition. Conductance of the mutated channels is slightly higher than the wild type maltoporin. Apparently the mutagenesis did not modify the asymmetry in ion conduction, suggesting that the mutations W74A and F227A do not affect the unidirectional insertion of the maltoporin channels into artificial membranes. Effect of the Mutations W74A and F227A on Maltodextrin Binding—After showing the directional incorporation through the asymmetry in conductance, we titrated maltohexaose molecules on one side only to probe the orientation of reconstituted maltoporin channels. Typical current recordings obtained from single wild type, W74A, and F227A maltoporin channels in the presence of one-side addition of maltohexaose are shown in A, B, and C of Fig. 3, respectively. We observed transient blockade events whatever the side of sugar addition was. Kinetic analysis shows that the power spectral density of the fluctuations in the current through individual maltoporin mutants have a Lorentzian form (Fig. 4). Similar behavior has already been reported for the wild type maltoporin channels (17Bezrukov S.M. Kullman L. Winterhalter M. FEBS Lett. 2000; 476: 224-228Crossref PubMed Scopus (70) Google Scholar). The rate constants governing the open-close sequences experienced by a given channel can then be determined using Equation 2 by means of a linear plot of the reciprocal relaxation times versus maltohexaose concentrations. The on and off rate constants are reported in Table I. The first observation is that the apparent dissociation rate constant does not depend on the side of sugar addition. The mutation W74A leads to an increase of the off rate comparing with the wild type channels, whereas a significant decrease is found for the modification F227A. This result is directly related to the longer average sugar residence time in F227A channels as observed in Fig. 3. At +150 mV, maltohexaose stays 1.1, 0.7, and 1.7 ms on average inside wild type, W74A, and F227A maltoporin channels, respectively. The second observation is that the mutation W74A introduces a pronounced on rate asymmetry with the side of sugar addition, whereas the asymmetry tends to be reduced in F227A. koncis is smaller than kontrans by factors of 9.4 and 2.3 for W74A and F227A channels, respectively. A comparison of the on rate constants with wild type maltoporin channels reveals a higher contribution of Trp74 than Phe227. The replacement F227A displays a small reduction of the on rate at the trans-side, whereas a small increase is observed at the cis-side. Mutant W74A shows a major reduction of the on rate constant at the cis-side compared with wild type by a factor of 3.9 at +150 mV.Fig. 4Spectral analysis of maltohexaose-induced fluctuations in the current. The power spectral densities of the current noise through W74A channels (A) and F227A channels (B) show a single Lorentzian form, implying that sugar binding to W74A and F227A channels is still a simple two-state model with no memory effect. They also indicate that W74A channel is highly asymmetric with the side of sugar addition. The applied voltage was +150 mV, and the maltohexaose concentration was 60 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IApparent kinetic and thermodynamic constants for maltohexaose binding to wild type maltoporin and point mutants W74A and F227ASide of sugar additionWild typeW74AF227AkonkoffKkonkoffKkonkoffK×106m-1·s-1s-1m-1×106m-1·s-1s-1m-1×106m-1·s-1s-1m-1cis2.793528900.713005403.25505820trans9.3855108806.6168039307.260012000 Open table in a new tab The kinetics of maltohexaose binding through wild type maltoporin channels has been shown to be sensitive to the external voltage with higher influence on the apparent dissociation rate of bound substrates (17Bezrukov S.M. Kullman L. Winterhalter M. FEBS Lett. 2000; 476: 224-228Crossref PubMed Scopus (70) Google Scholar, 18Kullman L. Winterhalter M. Bezrukov S.M. Biophys. J. 2002; 82: 803-812Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 19Schwarz G. Danelon C. Winterhalter M. Biophys. J. 2003; 84: 2990-2998Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). This binding property has been also used to detect the directional insertion of wild type maltoporin. We studied whether the mutations W74A and F227A influence this voltage dependence by determining the individual off rate constants, koffcis and kofftrans, at ±75 and ±150 mV from one-sided sugar addition experiments (see "Materials and Methods"). The results are presented in Fig. 5. Elementary dissociation rate constants of maltohexaose binding to wild type and mutated channels exhibit the same voltage dependence. Off rates are always higher at negative voltages. This voltage-induced asymmetry is more pronounced at high applied potentials and predominantly concerns kofftrans. About 50–60% difference in kofftrans is seen at –150 mV versus +150 mV for all three channels studied. Asymmetrical Addition of M6-ANDS—In a second series of measurements we probe the orientation using an asymmetric substrate. Covalent linkage of a bulky ANDS substituent at the reducing end of maltooligosaccharides strongly increases the inherent asymmetry of substrates. The mechanism of M6-ANDS transport through maltoporin channels was investigated at the single protein level by reconstituting one wild type maltoporin trimer into lipid bilayers. Further evidence of the asymmetry of the channel is observed after one-sided sugar addition (18Kullman L. Winterhalter M. Bezrukov S.M. Biophys. J. 2002; 82: 803-812Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 19Schwarz G. Danelon C. Winterhalter M. Biophys. J. 2003; 84: 2990-2998Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Typical recordings of maltohexaose-mediated current fluctuations are shown in Fig. 3A. It is interesting to note that the natural substrate closes the channels independently of which side it enters. In clear contrast to maltohexaose, Fig. 6A shows that one-sided addition of M6-ANDS generates conductance interruptions by multiples of one-third of the initial value only for substrate molecules entering from the cis- side, i.e. the side of maltoporin addition. On the other hand, no modification occurs in the current through maltoporin channels when M6-ANDS is added to the trans-side of the membrane. When maltoporin is injected to the trans-side, open channel conductance asymmetry is inverted, and the side of M6-ANDS addition that generates blockade events becomes the trans-side (data not shown). Importantly, the side of blockade events is independent of the sign of the external voltage, demonstrating that the introduction of two negative charges on the substrate molecule is not at the origin of the directional binding. Interestingly, the averaged residence time of a bound maltohexaose molecule, τr, is 1.1 ms, whereas M6-ANDS closes channels for about 5.0 ms. We verified that the presence of M6-ANDS to either side of the membrane does not modify channel permeability for natural substrates.