Article pubs.acs.org/JPCC
Protein Ion Channels as Molecular Ratchets. Switchable Current Modulation in Outer Membrane Protein F Porin Induced by Millimolar La3+ Ions Carmina Verdiá-Báguena, María Queralt-Martín, Vicente M. Aguilella, and Antonio Alcaraz* Laboratory of Molecular Biophysics, Department of Physics, Universitat Jaume I, 12080 Castellón, Spain ABSTRACT: The quest for innovative tunable nanodevices has mainly focused on switches that modulate their properties through engineered conformational changes. We propose here an alternative route that takes advantage of the crucial role that trace elements play in biological nanosystems. To this end, the effect of lanthanum, a high-valence rare-earth metal, known as blocker and modulator of many ion channels, has been studied in a wide, weakly selective biological pore, the bacterial porin outer membrane protein F (OmpF). We show that millimolar concentrations of lanthanum chloride have a dramatic impact on OmpF, reducing the conductance for positive but not for negative applied voltages, thus inducing switchable, reversible ion current rectification. By applying an external wave to this lanthanum-induced diode, we show that the system can consistently perform like a wave rectifier at considerably higher frequencies than previous nanofluidic diodes. This finding may be the starting point to develop molecular ratchets suitable for a variety of engineering applications.
■
INTRODUCTION Ion channels are membrane protein complexes present in all living organisms that communicate from the cell with the extracellular world.1 The comprehension of the mechanisms by which ion channels regulate the transport of molecules and the electric signal transduction at the molecular level is crucial for building ionic circuits in the emerging field of nanofluidics.2 Ion channels are not merely an inspirational source of synthetic biomimetic materials: the manipulation of biological membranes and ion channels to take advantage of their “sensing” properties has been particularly successful in a variety of biotechnological and analytical applications.3−6 Particular attention has been paid to ion channels that rectify current, allowing the flow of charge carriers predominantly in one direction and thus performing like liquid diodes.2,4 There are a number of ion channels that inherently display asymmetric conductance. A remarkable example is the family of inwardrectifying potassium channels in cardiac and neuronal cells. Other biological pores that display ohmic conduction have been artificially engineered to behave like enhanced diodes, looking for a tunable, flexible response.4 Gramicidin-A, the bacterial porin OmpF, and α-hemolysin are examples of biological pores that may show current rectification under specific conditions.4,7,8 These three channels have been extensively characterized for years given that they can be easily reconstituted in lipid membranes.9 Here we probe the transport properties of the outer membrane protein F (OmpF), a general diffusion porin located at the outer membrane of Escherichia coli (E. coli). It forms water-filled, voltage-gated channels that are responsible for the © 2012 American Chemical Society
uptake of small molecular mass nutrients and the removal of waste products.10−13 Despite its nonsymmetrical structure, and the fact that the fixed charge is also unevenly distributed along the pore, the channel shows no noticeable rectification.14 Interestingly, we recently reported that by reconstituting OmpF channels in membranes flanked by solutions of different pH a biological nanofluidic diode can be obtained.7 The measurements performed at single-channel level showed that the electric current is controlled by the protein fixed charge, and it can be tuned by adjusting the local pH, enabling a higher degree of control over the transport properties of the channel. Unfortunately, the extreme pH conditions required to obtain significant rectification pose some difficulties in using this nanofluidic diode in technological applications. A later study carried out by Miedema and colleagues15 emphasized the idea that an asymmetric charge distribution along the pore could yield channel rectification. They performed a series of sitedirected mutations in the OmpF porin so that the channel showed rectification without any external asymmetric conditions. This approach had some serious drawbacks: a large number of mutations in the protein were needed so that very often the channel could not fold properly, and hence it could not be obtained a fully functional channel.15 When attempting to investigate other strategies, it is worth considering that current rectification can alternatively be caused by the blocking of the pore by other ions, often multivalent Received: November 9, 2011 Revised: February 2, 2012 Published: February 27, 2012 6537
dx.doi.org/10.1021/jp210790r | J. Phys. Chem. C 2012, 116, 6537−6542
The Journal of Physical Chemistry C
Article
added an aliquot of a 50 mM LaCl3 solution of pH 6 to obtain the final concentration desired (typically, 5−20 mM). The quantity of LaCl3 solution added was enough to ensure a fast homogenization of the mixing but not to change significantly the effective concentration of KCl.
