Ligand Binding to Nicotinic Acetylcholine Receptor Investigated by

Ligand binding to the nicotinic acetylcholine receptor is studied by surface plasmon resonance. Biotinylated bun- garotoxin, immobilized on a ...
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Anal. Chem. 1999, 71, 3157-3165

Ligand Binding to Nicotinic Acetylcholine Receptor Investigated by Surface Plasmon Resonance Dietmar Kro 1 ger,† Ferdinand Hucho,‡ and Horst Vogel*,†

Laboratoire de Chimie Physique des Polyme` res et Membranes, Ecole Polytechnique Fe´ de´ rale de Lausanne, CH-1015 Lausanne, Switzerland, and Institut fu¨r Biochemie, Freie Universita¨t Berlin, Thielallee 63, D-14195 Berlin

Ligand binding to the nicotinic acetylcholine receptor is studied by surface plasmon resonance. Biotinylated bungarotoxin, immobilized on a streptavidin-coated gold film, binds nicotinic acetylcholine receptor both in detergentsolubilized and in lipid vesicle-reconstituted form with high specificity. In the latter case, nonspecific binding to the sensor surface is significantly reduced by reconstituting the receptor into poly(ethylene glycol)-lipid-containing sterically stabilized vesicles. By preincubation of a bulk nicotinic acetylcholine receptor sample with the competing ligands carbamoylcholine and decamethonium bromide, the subsequent specific binding of the receptor to the surface-immobilized bungarotoxin is reduced, depending on the concentration of competing ligand. This competition assay allows the determination of the dissociation constants of the acetylcholine receptor-carbamoylcholine complex. A KD ) 3.5 × 10-6 M for the detergent-solubilized receptor and a KD ) 1.4 × 10-5 M for the lipid vesicle-reconstituted receptor are obtained. For decamethonium bromide, a KD ) 4.5 × 10-5 M is determined for the detergent-solubilized receptor. This approach is of general importance for investigating ligandreceptor interactions in case of small ligand molecules by mass-sensitive techniques. Surface-sensitive techniques, in particular optical techniques, have gained increasing importance as bioanalytical tools for investigating molecular interactions.1,2 The underlying principle of surface-sensitive detection is to immobilize one component of a molecular reaction system on a sensor surface and monitor in situ the binding of the complementary partner to the surface without any additional separation step. The functionalization of the sensor surface plays a crucial role in the case of biopolymers: The immobilized component must retain its native conformation to enable specific binding; on the other hand, nonspecific binding (NSB) must be suppressed. Surface plasmon resonance (SPR) is an ideally suited surface-sensitive technique both for controlling the formation of molecular assemblies on sensor surfaces and for on-line and label-free detection of biomolecular * Corresponding author: (tel) (41) (21) 693 31 55; (fax) (41) (21) 693 61 90; (e-mail) [email protected]. † Ecole Polytechnique Fe ´ de´rale de Lausanne. ‡ Freie Universita ¨t Berlin. (1) Ulman, A. An Introduction to Ultrathin Organic Films: From LangmuirBlodgett to Self-Assembly; Academic Press Inc.: San Diego, 1991. (2) Robinson, G. Sens. Actuators B 1995, 29, 31-36. 10.1021/ac9814391 CCC: $18.00 Published on Web 06/24/1999

