pubs.acs.org/Langmuir © 2009 American Chemical Society
Simultaneous Optical and Electrical Single Channel Recordings on a PEG Glass Toru Ide,*,† Yuko Takeuchi,† Hiroyuki Noji,‡ and Kazuhito V. Tabata‡ †
‡
Soft Biosystem Group, Laboratories for Nanobiology, Graduate School of Frontier Biosciences and Department of Biomolecular Energetics, The Institute of Scientific and Industrial Research, Osaka University, Suita, Osaka, Japan Received December 3, 2009
Single molecule imaging of working ion-channels is much more difficult than that of water-soluble proteins because of the fragile nature of membranes and lateral diffusion of particles in the membranes, which does not allow fluorescent contamination for optical single channel recording. In this report, we reconstituted maxi-potassium channels from porcine uterine smooth muscle into artificial planar bilayers formed on poly(ethylene glycol) (PEG) modified glass and performed simultaneous optical and electrical recording of the single channels. The channels were immobilized in the membranes by anchoring to PEG molecules on the glass. The technique developed in this study should pave the way for single molecule pharmacology of ion-channels.
Introduction The application of single molecule imaging techniques has spread rapidly through many biological fields including the study of cytoplasmic protein and nucleotide dynamics.1 In contrast, application of these techniques to ion-channel proteins is far behind because it is much more difficult to image single ionchannel proteins while recording their functions simultaneously. This is because channel proteins express their function only when incorporated into a membrane, which has a very fragile nature. The purpose of our present study is to develop an experimental apparatus for simultaneous optical and electrical observations of single ligand bindings to a single channel protein, i.e., fusing single molecule imaging and single channel current recordings into a single experimental technique. Although single channel current recordings can directly measure current through a channel pore, the binding events of a single ligand have been inferred indirectly from multimolecule experiments. The ability to simultaneously measure single binding events and single channel current fluctuations is of great interest as it would enable us to open a new field of “single molecule pharmacology”. In 1999, we developed an experimental apparatus for simultaneous optical and electrical observation of single ion-channels by combining artificial lipid bilayer technique and single molecule imaging technique.2 There had been numerous studies on single channel recordings by the artificial bilayer technique3 and on single molecule imaging of membrane lipids in self-standing and solid supported bilayer membranes.4,5 However, we were the first to report the combination of these two technologies. Using this apparatus, we imaged several types of channels at the single *Corresponding author. E-mail:
[email protected], Fax: þ816-6879-4634. (1) Yanagida, T., Ishii, Y., Eds.; Single Molecule Dynamics in Life Science; Wiley-VCH: New York, 2008. (2) Ide, T.; Yanagida, T. Biochem. Biophys. Res. Commun. 1999, 265, 595–599. (3) Favre, I.; Sun, Y. M.; Moczydlowski, E. Methods Enzymol. 1999, 294, 287–304. (4) Schmidt, T.; Schutz, G. J.; Baumgartner, W.; Gruber, H. J.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2926–2929. (5) Schutz, G. J.; Schindler, H.; Schmidt, T. Biophys. J. 1997, 73, 1073–1080. (6) Ide, T.; Takeuchi, Y.; Yanagida, T. Single Mol. 2002, 3, 33–42. (7) Ide, T.; Takeuchi, Y.; Aoki, T.; Yanagida, T. Jpn. J. Physiol. 2002, 52, 429–434.
8540 DOI: 10.1021/la9045594
molecule level while simultaneously recording channel current.6,7 Borisenko et al. detected conformational changes of single gramicidin channels with a similar technique.8 For those studies, we developed a method for forming lipid bilayers on an agarose layer through which we could observe single fluorophores in the membrane with an objective type total internal reflection fluorescence (TIRF) microscope. With this type of membrane, we could constantly image the whole bilayer. However, because of thermal diffusion by channel proteins in the membrane, it was very difficult to detect interactions between a single ligand molecule and a single ion-channel. Therefore, immobilizing channel proteins in the bilayers is imperative for stable measurements of channel-ligand interactions. Even if the diffusion is confined to the visible field, immobilization is required for single ligand binding observations since it is likely that fluorophores conjugated to the channels will be photobleached during the wait time for ligand binding. One way to immobilize channel proteins is to make a membrane area very small.9-11 There, a membrane was formed at the tip of a pipet by the tip-dip method, and subsequent observed conformational changes in gramicidin channels were observed. However, their method is not suited to recording ligand-channel interactions because it is quite difficult to reduce background noise when there are many fluorescent ligands in the solution. For future studies on ligand-channel interactions at the single molecule level, we describe here a novel method for simultaneous optical and electrical observation of single ion-channels in which channel proteins are immobilized by poly(ethylene glycol) (PEG) molecules.