cations. To explore this possibility, we take advantage of the well-known role of La3+ ions in biological systems.16,17 Lanthanum is a high-valence rare-earth metal used as a substitute for calcium as a blocker of several nonselective ion channels such as those of the transient receptor potentialcanonical (TRPC) family.18 In fact, several studies have reported the blocking activity of lanthanum in such different systems as connexin hemichannels, murin frontal cortex networks, tobacco BY-2 cells, or the outward K+ channel.19−22 Moreover, lanthanum ions are also relevant to certain pathologies. Patients of hyperphosphatemia are treated with lanthanum carbonate, yielding abnormal amounts of the trivalent ion in the body.20 Here, we show and characterize a rectifying device built up using the wild-type OmpF protein: we obtain current rectification by adding millimolar concentrations of lanthanum chloride (LaCl3) to the solution, when reconstituting the channel into planar lipid membranes of diphytanoyl-phosphatidylcholine (DPhPC). We demonstrate that the channel conductance decreases by more than 50% under positive voltages applied at the side of protein addition (the cis side), in solutions of monovalent salts with millimolar concentrations of LaCl3. However, there is not any significant change in ion current at negative potentials. In addition, we found that this partial blockade of ion conduction across the channel is reversible and asymmetric, in the sense that lanthanum addition on the (trans) side opposite to the protein insertion has no effect on current rectification. To understand the role of lanthanum ions, we also performed selectivity experiments and found that millimolar amounts of lanthanum chloride are capable to invert the channel selectivity in KCl solutions, turning the channel into anion-selective.
■
RESULTS AND DISCUSSION Rectification Induced by Lanthanum. In the quest for alternative ways of obtaining tunable current rectification, we decided to investigate the possible blocking of the pore by multivalent cations. Recent studies performed in single conical nanopores showed that the presence of small amounts of divalent ions could change the internal electric potential of the nanopores, thus yielding current rectification.25,26 The transient binding of calcium ions induced voltage-dependent fluctuations, which were observed in current−voltage curves as a decrease in the magnitude of ion current with an increase in the magnitude of applied voltage.26 We speculated that something similar could be possible here, having in mind that divalent ions have been reported to interact tightly with some amino acid residues of the OmpF channel. Indeed, a recent publication of a 1.6 Å OmpF structure in 1 M MgCl2 showed that one Mg2+ is bound in the narrow central constriction of the channel.27 Aiming to investigate the role of Mg2+ as a potential blocker, we recorded I−V curves for a single OmpF channel inserted at 0.1 M KCl and millimolar concentrations of MgCl2 (up to 50 mM), as shown in Figure 1a. Unfortunately, the interaction between Mg2+ cations and the OmpF constriction is not strong enough to produce current asymmetry. Thus, we decided to
■
EXPERIMENTAL METHODS Wild-type OmpF, kindly provided by Dr. S. Bezrukov (NIH, Bethesda, MD, USA), was isolated and purified from an E. coli culture. Planar membranes were formed by the apposition of monolayers23 across orifices with diameters of 70−100 μm on a 15 μm thick Teflon partition using DPhPC. The orifices were pretreated with a 1% solution of hexadecane in pentane. An electric potential was applied using Ag/AgCl electrodes in 2 M KCl, 1.5% agarose bridges assembled within standard 250 mL pipet tips. The potential was defined as positive when it was higher on the side of the protein addition (the cis side of the membrane chamber), whereas the trans side was set to ground. An Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) in the voltage−clamp mode was used to measure the current and applied potential. The chamber and the head stage were isolated from external noise sources with a double metal screen (Amuneal Manufacturing Corp., Philadelphia, PA, USA). The pH was adjusted by adding HCl or KOH and controlled during the experiments with a GLP22 pH meter (Crison). Except where noted, measurements were obtained at T = 23 ± 1.5 °C. The average open channel conductance was obtained from the current measurement at an applied potential of ±100 mV in symmetrical salt solutions. The reversal potential was measured as the potential needed to achieve zero current when one or several channels were inserted into the bilayer. It was corrected with the liquid junction potential calculated from Henderson’s equation, as described in details elsewhere.24 The lanthanum salt, LaCl3 (Panreac, 98% pure), was added at millimolar concentrations at one or both sides of the membrane as follows: once one channel was inserted, we
