Functional Reconstitution of Protein Ion Channels into Planar

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NANO LETTERS

Functional Reconstitution of Protein Ion Channels into Planar Polymerizable Phospholipid Membranes

2005 Vol. 5, No. 6 1181-1185

Devanand K. Shenoy,*,† William R. Barger,‡ Alok Singh,† Rekha G. Panchal,§ Martin Misakian,| Vincent M. Stanford,⊥ and John J. Kasianowicz| Center for Bio/Molecular Science and Engineering, NaVal Research Laboratory, 4555 OVerlook AVenue SW, Washington, D.C. 20375, Geo-Centers Inc., 7 Wells AVenue, Newton, Massachusetts 02459, U. S. Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Frederick, Maryland 21702-5011, AdVanced Chemical Sciences Laboratory, 227/A251, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8313, and Information Technology Laboratory, 225/A231, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8940 Received March 11, 2005; Revised Manuscript Received April 13, 2005

ABSTRACT We demonstrate that polymerizable planar membranes permit reconstitution of protein ion channels formed by the bacterial toxins Staphylococcus aureus r-hemolysin (rHL) and Bacillus anthracis protective antigen 63. The rHL channel remained functional even after membrane polymerization. Surface pressure measurements suggest that the ease of forming membranes depends on membrane surface elasticity estimated from Langmuir− Blodgett monolayer pressure−area isotherms. The ability to stabilize nanoscale pores in robust ultrathin films may prove useful in single molecule sensing applications.

Ion channels provide the molecular basis for nerve activity,1 mediate the selective transport of ions2 and macromolecules,3 link cells to form functioning tissues,4 and act as lethal toxins.5,6 Much of our understanding of channel activity has been achieved using planar lipid bilayer membranes. However, nearly four decades since this in vitro system was developed,7,8 liquid-crystalline membranes limit the use of protein nanopores for technological applications because of the weak intermolecular interactions that stabilize phospholipid membranes. Recently, it was shown that channels could act as components of sensors to detect a variety of analytes including ions and small molecules,9-12 polynucleotides,13 and proteins.14 Planar lipid membranes,7,8 phospholipid bilayers that span small apertures, provide a convenient platform for most of these studies. In a pioneering effort by Tom Fare et al.,15 it was shown that alamethicin and calcium channels could be functionally incorporated into asymmetric * Correspondence and requests for materials should be addressed to D.K.S ([email protected]) or J.J.K ([email protected]). † Center for Bio/Molecular Science and Engineering, NRL. ‡ Geo-Centers Inc. § U. S. Army Medical Research Institute of Infectious Diseases. | Advanced Chemical Sciences Laboratory, NIST. ⊥ Information Technology Laboratory, NIST. 10.1021/nl050481q CCC: $30.25 Published on Web 05/07/2005

© 2005 American Chemical Society

bilayers on porous supports. Subsequently, in a seminal study, Cornell and colleagues11 demonstrated that derivatized gramicidin channels embedded in a complex solid support membrane could be used to detect biomolecules in aqueous solution. More recently, R-hemolysin channels16 were also reconstituted into membranes on solid supports. Supported bilayer membranes, however, pose several limitations for ion channel-based sensor applications. First, the solid support, which also acts as one of the electrodes, provides access to only one side of the membrane. Second, the distance between the support and the adjacent membrane surface may not provide sufficient space for some channels to functionally reconstitute.16 Third, because one of the membrane surfaces is inaccessible to bulk fluid, ions, analytes, and polymers can accumulate in the region between the solid support and the membrane. In contrast, unsupported planar lipid membranes provide access to both sides of the bilayer membrane and are better suited for single molecule sensing applications. Earlier work on membranes formed from polymerizable lipids either on solid supports15,17,18 or as liposomes19,20 demonstrated that these thin films can be made significantly more robust after polymerization. Subsequently, patterned membranes have been formed on solid supports using photopolymerizable lipids.21 One might therefore expect that

Figure 1. The chemical structures of the lipids used in this study. These include two different polymerizable molecules DC8,9PC [1,2bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine, in chloroform at either 2 or 10 mg/mL (w/v)], PTPE [1-palmitoyl-2-10,12 tricosadiynoyl-sn-glycero-3-phosphoethanolamine, in benzene at 10 mg/mL (w/v)], and the non-polymerizable DiPhyPC [1,2-diphytanoyl-sn-glycero-3-phosphocholine, in pentane or benzene at 10 mg/mL (w/v)]. All of the lipids were purchased from Avanti Polar Lipids (Alabaster, AL). DC8,9PC was also synthesized in our laboratory.