© 1999 American Chemical Society

interactions.3,4 SPR has been widely used to investigate watersoluble molecules, e.g., antigen-antibody interactions,5 epitope mapping,6 or DNA-protein interactions,7 to mention a few. We (for review, see ref 8) and others9-12 have recently tethered lipid bilayers to sensor surfaces for the investigation of interactions between ligand molecules in the bulk and membranes or membrane receptor proteins on the support. Here we present a competitive approach by immobilizing a ligand on the sensor surface and observing subsequently the specific binding of the membrane receptor in the presence of a certain amount of ligand in bulk solution. This approach is of interest when ligands are too small (relative molecular mass Mr typically several hundred daltons) to be measured directly upon binding to their immobilized receptors (Mr typically several hundred kilodaltons). The present “inverse” assay is ∼1000 times more sensitive then the direct binding. These membrane receptor-ligand interactions play a crucial role in transmembrane signaling; membrane proteins such as channel-forming receptors or G-protein-coupled receptors are important targets for therapeutic agents. Therefore, novel techniques for investigating the molecular interactions with such receptors are equally important both for elucidating the underlying molecular mechanisms of signal recognition and transduction and for the finding of new therapeutic agents.8 To reduce the complexity of natural membrane systems, it is often advantageous to work with purified membrane receptor proteins, either solubilized in detergents or reconstituted into artificial lipid membranes. This leads to a significantly increased receptor density on the sensor surface, enhancing the sensitivity for ligand detection. Since solubilization and reconstitution might (3) Knoll, W. MRS Bull. 1991, 56, 29-39. (4) Jo ¨nsson, U.; Malmqvist, M. In Advances in Biosensors; Turner, A., Ed.; JAI Press: London, 1992; Vol. 2, pp 291-336. (5) Mayo, C. S.; Hallock, R. B. J. Immunol. Methods 1989, 120, 105-114. (6) Johne, B.; Gadnell, M.; Hansen, K. J. Immunol. Methods 1993, 160, 191198. (7) Nilsson, P.; Persson, B.; Uhlen, M.; Nygren, P.-A. Anal. Biochem. 1995, 224, 400-408. (8) Heyse, S.; Stora, T.; Schmid, E.; Lakey, J. H.; Vogel, H. Biochim. Biophys. Acta 1998, 1376, 319-338. (9) Brink, G.; Schmitt, L.; Tampe´, R.; Sackman, B. Biochim. Biophys. Acta 1994, 1196, 227-230. (10) Striebel, C.; Brecht, A.; Gauglitz, G. Biosens. Bioelectron. 1994, 9, 139146. (11) Naumann, R.; Jonczyk, A.; Kopp, R.; van Esch, J.; Ringsdorf, H.; Knoll, W.; Gra¨ber, P. Angew. Chem., Int. Ed. Engl. 1995, 34, 2056-2058. (12) Salamon, Z.; Hazzard, J. T.; Tollin, G. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6420-6423.