Figure 1. Current rectification in OmpF channel when millimolar concentrations of multivalent cations are added to a 0.1 M KCl symmetrical solution at pH 6: (a). single-channel current−voltage curve after addition of small amounts of MgCl2 to a 0.1 M KCl solution; (b) current−voltage curves from single-channel recordings in different LaCl3 concentrations. Inset: comparison of the change in channel conductance measured under positive and negative bias (±100 mV) for several minute concentrations of LaCl3. 6538
dx.doi.org/10.1021/jp210790r | J. Phys. Chem. C 2012, 116, 6537−6542
The Journal of Physical Chemistry C
Article
explore an alternative way, taking advantage of the well-known blocking activity of La3+ ions in ion channels.16,17 Figure 1b shows the effect of lanthanum ions on OmpF single-channel current. Data were obtained from averaged time series with a duration of several seconds, and the measurements were repeated at least three times in independent experiments to ensure reproducibility. In the control experiments, without LaCl3, the I−V relationship was linear, confirming the ohmic behavior of the channel in monovalent salts.14 When LaCl3 was added symmetrically on both sides of the partition, the I−V curve became sublinear for positive potentials but remained unaltered for negative voltages. This means that, upon addition of LaCl3, the channel current becomes voltage-dependent and yields current rectification. Furthermore, successive additions of LaCl3 showed that the induced current asymmetry is also LaCl3 concentration dependent. This dependence saturates when the lanthanum concentration is about 10 mM. This fact is clearly shown in the inset of Figure 1b: the conductance (at 100 mV) drops for positivebut not for negativepotentials, reaching a plateau for millimolar concentrations of LaCl3. Channel Current Fluctuations and Reversibility. In previous studies, it was reported that OmpF channel conductance can change due to a variety of reasons (salt concentration, pH, and lipid charge, etc).14,28 In particular, channel conductance is a strong function of pH; in 1 M KCl it decreases by approximately a factor of 3 as the pH is lowered from pH 5 to pH 1. This reduction of conductance is accompanied by pronounced open-channel low-frequency current noise. Random stepwise transients with amplitudes at ∼1/5 of the monomer conductance are major contributors to this noise.28 Following a similar reasoning, a justification of the reduction in channel conductance could be that the presence of lanthanum ions would induce a flickering between states, leading to a mean-current drop for positive potentials. If that were the case, we would observe an increase of noise amplitude in the current traces upon addition of LaCl3. To test that tentative explanation, we recorded current time series at high sampling frequencies, looking for changes in the current noise. A sample trace is shown in Figure 2a. After the addition of LaCl3 at 15 mM (a concentration high enough to obtain a detectable rectification; see Figure 1b), the visual aspect of the current−time series does not change and subsequent analysis reveals no difference in noise amplitude. Thus, we can dismiss the possibility that flickering between substates may be the cause of the current drop at positive bias. One could think that once La3+ is bound to the channel, it induces a “channel blockage” or a low conductance state that appears both at positive and negative applied voltage. But this is not the case. When millimolar concentrations of lanthanum chloride are added to a 0.1 M KCl symmetrical solution (Figure 2a), the current levels drop only under positive potentials. This blocking of the channel is reversible in the sense that does not induce permanent changes in the channel. As soon as the potential is switched to negative values, the channel displays at once the same conductance as in control experiments. This is essentially different from other reported phenomena that yield conductance reduction in OmpF. In fact, Figure 2 shows the difference between the predictable La3+-induced closure and the well-known gating of OmpF porin by high voltage that it is somewhat random. The high-voltage induced closure (displayed in Figure 2b) does not yield current asymmetry and is
Figure 2. OmpF single-channel current recordings: (a) current traces before and after the addition of 15 mM LaCl3 on both sides of the partition (for negative bias the current remains unchanged; for positive bias, current drops to one-third); (b) current traces without LaCl3 showing the characteristic stepwise gating of the pore at very high voltages (∼150 mV). Note that under negative voltage, only one monomer remains finally open, and the voltage switch does not reopen the two other closed monomers.