any synthetic polymerizable lipid might easily form a planar bilayer membrane across an aperture. However, we show here that this is not true. The results below suggest that the ease with which planar membranes form correlates with their surface modulus estimated from Langmuir-Blodgett isotherms. We also show that technologically relevant channels may be functionally incorporated into unsupported polymerizable bilayer membranes. We anticipate that polymerizable lipids may ultimately permit locking channels in ultrathin films for a wide variety of biotechnological and analytical applications.11,14,17,18,22,23 Lipid bilayer membranes were formed in a poly(tetrafluoroethylene) (PTFE) chamber9 using a variation of a technique devised by Montal and Mueller.8 Briefly, membranes were formed across 80 µm to 100 µm diameter holes in a PTFE film partition (25 µm thick) that divides the chamber in two halves. Details of the experimental methods for forming planar lipid bilayer membranes and reconstituting poreforming toxins into membranes are described elsewhere.10 All experiments were performed at room temperature, i.e., (22.5 (1) °C. Three criteria were used to confirm that the polymerizable lipids used in this study (DC8,9PC [1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine], PTPE [1-palmitoyl,210,12-tricosadiynoyl-sn-glycero-3-phosphoethanolamine], and diPhyPC [1,2-diphytanoyl-sn-glycero-3-phosphocholine]) formed planar bilayer membranes. First, the capacitively coupled current increased by an amount expected when the membrane was formed. Second, the membranes could be ruptured by applying dc potentials with magnitudes >200 mV. Third, the subsequent addition of several pore-forming toxins to the aqueous phase bathing one side of the membrane caused spontaneous ion channel formation. For example, following the formation of a PTPE (Figure 1) bilayer membrane, ∼0.4 µL of 1 mg/mL Bacillus anthracis protective antigen 635 (PA63) was added to one-half of the 1182

Figure 2. (A) PTPE bilayer membranes permit the functional reconstitution of the pore-forming toxin Bacillus anthracis protective antigen 63 (PA63). The ionic current recording illustrates the spontaneous and stepwise formation of nanometer-scale proteinaceous pores. The channels formed after adding ∼0.2 µg of PA63 monomer to the aqueous phase bathing one side of the membrane (herein called cis), but the first passage time for PA63 channel insertion was highly variable. The applied dc potential was + 50 mV and a positive applied potential drives cations from the cis to the opposite (i.e., trans) side. The solution contained 0.1 M KCl, 5 mM EDTA, pH 6.6. The inset shows the current-voltage relationship for a PTPE membrane containing the conductance equivalent of about fifty PA63 channels. (B) Polymerization of a PTPE bilayer membrane monitored by capacitance change. Illumination of the membrane with a pen ray UV lamp for 30 min (50% duty cycle, 30 s on, 30 s off) causes a monotonic increase in the membrane capacitance. The inset demonstrates that even after UV illumination of a PTPE membrane, the pore-forming toxin Staphylococcus aureus R-hemolysin causes step increases in the ionic current.

chamber (herein identified as the cis compartment) while stirring. As shown in Figure 2A, the formation of individual PA63 ion channels is indicated by characteristic step increases in the ionic current for a fixed value, +50 mV, of the applied dc potential. The inset in Figure 2A illustrates the current-voltage relationship for the conductance equivalent of ∼50 PA63 channels. The shape of the I-V relationship is nonlinear and rectifying, as we observed for PA63 channels in membranes formed with DiPhyPC (Figure 1). The results in Figure 2B demonstrate that the capacitance of a PTPE planar membrane increases (typically between 6% and 40%) upon illumination with UV light. Generally, the longer the exposure time, the greater the increase in the membrane capacitance. Control experiments showed that the capacitance of DiPhyPC membranes increased by less than 5% under similar conditions. The pore-forming toxin Staphylococcus aureus R-hemolysin (RHL)6 spontaneously forms channels from seven identical monomers24,25 in liquid crystalline PTPE membranes (vide infra). Interestingly, despite the change in the membrane physical state upon UV irradiation, the ion current time series in Figure 2B (inset) demonstrates that RHL forms channels in a polymerized PTPE membrane. Most likely, despite the change in membrane capacitance, some of the lipid in that ultrathin film was still in the liquid-crystalline state. Indeed, in the absence of pore-forming proteins, we often observed transient spikes in the ionic current through the membrane after more intense UV illumination. This artifact may be caused by defects (e.g., geometric mismatches) in the interface between clusters of lipid molecules. Nano Lett., Vol. 5, No. 6, 2005