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influence the functional integrity of membrane receptor proteins, the choice of detergents and lipids is a critical step.13-15 In the present study, the nicotinic acetylcholine receptor (nAchR) is chosen as a representative example of a membrane receptor forming a ligand-gated ion channel. It is available in large quantities from electric organs of rays and is known to be structurally similar to the nAchR in the muscle and the brain of humans.16 This transmembrane protein occurs naturally in a dimeric form with a relative molecular mass of Mr ) 580 kDa. A functional monomer is formed by five homologous subunits of stoichiometry R2βγδ; each of the individual subunits is supposed to comprise four membrane-spanning segments.17,18 Acetylcholine is the natural agonist for the nAchR; once bound, the receptor opens a cation-selective ion channel within its transmembraneous portion. Each nAchR comprises two binding sites for acetylcholine located at clefts between each of the two R and a neighboring subunit; consequently the two binding sites are not identical and reveal different affinities for some ligands.19,20 The nAchR is an allosteric protein modulated by various ligands acting on either side of the membrane, as well as on the level of the lipid-protein interface.21 Upon prolonged binding of agonists, the receptor undergoes a structural transition; the channel closing is accompanied by an affinity increase for the ligand. This process is known as desensitization.22,23 Besides the number of drugs and therapeutic agents that target the nAchR, many different toxins interact highly specifically with the receptor. Examples include R-bungarotoxin (BgTx) of the banded krait Bungarus,24-27 R-najatoxin of the cobra Naja,24-27 and conotoxins produced by marine snail Conus.28 Because these neurotoxins have evolved to act with high specificity against nAchR, they have been extensively used for affinity purification of the nAchR29 and investigating the receptor.25-27,30-33 The present approach of immobilizing biotinylated bungarotoxin on a streptavidin-coated sensor surface has been chosen for the following reasons. Since SPR sensitivity is directly related to (13) Schu ¨ rholz, T.; Kehne, J.; Gieselmann, A.; Neumann, E. Biochemistry 1992, 31, 5067-5077. (14) Helenius, A.; Simons, K. Biochim. Biophys. Acta 1975, 415, 29-79. (15) Montal, M.; Labarca, P.; Fredkin, D. R.; Suarez-Isla, B. A. Biophys. J. 1984, 45, 165-174. (16) Taylor, P.; Abramson, S. N.; Johnson, D. A.; Valenzuela, C. F.; Herz, J. Ann. N. Y. Acad. Sci. 1991, 625, 568-587. (17) Devillers-Thie´ry, A.; Galzi, J. L.; Eisele´, J. L.; Bertrand, S.; Bertrand, D.; Changeux, J. P. J. Membr. Biol. 1993, 136, 97-112. (18) Hucho, F.; Tsetlin, V. I.; Machold, J. Eur. J. Biochem. 1996, 239, 539-557. (19) Conti-Tronconi, B. M.; Tang, F.; Walgrave, S.; Gallagher, W. Biochemistry 1990, 29, 1046-1054. (20) Rauer, B.; Neumann, E.; Widengren, J.; Rigler, R. Biophys. Chem. 1996, 58, 3-12. (21) Heidmann, T.; Changeux, J. P. Annu. Rev. Biochem. 1978, 47, 317-357. (22) Weiland, G.; Taylor, P. Mol. Pharmacol. 1978, 15, 197-212. (23) Ochoa, E. L. M.; Chattopadhyay, A.; McNamee, M. G. Cell. Mol. Neurobiol. 1989, 9, 141-178. (24) Lee, C. Y. Snake Venoms; Springer: Berlin, 1979. (25) Weber, M.; Changeux, J. P. Mol. Pharmacol. 1974, 10, 1-14. (26) Weber, M.; Changeux, J. P. Mol. Pharmacol. 1974, 10, 15-34. (27) Weber, M.; Changeux, J. P. Mol. Pharmacol. 1974, 10, 35-40. (28) McIntosh, M.; Cruz, L. J.; Hunkapiller, M. W.; Gray, W. R.; Olivera, B. M. Arch. Biochem. Biophys. 1982, 218, 335-341. (29) Ringler, P.; Kessler, P.; Me´nez, A.; Brisson, A. Biochim. Biophys. Acta 1997, 1324, 37-46. (30) Hucho, F. Angew. Chem., Int. Ed. Engl. 1995, 34, 39-50. (31) Rogers, K. R.; Eldefrawi, M. E.; Menking, D. E.; Thompson, R. G.; Valdes, J. J. Biosens. Bioelectron. 1991, 6, 507-516. (32) Sine, S.; Taylor, P. J. Biol. Chem. 1979, 254, 3315-3325. (33) Schmidt, J.; Raftery M. A. Anal. Biochem. 1973, 52, 349-354.