not immediately reversible. OmpF is a trimeric channel, and the three monomers are identical and functionally independent. An increase of the applied voltage leads to a channel closure that can be total (the three monomers at the same time), partial, or even sequential, as shown in Figure 2b. Each closure is triggered either by high, positive, or negative potentials and cannot be restored straightforwardly: once the monomer is closed, for example, by a positive bias, reversing the polarity does not always restore the open monomer conductance, but the pore could remain some time with the same conductance state (as is seen in Figure 2b where eventually only one monomer remains open) or even fluctuate between different states. A reversible conductance regulation is essential in the quest for fluctuation-driven transport via ratchet effect. Flashing ratchets are associated with nonequilibrium perturbations in temporally and/or spatially asymmetric systems.25 Those nonequilibrium fluctuations, which could be generated externally or by a chemical reaction far from equilibrium, can bias the random motion of a particle in an anisotropic medium without any net driving force.29 Interestingly, the ratchet mechanism constitutes a general design principle for implementing mechanical amplification in engineering applications.30 Thus, lanthanum-induced asymmetry may be the starting point to engineer a molecular ratchet using OmpF channel. This is clearly shown in Figure 3. For one tilting direction (negative voltage) the force required to move the ions through the pore is smaller than in the other direction (positive voltage) in which La3+ ions partially ‘‘block’’ the current. When the applied voltage across the membrane fluctuates with a zero mean, a net flow of ions (ion pump) could be observed.31 In addition, the ratchet mechanism observed in Figure 3 for an input signal of 1 Hz clearly shows that this frequency is lower than the frequencies of La3+ binding and relaxation processes. Similar results can be obtained with voltage waves of higher frequencies (10, 20, and 40 Hz are shown in Figure 3), even up to 100 Hz. This is an outstanding result in comparison with 6539
dx.doi.org/10.1021/jp210790r | J. Phys. Chem. C 2012, 116, 6537−6542
The Journal of Physical Chemistry C
Article
Figure 3. Molecular ratchet obtained with the addition of 5 mM LaCl3 at both sides of the membrane cell where two OmpF channels were inserted. The output current (bottom) is the response to a square wave input of ±100 mV (top) and shows the characteristic reversible rectification of a molecular diode. The electrolyte was KCl 0.1 M at pH 6.
previous rectifying devices: In the case of synthetic nanopores the ratchet mechanism has only been reported in the frequency range 0.005−0.1 Hz.31,32 In engineered α-hemolysin pores inserted in droplet interfacial bilayers, the rectification efficiency degrades substantially above 0.5 Hz.4 In contrast, we have shown here that a biological nanopore can be unsophisticatedly manipulated (without the aid of mutagenesis or targeted chemical modification) to surpass such severe frequency limitations. This could be essential to build liquid nanodevices that resemble traditional electronic circuits based on semiconductors,4 a capital finding for nanotechnological applications, such as bioelectronics, drug delivery, and single-molecule analysis.9,33 Two main reasons make this rectifying channel a good candidate for diode-like applications: first, its rectification properties are achieved just modifying slightly the conditions of the electrolyte solution; second, OmpF is a protein channel that, similar to Gramicidin-A and α-hemolysin, can be easily inserted in many different types of lipid environments. This feature has been crucial to produce robust synthetic scaffolds necessary for technological applications using solid supported membranes, tethered bilayer lipid membranes, or polymerizable lipids.7 Asymmetrical Channel Blockade and Rectification Ratio. The conductance reduction observed in OmpF channel upon addition of millimolar concentrations of LaCl3 is asymmetrical with regard to voltageit appears only when applying positive potentialsalthough LaCl3 is added at both sides of the channel. Aiming to get further insight on the interaction between La3+ ions and the protein, we studied the channel current in 0.1 M KCl solutions upon addition of 5 mM LaCl3 only on one side (either cis or trans) of the partition. The results, shown in Figure 4, are represented in terms of the rectification ratio, defined as the magnitude of the quotient between currents measured under opposite polarities. We observed that addition of LaCl3 only on the trans side yields no rectification (I−V/I+V = 1) regardless of the magnitude of the applied voltage. Conversely, when lanthanum was added on the cis side, the rectification ratio became greater than unity and also increased with the applied voltage. Therefore, current rectification is only displayed when La3+ ions flow in the direction from the cis to the trans side. The inset of Figure 4 compares the asymmetry achieved when LaCl3 is added on both sides of the partition and only on the cis side. It reveals that the presence of lanthanum at the trans side of the membrane does not contribute significantly to
Figure 4. Rectification ratio of current−voltage curves in OmpF is strongly dependent on the side of the channel where LaCl3 is added. In all experiments 5 mM LaCl3 was added to a 0.1 M KCl symmetrical solution at pH 6.