The ionic current recordings in Figure 2 demonstrate that both B. anthracis PA63 and S. aureus RHL form ionic channels in PTPE membranes. The stepwise increases in ionic current are consistent with the single-channel conductances for RHL9,24 and PA635 in non-polymerizable lipid membranes. Similar ion channel reconstitution experiments were performed with the polymerizable lipid DC8,9PC (Figure 1). Unfortunately, only 5% (2 of 18) of the attempts to form a highly insulating membrane were successful. In contrast, PTPE always formed high impedance membranes. Also, in one experiment with DC8,9PC, the membrane was not stable even for moderate values of the dc applied potentials (i.e., V > 100 mV). These issues notwithstanding, RHL was still able to functionally reconstitute into DC8,9PC membranes (data not shown). To correlate the ease of forming planar membranes with the mechanical properties of the polymerizable lipid membrane, we measured the surface compressional modulus from Langmuir-Blodgett (LB) isotherms of lipid monolayers. The surface pressures of the monolayers for each lipid (DC8,9PC, PTPE, and DiPhyPC) were determined at 22 °C with a NIMA model 611MC Langmuir-Blodgett trough using the Wilhelmy plate method. The monolayers were spread from chloroform solutions (∼0.452 to 0.550 mg/mL lipid/CHCl3, w/v) at the air-water interface of an area ∼85 cm2. After waiting 10 min for the chloroform to evaporate, the surface pressure was measured continuously as the monolayer trough barriers were compressed at 5 cm2/min. For studies of the polymerization of the monolayers, a few microliters of a solution of PTPE in chloroform were spread at the air-water interface. The film was then compressed to a surface pressure of ∼31 mN/m. The film was subsequently exposed to radiation from a UV light source (UVP, M/N UVS-28, rated at ∼1 mW/cm2 at 7.6 cm) that was held ∼13 cm from closest point of the film surface. The LB isotherms of the lipid monolayers at the air-water interface are shown in Figure 3A. DC8,9PC has a significantly steeper isotherm at high pressures than do PTPE and DiPhyPC. The results suggest that DC8,9PC monolayers are more solid-like, whereas the other two lipid monolayers are more liquid-crystalline-like. Figure 3A illustrates the surface pressure-loge(A) isotherms for all three lipids used here. To determine the stiffness of these insoluble monolayers at the air-water interface, we estimated the surface compression modulus from the LB isotherms using the expression M ) -dΠ/d loge(A) where Π is the surface pressure and A is the area per lipid molecule.26 Greater values of M correspond to stiffer films. For pure straight-chain fatty acids, Π vs. loge(A) graphs usually consist of two or more straight-line segments. A linear least-squares fit to the above equation using the data between 15 mN/m and 40 mN/m (35 mN/m for PTPE) suggests that the film stiffness follows the sequence DC8,9PC > DiPhyPC > PTPE. For comparison, a 24-carbon, straight-chain fatty acid has two characteristic moduli: ∼600 Nano Lett., Vol. 5, No. 6, 2005

Figure 3. (A) Pressure-area isotherm of monolayers formed by the lipids used in this study (DC8,9PC, PTPE, and DiPhyPC). The solid lines through each of the isotherms are the results of a linear least-squares fits of an equation for the surface compressional modulus to the film pressure data. Apparently, DC8,9PC is approximately 2 and 3 times as stiff as DiPhyPC and PTPE, respectively. (B) The increase in surface pressure of a PTPE monolayer caused by UV illumination. A PTPE monolayer was spread at the air-water interface and subsequently compressed to a film pressure of Π ∼ 31 mN/m. At constant barrier separation, the monolayer was then exposed to a constant UV irradiation. The film pressure increased to a limiting value (∼42 mN/m) within ∼10 min. Control experiments with a monolayer formed from DiPhyPC showed no change in film pressure.

