Reconstitution of Nicotinic Acetylcholine Receptors into Gel-Protected

Mar 12, 2004 - J. A. Beddow, I. R. Peterson,* J. Heptinstall, and D. J. Walton. Centre for Molecular and Biomolecular Electronics, Coventry University...
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Anal. Chem. 2004, 76, 2261-2265

Reconstitution of Nicotinic Acetylcholine Receptors into Gel-Protected Lipid Membranes J. A. Beddow, I. R. Peterson,* J. Heptinstall, and D. J. Walton

Centre for Molecular and Biomolecular Electronics, Coventry University SE, Priory Street, Coventry, CV1 5FB, U.K.

We report the functional reconstitution of nicotinic acetylcholine receptors into gel-protected bilayer lipid membranes using two different methods. In the first case, reconstitution was achieved by direct membrane formation from an emulsion of glycerol monooleate, hexane, and a membrane receptor extract. In the second case, incorporation was achieved via the fusion of vesicles from a preparation of membrane-bound receptors into preformed membranes after diffusion through the protective front gel layer. Measurement of the dc conductivity of the membranes in the presence of either acetylcholine or r-bungarotoxin was used to test for the functional activity of incorporated receptors. The opening of single transmembrane channel in a bilayer lipid membrane can be detected electrically by the resulting increase in membrane conductivity.1 Many millions of ligand-gated channel proteins can be accommodated into a small area of membrane. There is evidence that the transmembrane channel opens slightly in response to the binding of even a single specific ligand molecule.2 As the operation of individual receptors is independent and the currents through them combine linearly, the resulting change in membrane conductivity provides a signal proportional to the ligand concentration over many orders of magnitude3. Thus, biosensors based on ligand-gated ion channels incorporated into a suitable artificial bilayer lipid membrane should be capable of very high sensitivity, selectivity, and dynamic range. Biosensors based on this principle were first proposed4 in the early 1980s. Implementations have been demonstrated by many groups,5-10 and the field has been reviewed.11 However, the * Corresponding author. Current address: MembraSense Ltd., Technocenter, Puma Way, Coventry CV1 2TX, U.K. Tel: +44 24 7623.6970, Fax: +44 1926 748642. E-mail: [email protected]. (1) Tank, D. W.; Huganir, R. L.; Greengard, P.; Webb, W. W. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 5129-5133. (2) Forman, S. A.; Miller, K. W. Biophys. J. 1988, 54, 149-158. (3) Nelson, N.; Anholt, R.; Lindstrom, J.; Montal, M. Proc. Natl. Acad. Sci. U.S.A.. 1980, 77, 3057-3061. (4) Thompson, M.; Krull, U. J.; Venis, M. A. Biochem. Biophys. Res. Commun. 1983, 110, 300-304. (5) Eldefrawi, M. E.; Sherby, S. M.; Andreou, A. G.; Mansour, N. A.; Annau, Z.; Blum, N. A.; Valdes, J. J. Anal. Lett. 1988, 21, 1665-1680. (6) Yager, P.; Dalziel, A. W.; Georger, J.; Price, R. R.; Singh, A. Biophys. J. 1988, 51, A143-A143. (7) Sugao, N.; Sugawara, M.; Minami, H.; Uto, M.; Umezawa, Y. Anal. Chem. 1993, 65, 363-369. (8) Eray, M.; Dogan, N. S.; Liu, L.; Koch, A. R.; Moffett, D. F.; Silber, M.; Van Wie, B. J. Biosens. Bioelectron. 1994, 9, 343-351. (9) Nikolelis, D. P.; Theoharis, G. Bioelectrochemistry 2003, 59, 107-112. 10.1021/ac0350514 CCC: $27.50 Published on Web 03/12/2004