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the mass of the component binding to the surface, we immobilized the small ligand on the sensor surface in order to obtain an intrinsic signal amplification by the subsequent binding of the large nAchR dimer. Among several possible ligands, we selected R-bungarotoxin (Mr ) 8 kDa) as the surface-immobilized ligand. This 74 amino acid long polypeptide is known to retain its binding properties to nAchR upon immobilization.34,35 Here, biotinylated BgTx (biot-BgTx) is bound to streptavidin which is covalently immobilized on the sensor surface. In this approach, the surface concentration of BgTx can be easily controlled. Furthermore, because many different ligands can be biotinylated and directly compared, this method is of general interest for the investigation of receptor molecules. A prerequisite for investigating biomolecular interactions by the “label-free” SPR method is the efficient suppression of NSB to the sensor surface. In the case of detergent-solubilized nAchR, the presence of the detergent considerably reduces the NSB to surfaces. In the case of lipid vesicle-reconstituted receptor, we nearly completely suppressed the NSB by incorporating amphipatic poly(ethylene glycol)-lipids (PEG lipids) into the vesicles. The lipid moiety is embedded into the lipid bilayer thus anchoring the PEG chain in the membrane. The PEG polymer chains are thought to perform fluctuations between different conformations of the PEG chains in the solvent. Simulations demonstrate that the hydrophilic and flexible polymer can create a dense sterical and hydrophilic barrier,36-40 which in the present case should prevent direct contact between the vesicles and, for example, solid surfaces or proteins, thus leading to significantly reduced nonspecific binding of these so-called sterically stabilized vesicles (SSVs). EXPERIMENTAL SECTION Materials. Deionized water (specific conductivity 18 MΩ‚cm (Nanopure)) and solvents of UV quality were used. 16-Mercaptohexadecanoic acid, 11-mercaptoundecanol, and 1,2-dimyristoylsn-glycero-3-phosphatidic acid [poly(ethylene glycol)] ester (Mr PEG ) 750, DMPA-PEG750) were synthesized according to standard protocols.8,40 Streptavidin was purchased from Calbiochem (La Jolla, CA); biotinylated bungarotoxin (biot-BgTx) was from Molecular Probes (Leiden, The Netherlands); eserin, R-bungarotoxin, decamethonium bromide, conotoxin M1, and conotoxin SI were from Sigma (St. Louis, MO); carbamoylcholine was from Research Biochemicals International (Natick, MA); n-octyl-β-Dglucopyranoside (OG) was from Alexis (La¨ufelfingen, Switzerland); cholesterol (Chol), CHAPS, HEPES, acetylcholine, and EDTA were from Fluka (Buchs, Switzerland); 1,2-dioleoyl-snglycero-3-phosphatidylcholine (DOPC), 1,2-dioleoyl-sn-glycero-3phosphatidylglycerol (DOPG), 1-palmitoyl-2-oleoyl-sn-glycerophosphoethanolamine-N-[poly(ethyleneglycol)] (Mr PEG ) 2000 and 5000, POPE-PEG2000, POPE-PEG5000) were from Avanti Polar (34) Chen, L.; Martin, G. B.; Rechnitz, G. A. Anal. Chem. 1992, 64, 3018-3023. (35) Quinn, A.; Harrison, R.; Jehanli, A. M.; Lunt, G. G.; Walsh, S. J. Immunol. Methods 1988, 107, 197-203. (36) Lasic, D. D.; Papahadjopoulos, D. Science 1995, 267, 1275-1276. (37) Szoka, F.; Papahadjopoulos, D. Annu. Rev. Biophys. Bioeng. 1980, 9, 467508. (38) Du, H.; Chandaroy, P.; Hui, S. W. Biochim. Biophys. Acta 1997, 1326, 236248. (39) Allen, T. M.; Chonn, A. FEBS Lett. 1987, 223, 42-46. (40) Lasic, D. D., Martin, F., E. Stealth Liposomes; CRC Press: Boca Raton, 1995.