mN/m above film pressures of 25 mN/m (upright, solid-like phase) and ∼120 mN/m below 25 mN/m (tilted, liquid-like phase). The different estimated values of the surface compression modulus (i.e., 300, 150, and 105 mN/m for monolayers of DC8,9PC, DiPhyPC, and PTPE, respectively) most likely reflect the differences in the chemical structures of these three lipids (Figure 1). For example, DC8,9PC molecules might pack more closely than those of DiPhyPC or PTPE because both of its hydrocarbon tails are identical and can therefore interact relatively strongly through dispersive forces. In contrast, DiPhyPC has methyl groups on every fourth carbon of the two main chains (i.e., it is a branched chain compound) that confer fluidity to the chains. Similarly, PTPE has one chain derived from palmitic acid (a 16-carbon straight acyl chain), and the other chain derived from 11,12-tricosenoic acid (a 23 carbon kinked chain). The second chain, which is identical to either of the two chains of DC8,9PC, has a bend in the middle due to the diacetylene group. In both DiPhyPC and PTPE, steric hindrance prevents the close packing of the hydrocarbon tails and should therefore form more liquidlike monolayers than would DC8,9PC. An independent measure of polymerizable phospholipid (PL) polymerization was obtained by monitoring the surface pressure of a compressed monolayer film of PL molecules at the air-water interface of a LB trough. Figure 3B shows that UV irradiation increases the film surface pressure of a PTPE monolayer until a plateau value is reached. Because the film area is fixed, the increase in surface pressure is due to an increase in the area per lipid molecule. As noted above, phospholipid diacetylenic polymerization causes the molecules to tilt, thereby increasing both the molecular area and the surface pressure. The mechanism of UV-induced polymerization in diacetylenic phospholipids is known.19,20 The triple bonds in the 1183

Figure 4. Functional reconstitution of a single R-hemolysin ion channel in a PTPE bilayer membrane. (A) In the absence of single stranded DNA, the ionic current through a single RHL channel is quiescent. Adding ∼40 µL of 50-nucleotide long poly(thymidine) (poly[dT]50), indicated as +ssDNA) to the cis side of the chamber causes transient current blockades that occur at random intervals.13 Following UV illumination of the PTPE membrane, the polynucleotide-induced current blockades persisted. (B) Five characteristic poly[dT]50-induced ionic current blockades that occurred after UV illumination. In this subset of events, there appear to be only three predominant states (fully open, R and β). Here, there are three examples of intraevent transitions from state R to state β. (C) Time series for a set of many poly[dT]50 transit events before (left) and after (right) UV illumination. The three most likely states illustrated in (B) are clearly observed in this larger set of data. The density of the plot after UV irradiation appears greater because the number of data points is greater.

hydrocarbon chains (e.g., in PTPE) are replaced with double bonds with hydrocarbon chains of adjacent molecules. This may cause a change in the membrane dielectric constant and thus the membrane capacitance. However, the capacitance is inversely proportional to the film thickness. Thus, the UVinduced increase in PTPE membrane capacitance, Cm (Figure 2B), is probably due to a decrease in the membrane thickness. If this effect arises solely from a tilt of the PTPE molecules, then a 20% increase in Cm would correspond to a 36 degree tilt in the lipid hydrocarbon tails, assuming that the lipids are rigid rods and their long axes are initially oriented perpendicular to the plane of a 4 nm thick membrane. It was shown earlier that individual molecules of singlestranded RNA and DNA can be driven electrophoretically through a single RHL channel in DiPhyPC (nonpolymerizable) planar membranes.13,14 In this study, we wanted to determine if channels in the membrane remain functional after lipid polymerization. The result in Figure 4A shows that the single-channel current that flows through the RHL 1184