© 2004 American Chemical Society

practical application of membrane-based biosensors has been largely restricted by the fragility of artificial membranes, which typically last only a few hours. A number of techniques are under investigation to prepare membranes with improved durability.12 In the approach of our group, bilayer lipid membranes are formed by self-assembly from a “spreading solution” of the lipid in a hydrophobic solvent, which is dispensed between two layers of agarose hydrogel.13 The gel layers provide protection from dehydration and mechanical disruption, while still allowing direct contact with an electrolyte and molecular access to the membranes by diffusion through the gel.14 Bilayer lipid membranes in the gelprotected configuration have been shown to respond to the peptide antibiotics valinomycin and gramicidin in a way similar to that of natural membranes15,16 It has also been demonstrated that they can be used to detect the insertion of transmembrane pores created by activation of the complement system of human blood17,18 In the present paper, we report the functional incorporation of nicotinic acetylcholine receptors (nAChR) into gel-protected bilayer lipid membranes. The nicotinic acetylcholine receptor was chosen for the investigation because of the availability of a rich source suitable for extraction in the electric organs of the electric ray Torpedo marmorata. It is also the best-characterized ligandgated channel protein and has been widely used in both studies of reconstitution into bilayer lipid membranes (BLMs)19-21 and construction of a number of prototype biosensors5,22,23 (10) Cornell, B. A.; BraachMaksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Weiczorek, L.; Pace, R. J. Nature 1998, 387, 580-583. (11) Trojanowicz, M. Fresenius J. Anal. Chem. 2001, 371, 246-260. (12) Tien, H. T.; Ottova, A. L. Electrochim. Acta 1998, 43, 3587-3610. (13) Costello, R. F.; Peterson, I. R.; Heptinstall, J.; Byrne, N. G.; Miller, L. S. Adv. Mater. Opt. Electron. 1998, 8, 47-52. (14) Beddow, J. A.; Peterson, I. R.; Heptinstall J.; Walton, D. J. J. Electroanal. Chem. 2003, 544, 107-112. (15) Costello, R. F.; Peterson, I. R.; Heptinstall, J.; Walton, D. J. Biosens. Bioelectron. 1999, 14, 265-271. (16) Beddow, J. A.; Peterson, I. R.; Heptinstall, J.; Walton, D. J. Proc. SPIE 2001, 4414, 62-69. (17) Costello, R. F.; Evans, S. W.; Evans, S. D.; Peterson,I. R.; Heptinstall, J. Enzyme Microb. Technol. 2000, 26, 301-303 (18) Beddow, J. A.; Peterson, I. R.; Dwenger, C.; Annamenani, R.; Bion, J. F.; Heptinstall, J.; Walton, D. J. Clin. Diag. Lab. Immunol., submitted. (19) Montal, M. J. Membr. Biol. 1987, 98, 101-115. (20) Schindler, H.; Quast, U. Proc. Natl. Acad. Sci. U.S.A.. 1980, 77, 30523056. (21) Schurholz, T.; Weber, J.; Neumann, E. Bioelectrochem. Bioenerg. 1989, 21, 71-81. (22) Rogers, K. M. Mol. Biotechol. 2000, 14, 109-129. (23) Eray, M.; Dogan, N. S.; Reiken, S. R.; Sutisna, H.; Van Wie, B. J.; Koch, A. R.; Moffett, D. F.; Silber, M.; Davis, W. C. Biosystems 1995, 35, 183-188.

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Figure 1. Elevation and plan cross sections of gel-protected membrane assembly between PCB electrodes: RE, reference electrode; WE, working electrode. Plan views: white, bare metal; black, insulating; gray, resist.