Lipids (Alabaster, AL), and Slide-a-Lyser was from Pierce (Rockford, IL). SPR sensor chips, HEPES-buffered saline (HBS) Nhydroxysuccinimide (NHS), N-ethyl-N′-dimethylaminopropylcarbodiimide (EDC), and ethanolamine were purchased from Biacore (Uppsala, Sweden). nAchR-enriched membranes of Torpedo california were prepared as described elsewhere.41 A specific antibody WF642 against the binding region of the nAchR was obtained from A. Maelicke (U. Mainz, Germany). Instrumentation. A commercial SPR instrument was used (Biacore 10004,43 (Uppsala, Sweden)); if not otherwise stated, all SPR experiments were carried out at a flow rate of 5 mL/min at 25 °C. Functionalization of Gold Surfaces. Self-assembly of 16mercaptohexadecanoic acid and 11-mercaptoundecanol was carried out on the bare gold surface of the SPR sensor chips J1 (Biacore) outside the instrument in an ethanol vapor-saturated chamber to avoid evaporation of the solvent. A solution containing 1 mM 16-mercaptohexadecanoic acid and 1.5 mM 11-mercaptoundecanol (mixture of 40 mL of stock solution 4 mM 16-mercaptohexadecanoic acid in 1:1 water-ethanol, 40 mL of stock solution 6 mM 11-mercaptoundecanol in ethanol, and 80 mL of ethanol) in 1:7 water-ethanol was applied to the sensing surface in 30-mL aliquots. The solution was renewed every 20 min, yielding a mixed self-assembled monolayer (SAM) after 80-100 min. After the SPR chip was inserted into the instrument, the SAM was functionalized by the following sequence: The sensor surface was flushed with HBS buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20, pH 7.4) with a flow rate of 10 mL/ min. Then 20 mL of 0.2 M EDC-0.05 M NHS solution was injected at a reduced flow rate of 5 mL/min, followed by 50 mL of 250 mg/mL streptavidin (in 1:3 water-HBS) and 35 mL of ethanolamine (1 M, pH 8.5) solution. Finally, BgTx was bound to this surface by flushing 2 min with a solution of 50 mg/mL biotBgTx in HBS. Solubilization of the nAchR. nAchR-enriched Torpedo membranes were shaken for 10-15 h at 4 °C with a 9-fold excess of 55 mM OG, 250 mM NaCl, and 20 mM NaPi, pH 7.4 (OG well above the critical micellar concentration, cmc ∼25 mM).44 The solubilized membranes were centrifuged 30 min at 100000g; the supernatant was carefully removed and stored for up to 2 days at 4 °C. The concentrations of the receptor preparations were determined by measuring the optical density at 280 nm using an extinction coefficient of 450 000 M-1 cm-1 for the receptor monomer as estimated from the protein amino acid sequence. Typical receptor concentrations were ∼500 nM, assuming that the solubilized protein was exclusively nAchR. Since an OG micelle is composed of ∼80 individual molecules,45,46 1 out of 2000 detergent micelles contained a receptor molecule. (41) Hertling-Jaweed, S.; Bandini, G.; Hucho, F. In Receptor Biochemistry. A Practical Approach; Hulme, E. C., Ed.; IRL Press: Oxford, 1990; pp 163176. (42) Schro¨der B.; Reinhardt-Maelicke S.; Schrattenholz A.; McLane K. E.; Kretschmer A.; Conti-Tronconi B. M.; Maelicke A. J. Biol. Chem. 1994, 269, 10407-10416. (43) Raether, H. In Physics of Thin Films; Hass, G., Francombe, M. H., Hoffmann, R. W., Eds.; Academic Press: New York, 1977; Vol. 9, pp 145-261. (44) Gonzales-Ros, J. M.; Paraschos, A.; Farach M. C.; Martinez-Carrion, M. Biochim. Biophys. Acta 1981, 643, 407-420. (45) Gould, R. J.; Ginsberg, B. H.; Spector, A. A. Biochemistry 1981, 20, 67766781. (46) Camm, E. L.; Green, B. R. Arch. Biochem. Biophys. 1982, 214, 563-572.

Reconstitution of nAchR into Lipid Vesicles. The lipids dissolved in chloroform were mixed in the desired molar composition (typically 80% DOPC, 10% Chol, and 10% DOPG). The solvent was evaporated by a gentle flow of nitrogen followed by vacuumdrying for 1.5 h. The dried lipid film was solubilized by adding 480 µL of buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, pH 7.4) and 270 µL of 10% (w/w) CHAPS, followed by ultrasonication (tip sonifier, 5 s, room temperature) with a final lipid concentration of 2 mg/mL. A 250-µL aliquot of enriched receptor membranes (1.9 mg/mL receptor) was added, the suspension was gently shaken for 2 h and subsequently centrifuged for 30 min at 100000g to remove nonsolubilized material. Lipid vesicles were formed by dialyzing the supernatant against a 650-fold volume of buffer comprising 500 mM NaCl, 10 mM HEPES, 3 mM EDTA, and 1 mM NaN3, pH 7.4 in a Slide-a-Lyser, first for 30 min at room temperature, then 8-15 h, and finally 2-4 h, exchanging the dialyzing buffer at each step (modified method of Schu¨rholz et al.13). The dialyzed vesicles were stored for up to 2 days. Prior to SPR measurements, the vesicles have been centrifuged for another 30 min at 100000g to remove receptor aggregates originating from nonreconstituted receptors. If not otherwise stated, all steps were performed at 4 °C. The concentrations of the receptor incorporated into these vesicles were determined by measuring the optical density at 280 nm ( ) 450 000 M-1 cm-1) in the presence of a high excess of CHAPS to solubilize the vesicles and to reduce light scattering. Typical concentrations were 350 nM, assuming that the optical density of the sample was exclusively caused by nAchR. The vesicles showed a narrow size distribution of 18-20-nm diameter as determined by quasi-elastic light scattering using standard procedures. For typical surface areas of 0.6 nm2 per lipid molecule, one estimates that each vesicle contains about 4000 lipid and 0.5 receptor molecules on average. Determination of Dissociation Constants of nAchR. A competitive ligand-binding assay was used to determine the dissociation constants of the receptor complex with carbamoylcholine and decamethonium bromide. Both compounds compete with BgTx for the same binding site on the nAchR.22,24 Therefore a fixed amount of either detergent-solubilized or vesicle-reconstituted receptor was preincubated with a particular ligand, occupying a fraction of the available receptor ligand-binding sites. Subsequently purging this solution in a flow-through cell over the functionalized SPR chip leads to binding of the nAchR to the surface-bound BgTx in relation to the concentration of receptors comprising free (nonoccupied) ligand-binding sites. This concentration was determined by measuring the initial surface binding rate and comparing it to the kinetics of the surface binding of nAchR in the absence of competitor. This approach is valid if the reaction of receptor and surfacebound BgTx is mass transport controlled.47-50 Under our experimental conditions, the equilibrium between the receptor and the ligand in solution is practically not disturbed because less than 1% of the total number of solubilized receptor molecules within the volume of the flow cell is bound to the sensor surface. For a (47) Glaser, R. W. Anal. Biochem. 1993, 213, 152-161. (48) Karlsson, R.; Roos, H.; Fa¨gerstam, L.; Persson, B. Methods: A Companion to Methods in Enzymology 1994, 6, 99-100. (49) Eddowes, M. J. Biosensors 1987, 3, 1-15. (50) Sjo ¨lander, S.; Urbaniczky, C. Anal. Chem. 1991, 63, 2338-2345.