the rectification properties of the OmpF porin. Therefore, the experiments suggest that the addition of lanthanum chloride at the cis side of the membrane cell is a necessary and suf f icient condition to convert the OmpF channel in a rectifying system. Interestingly, the levels of current asymmetry induced by LaCl3 are comparable to the rectification values reported in several studies. For instance, Miedema et al. mutated some residues of the OmpF channel and obtained a rectification ratio ∼3 at 100 mV,15 which is very close to our result at the same voltage, ∼2.5. Likewise, the ratios achieved using other proteins such as modified Gramicidin A are similar, as is the case of Macrae et al. who got rectification ratios from 3.4 to 4.8, depending on the modification performed.9 In addition, these values are comparable to the ratios ∼2 (at 100 mV) obtained with synthetic, conical nanopores,34 so that the OmpF−LaCl3 system can be a valid alternative to those polymer-based or solid-state rectifying nanodevices. The distinctive feature of the OmpF−LaCl3 system as nanofluidic diode is the fact that the rectification properties may be easily tuned. We have shown that the LaCl 3 concentration and voltage regulate the current asymmetry. Motivated by previous studies of pH-modulated channel conductance28 and channel rectification,7 we have extended the study to the effect of the acidity of KCl solutions in the OmpF current asymmetry. Single-channel current measurements were performed in 0.1 M KCl solutions of different acidity before and after the addition of 5 mM LaCl3 under positive voltages. It was found 6540
dx.doi.org/10.1021/jp210790r | J. Phys. Chem. C 2012, 116, 6537−6542
The Journal of Physical Chemistry C
Article
that the ratio between the current after and before the addition of lanthanum (I+/Icontrol) approached unity when the acidity was increased. Figure 5 displays the ratio I+/Icontrol for V = 100
Figure 6. Inversion of OmpF cationic selectivity upon addition of millimolar concentrations of LaCl3 to KCl solutions at pH 6. Around 4−6 mM LaCl3 suffices to turn the OmpF channel into anion selective. Solid lines are drawn to guide the eye. Figure 5. Dependence of the OmpF current rectification on the acidity of the solution 0.1 M KCl + 5 mM LaCl3. Plot of the ratio between the current at +100 mV after and before LaCl3 addition, I+/Icontrol. Current rectification vanishes near pH 3.
higher KCl concentrations: 0.1/1 M. The successive addition of LaCl3 aliquots causes a reduction of the Erev, turning the channel first into an almost neutral pore and eventually reversing the channel selectivity when LaCl3 concentration is around 4−6 mM. The amount of LaCl3 that is needed to reverse channel selectivity depends on KCl absolute concentration. This result indicates that, together with the effect of inhibiting the channel selectivity, there is a subtle shielding produced by the increased amount of ions in the system. It is important to note that the effect shown in Figure 6 is essentially different from the inversion of selectivity in the OmpF channel already reported in chloride salts of different multivalent ions.35,24 In those studies, no monovalent cations were present, so that the multivalent cations were the only ones responsible for the cation flux. The drawback of those conditions is the large diffusion potential originated by the concentration gradients of LaCl3 (due to the different mobilities of La3+ and Cl−) that hinder the elucidation of the actual sources of the observed ion selectivity.35 In the present work we overcome such difficulties showing that trace amounts of multivalent cations superimposed on a supporting 1:1 electrolyte solution provoke huge effects. This evidences the strong interaction between this trivalent salt and the OmpF protein. Finally, it is worth mentioning that we performed Erev measurements using MgCl2 instead of LaCl3 to check whether the divalent ions were able to produce the same selectivity inversion as LaCl3. We found that MgCl2 at millimolar concentrations (up to 50 mM) did not change significantly the Erev, and no selectivity inversion was found.