The two methods used for incorporation of nAChR are similar to methods used with other BLM configurations.24 In the first and most direct route, the hydrophobic spreading solvent contains both lipid and receptor, and the latter is incorporated in the membrane as it self-assembles. Since the continuous phase of the electric organ extract is aqueous, an initial processing step resembling the inversion of an emulsion25,26 is required to transfer the receptors to a hydrophobic solvent. We report here an attempted implementation of this route. The second demonstrated method for receptor incorporation involves the fusion of receptor membrane material with readyformed glycerol monooleate (GM) membranes. Here no modifications of the membrane or receptor material are necessary, but the nAChR must first diffuse through the supporting gel layers. EXPERIMENTAL SECTION Glycerol, NaOH, NaH2PO4, and KCl were all AnalaR grade and obtained from Fisher Scientific. Acetylcholine chloride, fluorescein isothiocyanate (FITC)-R-bungarotoxin, NaN3, iodoacetamide, low electroendosmotic type 1-A agarose, 1-monooleoyl-rac-glycerol (99%), phenylmethanesulfonyl fluoride (PMSF), and HPLC grade hexane were obtained from Sigma-Aldrich. Ultrapure water was obtained from an Elgar recycling deionizer-filter unit giving a specific resistance of 18 MΩ‚cm. GM spreading solution was made up at a concentration of ∼20 g/L in hexane. The electrolyte used was a solution of 0.1 M KCl and 10 mM NaH2PO4 adjusted to pH 7.4. The homogenization buffer used for the nAChR extraction procedure was a solution of 10 mM sodium phosphate, 10 mM NaN3, 1 mM PMSF, and 5 (24) Miller, C. Ion Channel Reconstitution; Plenum: New York, 1986. (25) Campbell, I.; Norton, I.; Morley, W. Neth. Milk Dairy J. 1996, 50, 167. (26) Ruckenstein, E. Langmuir 1997, 13, 2494.

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mM iodoacetamide. Its pH was adjusted to 7.5 by the addition of 0.1 M NaOH. Septums were prepared from 10-µm-thick poly(tetrafluoroethylene) (PTFE) film (Goodfellows, UK). This was cut into squares of around 2 cm × 2 cm and central perforations of 100-200 µm in diameter were made in the squares of film using an electric spark. The electrical spark was generated using a car ignition coil connected to a voltage supply. The size and uniformity of the perforations was checked using a binocular microscope (Vickers Instruments, ×30 magnification). The electrodes used for measuring the electrical properties of the membranes were based on PCBs. In the current configuration, using a bipotentiostatic circuit two Ag/AgCl reference and working electrodes are required on each side of the gel-protected membranes, as shown in Figure 1. The PCB electrodes, shown in the lower part of Figure 1, were produced by Circast Ltd. The Ag electrode regions on the PCBs were chlorided in a 0.1 M KCl solution at 9 V with a silver wire counter electrode. Both electrodes were first connected as the cathode for 5 s and then as the anode for 5 s. The smaller top electrode was connected to the bottom electrode via soldered fine wires. The bipotentiostat circuit was built with low-noise low-biascurrent integrated operational amplifiers as previously described.16 A feedback circuit ensured that the voltage sensed by the reference electrodes across the membrane was equal to the input voltage divided by 100. An output signal proportional to the membrane current and independent of drive voltage was derived from the voltage drop across a current sensing resistor in series with a working electrode. In both cases, minimal voltage offset and unconditional stability over a wide range of electrode impedance conditions were ensured by lead-lag techniques. The circuit

Figure 2. PCB membrane element connected via PCB edge connector to the membrane testing circuit.

and the PCB electrode assembly were located in adjacent diecast metal boxes fixed rigidly to each other and forming a Faraday cage, as shown in Figure 2. Agarose-electrolyte mixture was prepared by heating to boiling point 1.5 wt % agarose powder, 10 vol % glycerol, and the remainder buffered electrolyte solution. While the agaroseelectrolyte mixture was still liquid, typically 1 mL was pipetted onto a glass microscope slide together with a few plastic spacer strips of the desired thickness; a second microscope slide was then pressed down on top to expel excess mixture. The gels were allowed to set for 30 min at 5 °C and then the upper slide was carefully removed. Gels were cut into squares ∼1 cm on a side and removed from the bottom slide using tweezers. The electrode gel-protected membrane assembly was assembled by sequentially stacking a bottom PCB electrode, a 500 µm-thick bottom gel layer, a perforated PTFE square, 10 µL of spreading solution in the region of the PTFE perforation, a top gel of thickness 200-250 µm, and a top electrode assembly, with central perforation providing access to the top gel. The assembled membrane units were then plugged in to the bipotentiostat,16 and the conductance and capacitance of each measured by biasing with 50 mV dc or with a sinusoidal voltage of 20 mV ppk, 250 Hz, respectively. The bipotentiostat output signal, proportional to membrane current, was observed using a Thurlby Thandar Instruments 1906 multimeter or a Tektronix model 544 oscilloscope. The A/D output of voltmeter was connected via a RS232 port to a PC running in-house data capture software. Membrane vesicles containing nAChR were prepared from frozen electric organ tissue from T. marmorata according to the method of Lindstrom et al.27 Throughout the extraction procedure, all solutions and glassware were kept on ice. Protease inhibitors (27) Lindstrom, J.; Anholt, R.; Einarson, B.; Engel, A.; Osame, M.; Montal, M. J. Biol. Chem. 1980, 255, 8340-8350.