channel in a liquid-crystalline PTPE membrane is relatively large and quiescent (the single channel conductance is virtually indistinguishable from that in DiPhyPC membranes). The subsequent addition of 50-nucleotide long poly(thymidine) (poly[dT]50) to the cis side of the chamber causes short-lived blockades, and these blockades were present even after irradiating the system with UV light. Figure 4B illustrates the types of blockades caused by poly[dT]50 after irradiation by a pen ray lamp (UVP Light Sources, Upland, CA, M/N 11SC-2, rated at 1.9 mW/cm2 at 1.9 cm) that was held at a distance of approximately 2 cm from the membrane. The polynucleotide-induced current blockade levels are virtually identical to those before UV illumination. There are predominately three conducting states, fully open, partially blocked (state R) and nearly fully blocked (state β). Moreover, some of the blockades have a characteristic two-state pattern that starts in state R and then transitions to state β. These patterns are characteristic of poly[dT]100 entering and threading through the RHL channel in DiPhyPC membranes.14 Figure 4C illustrates a time series for a set of many poly[dT]50-induced current blockades before (left) and after (right) UV illumination. The most probable states, which are clearly evident, correspond to those illustrated in Figure 4B. To enable studies of the long term stability of these polymerized membranes that act as protein channel scaffolds, new methods of attaching or suspending lipid membranes across apertures on solid supports are needed. In the current free-standing membrane configuration, there is a torus of solvent at the Gibbs-Plateau border between the phospholipid bilayer membrane and the solid support (e.g., Teflon in this case) at the edges of the bilayer membrane contacting the aperture. The torus may pose two issues for the use of ion channels in real-world applications. First, membrane formation via either the “painted” or “solvent- free” techniques complicates the platform construction. Second, lipid bilayer membranes may not be sufficiently robust, perhaps because of the torus. We are trying to develop a new system to circumvent both issues. It is well-known that polymerizable lipids remain stable for long periods of time. Without an appropriate configuration of the type required to estimate the quantitative increase in stabilization, it is reasonable to assume that the polymerizable lipid system, with the functional channel reconstituted within it, will remain stable for long periods of time (the protein channel is known to be stable if the right conditions such as pH and temperature are provided). An additional potential source of instability in such systems could be the presence of phase-separated domains in polymerized bilayer membranes. As discussed earlier, we had significant difficulty using one of the commercially available polymerizable lipids (DC8,9PC). Specifically, even when it was not polymerized, this lipid formed membranes only ∼10% of the time. This is in stark contrast to the results we obtained with PTPE and with the nonpolymerizable lipid DiPhyPC (both form membranes 100% of the time when in the liquid-crystalline state). DC8,9PC presumably forms phase-separated domains which results in a nonhomogeneous Nano Lett., Vol. 5, No. 6, 2005

film, and this could be the reason for the difficulty we encountered in forming stable black lipid membranes. We are currently planning on setting up a Brewster angle microscope to study the formation of liquid crystalline or other thermodynamic phases within the polymerizing membrane on a LB trough. However, to study phase formation in situ (i.e., in a black lipid membrane with protein channel inserted in a bilayer apparatus) is significantly more challenging and will require a highly specialized experimental set up that is yet to be designed and constructed. The functional reconstitution of transmembrane proteins into polymerizable membranes15,18,27 creates an opportunity to produce robust synthetic scaffolds for protein channels leading to practical applications (e.g., analyte sensing). The results presented here represent the initial proof-of-principle measurements for integrating channels as biological sensing elements within robust membranes for chemical and biological sensing applications (e.g., detection of pathogens by DNA identification and analysis). Furthermore, such reconstitution is demonstrated using technologically relevant protein ion channels, e.g., the R-hemolysin channel that has shown promise as a “stochastic sensor” for small ions,9 small molecules,12 proteins,14 and individual polynucleotides10,28,29,30 and the anthrax protective antigen ion channel PA63.5 The ability to interrogate transmembrane protein nanopores in robust nanoscale membranes could be extremely beneficial to science and for technological applications. This work could impact a wide range of disciplines including chemistry, biochemistry, molecular biology, biophysics, biology, engineering at the nanoscale, and diagnostics and pharmaceutical screening. In the latter application, one can easily imagine screening for therapeutics against membrane-bound toxins by embedding the latter channels in a robust membrane. The research described here highlights a path for the development of devices based on stochastic sensing for single-molecule detection and identification. Acknowledgment. This work was performed in part with financial support from DARPA DSO, the nonmedical CB defense technology program managed by DTRA, the NIST Advanced Technology Program, the NIST “Single Molecule Manipulation and Measurement” program, and the NSF (NIRT grant CTS-0304062). The identification of commercial materials and their sources is made to adequately describe the experimental results. In no case does this identification imply recommendation by the participating organizations, nor does it imply that the material is the best available.

Nano Lett., Vol. 5, No. 6, 2005

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