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detailed discussion of this problem we refer to Piehler et al.51 Experimental Conditions. In competition experiments, the solubilized nAchR (330 nM in OG buffer) was mixed with a defined amount of ligand, either carbamoylcholine or decamethonium bromide (10 nM-50 mM). The solution was incubated for 8-12 min at RT before injecting 30 mL onto a BgTxfunctionalized surface. For calibration, pure receptor solution (20500 nM) was applied to a functionalized sensor surface. nAchR in SSVs was incubated for 10 min with solutions of 10-7-10-2 M carbamoylcholine before measurement on a lipidpresaturated BgTx surface. Calibration has been performed using receptor concentrations of 75-450 nM. Evaluation of the Binding. The kinetics of receptor binding to the sensor surface was analyzed according to a previously described method.51-54 We assume for the ligand concentrations used only one relevant ligand-binding site per receptor monomer, similarly to the case of acetylcholine binding to the receptor in solution.20 Since the receptor naturally occurs as a dimer, we have to consider two independent ligand-binding sites per receptor dimer. At the surface we regard the following reaction scheme: ka

When a ligand L reacts with a binding site R of the receptor in solution the equilibrium equation is

KD ) cRcL/cRL where KD is the equilibrium dissociation constant and cR, cL, and cRL are the equilibrium concentrations of the respective species. At equilibrium the concentration of free receptor binding sites (cR) is given by

cR )

c0R - c0L - KD + 2

cRRLL ) c2RL/2c0R

d

Here the nAchR dimer (indicated as RR) is assumed to initially interact with one molecule of BgTx on the sensor surface, forming a receptor-BgTx complex. That means, we assume identical association rates irrespective of whether the nAchR dimer has one or two free ligand-binding sites. ka is the rate constant of association and kd the rate constant of dissociation of the reaction. In a typical SPR experiment, the binding of the nAchR to the sensor surface is measured as a so-called SPR response signal I, which is directly proportional to the number of nAchR dimers bound to the surface. At the chosen experimental conditions, the nAchR solution flows above the sensor surface in a way that the surface reaction is mass transport controlled. The time course of the SPR signal I(t) reflects the nAchR binding to the sensor surface and can be described as

I(t) ) (r0/ks)(1 - e

)

(1)