were added to protect membrane proteins from proteases released during homogenization. The protease inhibitor PMSF is only stable for ∼1 h in aqueous conditions and therefore was made up fresh and added to homogenization buffer at the start of the procedure. A 5-10-g sample of frozen electric organ tissue was placed in 50-100 mL of homogenization buffer and allowed to partially defrost for 15-30 min. The tissue was then cut into roughly 1-cm3 chunks, and any obvious connective tissue and attached skin was removed. The tissue was homogenized using a top blade blender in roughly 2 volumes of homogenization buffer. This homogenization was repeated at least 4 times in 30-s bursts. The resulting homogenate was centrifuged at 3000 rpm in 10mL plastic centrifuge tubes (MSE Centaur 2). Any floating debris was removed from the supernatant after centrifugation using a Pasteur pipet. The supernatant was then pooled in 50-mL ultracentrifuge tubes. The pelleted material from the low-speed centrifugation was rehomogenized and recentrifuged, and the supernatant was again pooled together with the initial fractions. The pooled supernatant was then centrifuged at 11 krpm for 120 min at 4°C (MSE High-Spin 21). The resulting supernatant was then discarded, and the pellet was resuspended in a small volume (5-10 mL) of homogenization buffer. This was split into 1-mL aliquots and frozen in Eppendorf tubes at either -20 or -80 °C for overnight or long-term storage. The emulsions were prepared by adding 5-10 mg of GM to 50-100 µL of receptor extract in a 2-mL glass vial. This was vortexed for 30 s or until all the lipid was suspended. GM dissolved in hexane (20 mg/mL) was then added to this solution in 100-µL aliquots followed by vortex mixing until the total volume was 1000 µL. This solution was transferred to a 2-mL plastic Eppendorf tube and centrifuged at 10 krpm for 5 min. The clear bottom phase was removed using a syringe, and the solution was again vortexed and decanted back into a 2-mL glass vial. This solution was then used instead of the standard lipid in hexane as the membraneforming solution. The solution was mixed for 30 s prior to use as a spreading solution for membrane formation. For incorporation of nAChR by fusion, GM membranes were first prepared as detailed above. Each cell was checked both at formation and after 10 min for formation of a stable high-resistance membrane. Then 10-20 µL of aqueous membrane suspension was placed in the top well of the cell assembly, and the cell was left to stand for at least 30 min. After this time, excess remaining liquid was removed from the top well using paper tissue. The test membranes from both procedures were connected to the bipotentiostat with an applied transmembrane voltage of 50 mV dc; the output voltage signal was collected and recorded on a PC connected to the voltmeter. The membranes were left to stabilize for at least 10 min and then either 10-20 µL of electrolyte, 10 mM acetylcholine solution, or 1 unit/mL R-bungarotoxin solution was added to the unit and the membrane current was recorded for at least 10 min. After this period, excess remaining liquid was removed and a further drop of 10 mM acetylcholine solution or 1 unit/mL R-bungarotoxin solution was added depending upon the response under test. Analytical Chemistry, Vol. 76, No. 8, April 15, 2004

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In the lower trace shown in Figure 4, the R-bungarotoxin solution was added to a similarly prepared membrane at 15 s and then the ACh solution at 600 s. No increase in conductivity was observed over the period of the trace.