Materials and Methods Reagents. Diphytanoylphosphatidylcholine (DPhPC) was purchased from Avanti Polar Lipids, Inc. (USA), β-BODIPY 530/550 HPC from Molecular Probes (USA), aminopropyltrietoxysilane from Shin-Etsu Chemical (Japan), NHS-PEG-biotin (8) Borisenko, V.; Lougheed, T.; Hesse, J.; Fureder-Kitzmuller, E.; Fertig, N.; Behrends, J. C.; Woolley, G. A.; Schutz, G. J. Biophys. J. 2003, 84, 612–622. (9) Harms, G. S.; Orr, G.; Montal, M.; Thrall, B. D.; Colson, S. D.; Lu, H. P. Biophys. J. 2003, 85, 1826–1838. (10) Lu, H. P. Curr. Pharm. Biotechnol. 2004, 5, 261–269. (11) Lu, H. P. In Methods in Nano Cell Biology; Jena, B., Ed.; Elsevier: Amsterdam, 2009.
Published on Web 12/23/2009
Langmuir 2010, 26(11), 8540–8543
Ide et al.
Article
Figure 1. (a) Diagrammatic representation of the horizontal membrane apparatus. (b) Schematic diagram of a channel protein immobilized on the PEG-coated glass. from Nektar (USA), and agarose type VII, asolectin and gramicidin D from Sigma (USA). Cy5 monofunctional reactive dye was purchased from Amersham Pharmacia Biotech (Sweden). All other chemicals were commercial products of an analytical grade. Preparation of Porcine Uterine Vesicles. Vesicles containing BK channels were isolated from porcine uterine smooth muscle using a modification of the procedure of Toro et al.12 The heavy microsomal fraction was centrifuged on a discontinuous sucrose gradient. Membrane fractions were obtained from 20:25% and 25:30% sucrose interfaces. The final pellets were resuspended in 300 mM sucrose, 100 mM KCl, and 5 mM NaPIPES at pH 6.8. The antiBK channel antibody raised at the amino terminus was conjugated with Cy5 molecules using a Cy5monofunctional reactive dye kit. Free Cy5 was removed via a NAP-10 gel filtration column (GE Healthcare). The Cy5conjugated antibodies were incubated for 60 min at 22 °C with BK-channel containing vesicles . In order to remove unreacted antibodies, the mixture was washed twice with 200 volumes of the appropriate buffer following centrifugation. Preparation of Bovine Tracheal Vesicles. The bovine smooth muscle plasma membrane vesicles were prepared according to the method of Slaughter et al.13 Microscopy. The microscopy used has been previously described2 with slight modifications to the optical lenses and filters. Unless otherwise stated, images were recorded using an ICCD camera, an image intensifier (VS4-1845, Video Scope, USA) and an electron bombarded CCD camera (C7190-20, Hamamatsu Photonics, Japan), and stored on digital videotape. (x, y) coordinates of particles in each video frame were calculated by a personal computer using image analysis software (Scion Image, Scion Corp., USA). Glass Pipettes. Fine glass pipettes were made from glass capillary with outer and inner diameters of 1.5 mm and 0.9 mm, respectively, using a pipet puller (P-97, Sutter Instrument, USA). The pulled capillaries were processed with a microforge apparatus (MF-900, Narishige, Japan) such that the tip aperture was approximately 10 μm. The Bilayer Membrane Chamber. The bilayer apparatus consisted of two chambers (Figure 1). The upper chamber was made of a glass tube with an inner and outer diameter of 8 mm and 10 mm, respectively, and could be adjusted using a micromanipulator (PCS-5000, Burleigh Instruments, USA). On the bottom of the upper chamber, a thin polypropylene (PP) film (0.20.3 mm thick) with a small pore in the center was attached. The pore on the film was made as follows: A heated stylus was pushed (12) Toro, L.; Ramos-Franco, J.; Stefani, E. J. Gen. Physiol. 1990, 96, 373–394. (13) Slaughter, R. S.; Welton, A. F.; Morgan, D. W. Biochim. Biophys. Acta 1987, 904, 92–104.