Here the initial binding rate is r0 ) const ka[cRR + cRRL]. cRR indicates the equilibrium concentration of nAchR dimer with two free ligand-binding sites; cRRL is the equilibrium concentration of the nAchR dimer with one bound ligand. The constant term “const” in eq 1 relates the SPR signal of a particularly functionalized sensor surface to the surface concentration of the bound nAchR. ks ) ka[cRR + cRRL] + kd and t is the time in seconds, starting the association reaction at t ) 0. Fitting the experimental time course of the SPR signal by eq 1 yields r0 and using the calibration measurements [cRR + cRRL], the concentration of receptors with free ligand-binding sites in the bulk flow solution. (51) Piehler, J.; Brecht, A.; Giersch, T.; Hock, B.; Gauglitz, G. J. Immunol. Methods 1997, 201, 189-206. (52) Dunn, S. M. J.; Raftery, M. A. Biochemistry 1997, 36, 3846-3853. (53) Karlsson, R. Anal. Biochem. 1994, 221, 142-151. (54) Friguet, B.; Chafotte, A. F.; Djavadi-Ohaniance, L.; Goldberg, M. E. J. Immunol. Methods 1985, 77, 305-319.

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(2)

with c0L and c0R representing the total concentrations of ligand and receptor-binding sites. Only those receptors do not bind to the sensing surface where both ligand-binding sites of the dimer are blocked. We name this species RRLL and its equilibrium concentration cRRLL. If the two binding sites on the receptor dimer are independent,20 cRRLL can be calculated using a standard binomial distribution55 as

RR + BgTx y\ z RR-BgTx k

-ks(t-t0)

x

(c0R + c0L + KD)2 - c0Rc0L 4

(3)

The concentration of receptors that still can bind to the sensing surface is

c2RL c0R cRR + cRRL ) 2 2c0R

(4)

Taking into account that cRL ) cOR - cR, we obtain from eqs 2 and 4

cRR + cRRL )

[

c0R 1 c0R + c0L + KD 2 2cOR 2

x

]

2

(c0R + c0L + KD)2 - c0Rc0L 2c0R (5) 4

In competition experiments, the initial binding rates, which have been shown to depend linearly on the concentration of receptor dimers comprising free binding sites, are determined and related to a specific receptor concentration. These concentrations are fitted to eq 5 to obtain the equilibrium dissociation constant of the ligand-receptor interaction in solution. Nonspecific Binding of nAchR Vesicles. Four different nAchR vesicle solutions ((1-3) with and (4) without PEG-lipids) were prepared according to the described procedure. Solutions 1-3 were dialyzed at RT against the 650-fold volume of buffer comprising 10 mM HEPES, 3 mM EDTA, and 1 mM NaN3, pH 7.4. In solutions 1 and 2, the buffer additionally contained 500 mM NaCl, in (3) 500 mM NaCl and 1:10 cmc CHAPS. After 30 min, the buffers were renewed and the ionic strength of the dialysis buffer 1 was reduced from 500 to 300 mM NaCl. After 5 h, dialysis at 4 °C the buffers were exchanged again, the ionic (55) Stevens, F. J. Mol. Immunol. 1987, 24, 1055-1060.

Figure 2. Binding of streptavidin to activated (upper curve) and nonactivated (lower curve) mixed SAMs. Streptavidin is injected at (1); due to the lower refractive index of the solution a decrease in the SPR signal occurs. When switching back to the running buffer at (2), a jump appears in the opposite direction. Adsorption of streptavidin occurs between these two points; the overall binding is measured as the RU difference of buffer flowing across the surface before and after protein injection. The on-rate of protein adsorption is considerably increased upon activation of carboxy groups; the off-rate upon buffer wash is reduced. Figure 1. Schematic representation of the design of the sensor surface. A gold film is first covered with a mixed SAM, comprising 16-mercaptohexadecanoic acid and 11-mercaptoundecanol, to which streptavidin is then covalently coupled. Biot-BgTx is bound via the biotin tag to the streptavidin, resulting in a specifically funtionalized sensor surface for subsequent binding of nAchR. The receptor is either applied in detergent-solubilized (1) or vesicle-reconstituted form (2).