Figure 3. Trace showing membrane conductance change in response to addition of ACh solution and R-bungarotoxin solution to a membrane prepared from an emulsion of GM, hexane, and crude nAChR preparation.

Figure 4. Typical traces showing membrane conductance change in response to addition of ACh solution and R-bungarotoxin solution to GM membranes following fusion of nAChR membrane vesicles.

RESULTS The unmodified GM membranes had a typical membrane conductance of ∼100 pS. All membranes were left to stabilize for at least 10 min prior to further testing, and any that showed signs of poor stability such as a spontaneous increase in conductivity were discarded. In control experiments, no increase in membrane conductivity was observed following addition of the test solutions to unmodified GM membranes. Additionally, there was no change in membrane conductivity following addition of 0.1 M KCl electrolyte solution or R-bungarotoxin to the membranes with incorporated receptors. Figure 3 shows a trace of membrane conductivity against time for a membrane prepared from an emulsion of GM, hexane, and crude nAChR membrane extract. Following addition of 10 mM acetylcholine solution at 15 s, there is a large increase in membrane conductance that reaches a maximum of ∼3.5 µS within 400 s. Following this, there is some decrease in the conductance, which continues after the addition of R-bungarotoxin at 700 s. In comparison, no increase in membrane conductance was observed where R-bungarotoxin was added to a similarly prepared membrane prior to the addition of acetylcholine. Figure 4 shows a typical trace of membrane conductance against time for a GM membrane that had been exposed to the aqueous nAChR membrane extract for 30 min. The upper trace shows a gradual increase in membrane conductivity following addition of a 10 mM acetylcholine (ACh) solution at 15 s. The membrane conductivity increases from ∼150 pS initially to ∼400 pS within 10 min. There are several regions of high noise on the trace associated with electrical pickup as the metal of the hypodermic needle approaches the electrodes. The membrane conductivity then begins to decrease after the addition of R-bungarotoxin solution at 600 s. 2264

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DISCUSSION The results reported here are consistent with the incorporation of functional nAChR proteins into gel-protected bilayer lipid membranes. The methods used for incorporation of the receptors are very similar to methods used with other BLM configurations.24 However, the gel-protected membrane configuration provides a system for membrane study that is more robust and less difficult technically than other BLM configurations. The initial aim of the study was to demonstrate direct incorporation of nAChR at the membrane formation step. The main driving force for membrane formation is absorption of the lipid molecules at the high-energy interfaces between the agarose gels and the hydrophobic solvent. As expected, no resistive membranes were formed without the use of a hydrophobic solvent in the spreading solution. It was therefore necessary to incorporate the receptors into a suitable hydrophobic spreading solution. The spreading solution used was prepared from aqueous nAChR extract emulsified with lipid in hexane. A typical conductivity response of a membrane prepared using the emulsion is shown in Figure 3. There is a rapid increase in membrane conductivity after the addition of 10 mM ACh solution to the top gel of the membrane assembly. This is consistent with the opening of the nAChR channels as ACh diffuses through the top gel layer and binds to receptors embedded in the membrane. The maximum level of membrane conductance is reached within ∼450 s of addition of the ACh solution. If a single-channel conductance of ∼20 pS is assumed, the peak conductivity observed is equivalent to the opening of ∼175 000 channels.20 There is a gradual decrease in the measured conductivity after the addition of R-bungarotoxin solution to the membrane. This is consistent with inhibition of nAChR channel opening due to binding of the R-bungarotoxin to the receptors. In control experiments, there were no responses for membranes exposed to R-bungarotoxin before ACh. However, addition of R-bungarotoxin solution was not always sufficient to cause a reduction in membrane conductivity after exposure of membranes to ACh. It is therefore possible that the observed peak in response and decline is due to desensitization of receptors rather than the effect of R-bungarotoxin binding alone. There are several other reports of direct incorporation of membrane proteins into BLMs from direct spreading of solvent lipid solutions.28 Reiken et al. used a similar technique for incorporation of nAChR into bilayer lipid membranes.29 However, the reproducibility was poor.14,23 In the current work, only 1020% of the membranes prepared using the emulsion showed a conductivity response with ACh. The procedure is quite simple and can probably be improved to yield a greater number of functional receptor membranes. Spontaneous membrane failure in not uncommon but almost always occurs as either a very rapid drop in membrane resistance or a trace with a steadily increasing rate of increase in membrane (28) Montal, M.; Darszon, A.; Schindlers, H. Q. Rev. Biophys. 1981, 14, 1-79. (29) Reiken, S. R.; Van Wie, B. J.; Sutisna, H.; Moffett, D. F.; Koch, A. R.; Silber M.; Davis, W. C. Biosens. Bioelectron. 1996, 11, 91-102.