Langmuir 2010, 26(11), 8540–8543
on the film to make a projection. The top of the projection was shaved with a razor in order to make a circular pore of the appropriate diameter. This is the same method used to make an aperture for a conventional vertical bilayer, except that the projection was made taller than normal. The lower chamber consisted of a 35 mm glass dish with a hole 12 mm in diameter in the bottom. A coverslip was fixed over the hole with adhesive just prior to the experiment. The upper and lower chambers were connected to a patch clamp amplifier (CEZ-2400, Nihon-Kohden, Japan) through Ag-AgCl electrodes. The membrane potential was defined as the voltage of the upper solution with respect to the lower chamber, which was held at virtual ground. PEG Coating of a Coverslip. Coverslips (0.12-0.17 mm thick, Matsunami, Japan) were coated with PEG molecules in order to prevent strong interaction between the membrane and the glass surface. First, coverslips were incubated for 1 h at 90 °C in 2% (v/v) aminopropyl trietoxysilane to aminate the glass surface. Then, after wash with pure water the coverslips were reacted with NHS-PEG-biotin (10 mM NHS-PEG-biotin, 50 mM MOPS, pH 7.5) for 1 h at room temperature. The coverslips were washed with and stored in pure water at 4 °C. Several minutes before use in bilayer experiments, the coverslip was coated with avidin by putting a small volume of solution containing 50 μg/mL streptavidin, 2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, and 8.1 mM NaH2PO4, pH 7.4 onto the glass.
Formation of a Bilayer on PEG-Coated Glass (Figure 2). Figure 2 is a schematic representation of the procedures for forming bilayers on the PEG-glass. (1) First, appropriate recording solution was added to the upper and lower chamber. (2) Lipids dissolved in n-decane were added to the underside of the bottom of the upper chamber. A thick lipid membrane covered the aperture on the polypropylene film. (3) The upper chamber was plunged into the lower solution and moved downward until the thick membrane came into contact with the PEG-modified glass. (4) By slightly increasing the pressure in the upper chamber, the membrane began to expand resulting in the center of the membrane becoming a bilayer by contacting the two monolayers.2,6,14 This process of membrane-thinning is similar to those for conventional vertical15 and other types of bilayers.16,17 Alternatively, it is possible to form a bilayer on a PEG-coated glass by pressing a bilayer membrane that was preformed in an aqueous environment (30 ). Plunging a thick membrane into the lower solution, precisely regulating the hydrostatic pressure of the upper side of the membrane, and holding the membrane at a fixed height, the (14) Ide, T.; Ichikawa, T. Biosens. Bioelectron. 2005, 21, 672–677. (15) White, S. H. In Ion Channel Reconstitution; Miller, C., Ed.; Plenum Press: New York, 1986; pp 115-130. (16) Funakoshi, K.; Suzuki, H.; Takeuchi, S. Anal. Chem. 2006, 78, 8169–8174. (17) Thompson, J. R.; Heron, A. J.; Santoso, Y.; Wallace, M. I. Nano Lett 2007, 7, 3875–3878.
DOI: 10.1021/la9045594
8541
Article
Ide et al.