mV. The pH (symmetric in both solutions) modifies considerably the OmpF current rectification. Furthermore, a pH around 3 completely inhibits the asymmetric response of the pore in the presence of LaCl3 so that the current rectification can be totally canceled by adjusting the pH of the bathing solutions. The combination of the channel asymmetric partial blockade and its pH-tunable rectification properties open new possibilities to regulate channel conductance and the channel sensitivity to lanthanum cations simply by adjusting the pH of the surrounding solutions. In this sense, this nanofluidic diode has some advantages over other biological systems where the rectification ratio cannot be externally manipulated at will once the device is setup and the voltage is applied. Lanthanum-Induced Selectivity Inversion. The experiments reported so far show that millimolar concentrations of LaCl3 could induce current rectification in the OmpF channel. Another key property of ion channels is their selectivity, i.e., the ability to select ions either by their charge or by their specificity. OmpF is regarded as a weakly cation selective channel in physiological conditions.14,28 Earlier OmpF selectivity measurements35 in concentrated solutions of LaCl3 showed that the trivalent cations can cause an inversion of the channel cationic selectivity into anionic. So, we investigated whether the current reduction induced by millimolar LaCl3 could have a similar effect in the channel selectivity. The most popular way to assess the channel preference for anions or cations, i.e., its selectivity, is measuring the reversal potential, Erev, defined as the applied potential across the channel needed to get zero electric current when there is a concentration gradient between both solutions. We performed Erev measurements under a KCl salt gradient, before and after the symmetrical addition of LaCl3 on both sides of the channel. The applied Erev prevents the flow of potassium and chloride ions down their electrochemical potential gradient, so Erev measures the relative preference of the channel for K+ cations over Cl− anions, but not over La3+, because lanthanum concentration is the same on both solutions. Figure 6 shows two sets of reversal potential measurements where the ratio of KCl cis/trans concentrations is kept constant ([KCl]cis/[KCl]trans = 10) and the LaCl3 concentration in both solutions is symmetrically changed. The first series corresponds to a 15/150 mM KCl pair of solutions and the second one to
■
CONCLUSIONS
In this paper we showed that millimolar concentrations of lanthanum ions induce switchable current rectification in the OmpF channel, so that the system can consistently perform like a molecular ratchet at considerably higher frequencies than previous approaches. Furthermore, we disclosed the directional character of the current asymmetry by showing that the presence of LaCl3 in cis solution is a necessary and suf f icient condition to produce rectification. We also found that the channel rectification properties can be easily modulated by adjusting the pH of the bathing solutions so that in high-acidity solutions the current rectification can be totally canceled. Finally, we analyzed the effect of lanthanum in the ion selectivity of OmpF, revealing that millimolar concentrations of LaCl3 give rise to channel selectivity inversion. 6541
dx.doi.org/10.1021/jp210790r | J. Phys. Chem. C 2012, 116, 6537−6542
The Journal of Physical Chemistry C
■
Article
(29) Astumian, R. D. Science 1997, 276, 917−922. (30) Reichenbach, T.; Hudspeth, A. J. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 4973−4978. (31) Siwy, Z. S.; Fulinski, A. Am. J. Phys. 2004, 72, 567−574. (32) Siwy, Z. S.; Fulinski, A. Phys. Rev. Lett. 2002, 89, 198103. (33) Daiguji, H.; Oka, Y.; Shirono, K. Nano Lett. 2005, 5, 2274− 2280. (34) Schiedt, B.; Healy, K.; Morrison, A. P.; Neumann, R.; Siwy, Z. S. Nucl. Instrum. Methods Phys. Res., Sect. B 2005, 236, 109−116. (35) García-Giménez, E.; Alcaraz, A.; Aguilella, V. M. Phys. Rev. E: Stat., Nonlinear, Soft Matter Phys. 2010, 81, 021912.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +34964728044. Fax: +34964729218. E-mail: alcaraza@ fca.uji.es. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We acknowledge funding from the Spanish Ministry of Science and Innovation (MICINN Project FIS2010-19810) and Fundació Caixa Castelló-Bancaixa (Project No. P1-1A2009-13).