conductivity up until failure occurs. The stability of each membrane unit was checked both at formation and at least 10 min afterward. Any membranes showing a spontaneous increase in conductivity over time were discarded. The long-term stability of reconstituted membranes was not significantly different from plain GM lipid membranes, with 3040% of membranes still resistive after 24 h. However, after 24 h, none of the membranes containing receptors showed any response to the addition of ACh. This is likely to be due to either denaturation or degradation of the proteins in the artificial membrane environment. A number of factors may contribute to degradation of the receptors such as storage at room temperature and long-term exposure to the hydrophobic solvent, hexane. It is also likely that there is some contamination of the crude membrane material with proteases. Attempts to purify the receptors further by the use of sucrose gradient centrifugation did not lead to any obvious improvements in the reconstitution results. The purification procedures yield an enriched nAChR fraction, but there is still some contamination with other native membrane material. Alternative methods for greater purification of membranebound receptors commonly involve the use of detergents.25 This was avoided in the current work as it has been found that even very low levels of detergent contamination have an adverse effect on membrane stability. Figure 4 shows two membrane conductivity traces from the experiments on the fusion approach to receptor incorporation. In the upper trace, there is an increase of ∼200 pS in membrane conductance following the addition of the ACh solution, which is equivalent to the opening of ∼10 channels. Unfortunately the noise level is higher than any single-channel conductance on the traces so individual opening events are not distinguishable. Again there is some decrease in the measured conductivity after the addition of R-bungarotoxin solution to the membrane. In the lower trace in Figure 4, the R-bungarotoxin solution was added prior to ACh solution and there is no change in membrane conductance over time.

In comparison to the results from the emulsion experiments, it appears that a much smaller number of receptors are incorporated. Indeed, the fusion method is likely to be limited by a number of factors. Before the receptor membrane vesicles can fuse with the membranes they have to diffuse through the supporting agarose gel layer. In the present set of experiments, the gel layer was ∼200 µm thick and the material was only left to diffuse for 30 min. There may also only be a small driving force for fusion of membrane fragments to the preformed membranes. However, the fusion technique was more reproducible than the emulsion technique, with more than half of the membranes tested showing a conductivity response with ACh. This is probably due to differences in the treatment of the membrane receptor extract in the two techniques. As mentioned earlier, the emulsification procedure used was only rudimentary, so it is possible that the receptors were not uniformly distributed within the lipid and hexane emulsions. In addition, the treatment of the membrane receptors in the fusion technique is not as harsh, so that more of them may be left in a functional state after incorporation. CONCLUSION We have demonstrated the incorporation of functional ligandgated channels into a stable bilayer lipid membrane configuration using two different methods. Each method is appropriate for a effective mode of sensor use, in which the membrane element is customized for the detection of a specific analyte either during production or at the point of use. ACKNOWLEDGMENT J.A.B. gratefully acknowledges a postdoctoral research grant from the EPSRC (Grant 99316934).

Received for review February 9, 2004.

September

9,

2003.

Accepted

AC0350514

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