Figure 2. Schematic representation of the procedures for forming bilayers on the PEG glass. The dried PEG-coated coverslip was fixed with adhesive over the hole on the bottom of the chamber. Bilayers were formed on a PEG-coated coverslip as described in the Materials and Methods section. (1) First, recording solution was added to the chambers. (2) Lipids dissolved in n-decane were added to the underside of the bottom of the upper chamber. (3) The upper chamber was plunged into the lower solution and moved downward until the thick membrane came into contact with the PEGcoated glass. (4) By slightly increasing the pressure in the upper chamber, the membrane began to expand resulting in the center of the membrane becoming a bilayer. Alternatively, it is possible to form a bilayer on a PEG-coated glass by pressing a preformed bilayer membrane. (30 ) Regulating the hydrostatic pressure of the upper side of the membrane, the membrane thinned spontaneously to form a bilayer in an aqueous environment. membrane thinned spontaneously to form a bilayer. This process of thinning could be observed with a normal bright field microscope. Since these bilayers were inflated downward for the hydrostatic pressure difference, it was possible to contact whole bilayer area with the PEG-modified glass. Since there must be a water layer on the agarose layer, the two procedures are exactly the same from a microscopic viewpoint. There were no significant differences in channel properties such as single channel conductance and voltage dependency. Reconstitution of a Channel Protein into Bilayers. The channel in the vesicular membrane was transferred into the artificial bilayer by vesicle-fusion. In order to induce rapid fusion of the vesicle within the limited area of the bilayer, vesicles that had been osmotically loaded were puffed using a fine glass capillary positioned close to the membrane, as described previously.7
Determination of the Lateral Diffusion Constant from a Single-Molecule Trajectory. The mean square displacement (MSD) that is averaged over a trajectory at each time interval (Δt) was calculated from the trajectory of a particle. The diffusion constant D was calculated from the slope of the Δt - MSD plot by least-squares fitting.
Results and Disscussion BK-channels, which are known to have high single channel conductance, were fluorescently labeled using anti-NH2-terminus 8542 DOI: 10.1021/la9045594
antibodies that had been conjugated with cy5-dye molecules. The dye was conjugated by reacting NHS-cy5 with the antibody; the conjugation ratio was determined to be cy5/antibody = 1-10. Bilayer membranes were formed on PEG-coated glass as described above (see also Supporting Information). Channels were incorporated into the membrane by vesicle fusion. Figure 3a shows a fluorescence image of a uterine BK-channel in an artificial bilayer membrane that had been conjugated with antibodies at a conjugation ratio of 1.0. Figure 3b shows the time course of fluorescence intensity of the spot shown in part a. In this case, the total intensity was approximately four times the stepwise decrease that is caused by the photo bleaching of dyes attached to the antibody, which indicates that the channel was most likely labeled with four antibodies because the dye/protein of antibody was 1.0 and four homologous subunits assembled to form a channel. Although it is possible to observe single fluorophores in the membrane, as seen in this case, we labeled a channel protein with a number of dye molecules so that the channel could be easily detected. Knowing the x-y position of a channel in a membrane is both necessary and sufficient for our present purpose: to observe the interaction between a single channel protein and a single drug molecule. Figure 3c shows BK-channel immobilization upon anchoring to the glass surface through a PEG molecule. This figure represents thermal diffusion of a BK-channel in a bilayer membrane formed on a PEG-coated glass coverslip. Line segments in the figure show the trajectory of a single channel. Bilayer membranes were formed on PEG spacers with (þ) and without (-) anti-BK-channel antibodies as described in the Supporting Information. Trajectories of single channels were taken several minutes after the channels were incorporated by vesicle fusion. The diffusion coefficient, calculated from single channel trajectories, was D = 0.64 ( 0.57 μm2/s (n = 5) without anchoring to the glass via PEG. Although the channels moved rapidly in bilayers, this value is an order of magnitude smaller than that obtained in agarose supported bilayers in which channels move almost freely in the membrane.3 This is probably because of friction between the channel and PEG spacers. By contrast, the diffusion coefficient in Figure 3c (þ) was found to be much smaller (D < 0.01 μm2/s), showing that the channel was immobilized by anchorage through a PEG (Figure 1). Single channel current records (Figure 3d) indicate that the natural properties of BK-channels can also be measured using immobilized channels (see also Figure S2 in Supporting Information). Voltage dependence, which is the signature feature of BK-channels, was maintained when the channel was reconstituted into an artificial bilayer and immobilized by a PEG anchor. The immobilized channel was activated more at lower membrane potential; i.e., it was activated at depolarizing membrane potential, in agreement with native channel behavior. From the slope of the I-V curve, single channel conductance was determined to be 345 pS, which agrees well with conventional bilayer and patch-clamp experiments.18 In order to investigate the effect of a narrow ion-conduction pathway between a bilayer membrane and glass on the series resistance, we incorporated a number of gramicidin channels into bilayers formed in an aqueous environment and then pressed them against the PEG-coated glass surface. Alternatively, ionchannels were reconstituted into bilayers on the PEG-layer followed by moving the membrane upward to be apart from the PEG-layer. Regardless, pressing the membrane onto the PEG-layer did not cause significant changes in channel current (18) Magleby, K. L. J. Gen. Physiol. 2003, 121, 81–96.