■
REFERENCES
(1) Hou, X.; Guo, W.; Jiang, L. Chem. Soc. Rev. 2011, 40, 2835−2401. (2) Siwy, Z. S.; Howorka, S. Chem. Soc. Rev. 2010, 39, 1115−1132. (3) Bayley, H.; Cremer, P. S. Nature 2001, 413, 226−230. (4) Maglia, G.; Heron, A. J.; Hwang, W. L.; Holden, M. A.; Mikhailova, E.; Li, Q.; Cheley, S.; Bayley, H. Nat. Nanotechnol. 2009, 4, 437−440. (5) Macrae, M. X.; Blake, S.; Jiang, X.; Capone, R.; Estes, D. J.; Mayer, M.; Yang, J. ACS Nano 2009, 3, 3567−3580. (6) Wilson, N. A.; Abu-Shumays, R.; Gyarfas, B.; Wang, H.; Lieberman, K. R.; Akeson, M.; Dunbar, W. B. ACS Nano 2009, 3, 995−1003. (7) Alcaraz, A.; Ramírez, P.; García-Giménez, E.; López, M. L.; Andrio, A.; Aguilella, V. M. J. Phys. Chem. B 2006, 110, 21205−21209. (8) Macrae, M. X.; Blake, S.; Mayer, M.; Yang, J. J. Am. Chem. Soc. 2010, 132, 1766−1767. (9) Majd, S.; Yusko, E. C.; Billeh, Y. N.; Macrae, M. X.; Yang, J.; Mayer, M. Curr. Opin. Biotechnol. 2010, 21, 439−476. (10) Delcour, A. H. Front. Biosci. 2003, 8, d1055−d1071. (11) Nikaido, H. Microbiol. Mol. Biol. Rev. 2003, 67, 593−656. (12) Benz, R.; Schmid, A.; Hancock, R. E. W. J. Bacteriol. 1985, 162, 722−727. (13) Saint, N.; Lou, K. L.; Widmer, C.; Luckey, M.; Schirmer, T.; Rosenbusch, J. P. J. Biol. Chem. 1996, 271, 20676−20680. (14) Alcaraz, A.; Nestorovich, E. M.; Aguilella-Arzo, M.; Aguilella, V. M.; Bezrukov, S. M. Biophys. J. 2004, 87, 943−957. (15) Miedema, H.; Vrouenraets, M.; Wierenga, J.; Meijberg, W.; Robillard, G.; Eisenberg, B. Nano Lett. 2007, 7, 2886−2891. (16) Canavese, C.; Mereu, C.; Nordio, M.; Sabbioni, E.; Aime, S. Curr. Med. Chem. 2005, 12, 1631−1636. (17) Feng, L.; Xiao, H.; He, X.; Li, Z.; Li, F.; Liu, N.; Zhao, Y.; Huang, Y.; Zhang, Z.; Chai, Z. Toxicol. Lett. 2006, 165, 112−120. (18) Vazquez, G.; Wedel, B. J.; Aziz, O.; Trebak, M.; Putney, J. W. Jr. Biochim. Biophys. Acta 2004, 1742, 21−36. (19) Thompson, R. J.; Zhou, N.; MacVicar, B. A. Science 2006, 312, 924−927. (20) Gramowski, A.; Jügelt, K.; Schröder, O. H.; Weiss, D. G.; Mitzner, S. Toxicol. Sci. 2011, 120, 173−183. (21) Lachaud, C.; Da Silva, D.; Cotellea, V.; Thuleau, P.; Xiong, T. C.; Jauneau, A.; Brière, C.; Graziana, A.; Bellec, Y.; Faure, J.-D.; Ranjeva, R.; Mazars, C. Cell Calcium 2010, 47, 92−100. (22) Wang, L. H.; Jiang, N.; Zhao, B.; Li, X. D.; Lu, T. H.; Ding, X. L.; Huang, X. H. J. Biol. Inorg. Chem. 2010, 15, 989−993. (23) Montal, M.; Mueller, P. Proc. Natl. Acad. Sci. U. S. A. 1972, 69, 3561−3566. (24) Alcaraz, A.; Nestorovich, E. M.; López, M. L.; García-Giménez, E.; Bezrukov, S. M.; Aguilella, V. M. Biophys. J. 2009, 96, 56−66. (25) Siwy, Z. S.; Powell, M. R.; Kalman, E.; Astumian, R. D.; Eisenberg, R. S. Nano Lett. 2006, 6, 473−477. (26) Siwy, Z. S.; Powell, M. R.; Petrov, A.; Kalman, E.; Trautmann, C.; Eisenberg, R. S. Nano Lett. 2006, 6, 1729−1734. (27) Yamashita, E.; Zhalnina, M. V.; Zakharov, S. D.; Sharma, O.; Cramer, W. A. EMBO J. 2008, 27, 2171−2180. (28) Nestorovich, E. M.; Rostovtseva, T. K.; Bezrukov, S. M. Biophys. J. 2003, 85, 3718−3729. 6542
dx.doi.org/10.1021/jp210790r | J. Phys. Chem. C 2012, 116, 6537−6542