Langmuir 2010, 26(11), 8540–8543
Ide et al.
Article
Figure 3. (a) Fluorescence image of a cy5 labeled uterine BK-channel incorporated into a bilayer membrane. (b) Stepwise decrease in fluorescence intensity of the spot shown in part a. (c) Trajectories of a single BK-channel protein observed in the absence (-) and in the presence (þ) of antibodies. (d) Single channel records taken from the membrane shown in part c (þ).
(Figure S3(A) in Supporting Information). Therefore, the voltage drop due to a narrow ion-pathway between the membrane and the glass was not significantly large, meaning we can apply this measurement technique to channels having rather large channel conductance. In principle, our technique is applicable to all types of channels assuming they can be incorporated into an artificial bilayer membrane. However, it is necessary to check the channel properties after anchoring them to the glass surface because of potential changes in ion-conduction properties. For example, a bovine tracheal BK-channel, which was reconstituted into a bilayer with the intracellular side of the channel facing the upper side of the membrane, showed a decrease in conductance amplitude after the membrane was pressed against the PEG-coated glass (Figure S3(B) in Supporting Information). In that experiment, the channel was not anchored to the glass through PEGs. Interestingly, the single channel conductance could not be recovered by pulling the membrane from the PEG-layer, indicating that the decrease in the single channel conductance caused by pressing the membrane against PEG molecules was not due to an increase in series resistance. Although the exact reason for this decrease is unknown, it could be that mechanical stress caused by pressing the (19) Dopico, A. M.; Kirber, M. T.; Singer, J. J.; Walsh, J. V., Jr. Am. J. Hypertens. 1994, 7, 82–89. (20) Kawakubo, T.; Naruse, K.; Matsubara, T.; Hotta, N.; Sokabe, M. Am. J. Physiol. 1999, 276, H1827–H1838. (21) Mallouk, N.; Allard, B. Am. J. Physiol. Cell Physiol. 2000, 278, C473–C479. (22) Mienville, J.; Barker, J. L.; Lange, G. D. J. Membr. Biol. 1996, 153, 211–216. (23) Qi, Z.; Chi, S.; Su, X.; Naruse, K.; Sokabe, M. Mol. Membr. Biol. 2005, 22, 519–527.
Langmuir 2010, 26(11), 8540–8543
membrane onto the PEG-layer modified the properties of the channel protein since certain BK-channel have mechanosensitivity.19-24 In this study, we have developed a novel technique for simultaneous optical and electrical measurements of single ionchannel proteins, which allows us to measure the optical properties of single ion-channels much more easily than what we proposed previously.2,6 In this new technique, we need only observe a small area in the membrane because the channel is immobilized onto the glass. This also means that it is possible to measure rapid changes in single particle fluorescence such as fast association and dissociation of a fluorescently labeled drug to the channel using faster optical detectors such as an avalanche photodiode because noise from other parts of the membrane can be completely removed. Acknowledgment. The authors thank Dr. Peter Karagiannis for carefully revising the manuscript. This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Molecular and System Life Science, and Innovative nanoscience of supermolecular motor proteins working in biomembranes). Supporting Information Available: Figures showing a detailed analysis of single channel records. This material is available free of charge via the Internet at http://pubs.acs. org. (24) Yuan, C.; O’Connell, R. J.; Feinberg-Zadek, P. L.; Johnston, L. J.; Treistman, S. N. Biophys. J. 2004, 86, 3620–3633.
DOI: 10.1021/la9045594
8543