strength of dialysis buffer 1 was further reduced to 150 mM, and dialysis was completed for 10 h at 4 °C. To reduce NSB of lipids, the sensor surface was pretreated with a lipid mixture with a molar composition of 70% DOPG, 10% DOPG, 10% Chol, 3% DMPA-PEG750, 3% POPE-PEG2000, and 3% POPE-PEG5000 in a buffer of 150 mM NaCl, 10 mM HEPES, 3 mM EDTA, and 1 mM NaN3, pH 7.4. Each of the nAchR vesicle solutions was blocked with a high excess of BgTx and the NSB was measured on a BgTx-modified surface in the corresponding dialysis buffer. Subsequently the nonblocked vesicles (without addition of BgTx) were used, yielding nonspecific and total binding. Functionality of the Lipid Vesicle-Reconstituted nAchR. Stock solutions of SSVs were diluted 1:10 with a 0.1 M solution of carbamoylcholine or decamethonium bromide and incubated for 10 min at RT before measurement. To test the binding of acetylcholine, the receptor was first incubated for 15 min with 10-4 M eserin41 followed by a 10-min incubation with 10-2 M acetylcholine. RESULTS AND DISCUSSION Surface Assembly. Figure 1 depicts schematically the design of the sensing surface. The bare gold surface was covered with a protecting SAM comprising a mixture of 11-mercaptoundecanol and 16-mercaptohexadecanoic acid. The 16-mercaptohexadecanoic acid is required for further functionalization; 11-mercaptoundecanol serves to reduce the surface concentration of the longer 16-mercaptohexadecanoic acid enabling a better surface accessibility to the carboxy groups. By this approach, the number of

possible binding sites on the surface is controlled by changing the ratio of the two self-assembling components.56 The carboxy groups of the SAM were activated with EDCNHS yielding activated esters,57,58 which were subsequently reacted with the free amino groups of streptavidin. In Figure 2 we compare the adsorption properties of streptavidin on (EDCNHS) activated and nonactivated SAMs. From the pronounced faster binding to the activated surface and the stability of the formed layer against washing, we conclude that streptavidin was covalently immobilized on the sensor surface. For competition experiments, the amount of surface immobilized streptavidin typically corresponded to 2500 resonance units (RU). The streptavidin interlayer shields the nAchR from the mixed self-assembled monolayer on which nAchR has a comparatively high nonspecific binding (results not shown). The surfaces were then functionalized by binding biot-BgTx; the SPR response was ∼800 RU corresponding to a ratio of 2.4 BgTx molecules per streptavidin molecule. This value seems reasonable considering the cuboidal shape of streptavidin with two times two opposing binding sites. Finally, the binding of detergent-solubilized or vesicle-reconstituted nAchR was measured on these functionalized sensor surfaces. For measuring nAchR reconstituted in lipid vesicles, our functionalized planar surface is advantageous compared to many other sensing surfaces published so far. Very often traditional sensor surfaces comprise a dense polymer network of typically 100-200nm thickness,58 forming a three-dimensional sensing interface. Vesicles with a dimension of 20 nm (plus the bulky PEG chains) might be hindered from entering the network and approaching the most sensitive area43 near the gold surface. In our case, the (56) Keller, T. A.; Duschl, C.; Kro¨ger, D.; Se´vin-Landais, A. F.; Cervigni, S. E.; Dumy, P.; Vogel, H. Supramol. Sci. 1995, 2, 155-160. (57) Hermanson, G. T., Mallia, A. K., Smith, P. K., Eds. Immobilized Affinity Ligand Techniques; Academic Press: San Diego 1992. (58) Liedberg, B.; Johansen, K. In Affinity Biosensors: Techniques and Protocols; Rogers, K. R., Mulchadani, A., Eds.; Methods in Biotechnology Vol. 7; Humana Press Inc.: New York, 1998.

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vesicles can bind without steric hindrance to the functionalized surface at distances where SPR has the highest sensitivity. Specificity of the nAchR-Ligand Interaction. To probe the contrast between specific and nonspecific binding of the OGsolubilized nAchR to the functionalized sensor surface, two different tests have been performed. Figure 3 shows the binding on a streptavidin-modified surface before and after functionalization with biot-BgTx. The NSB on nonmodified streptavidin surfaces was negligible, whereas after functionalization, a drastic increase in the SPR signal was monitored indicating changed surface properties. To test whether this is due to a specific interaction, the nAchR was preincubated with BgTx in bulk solution to block the specific receptor binding sites prior to applying the receptor to the sensor surface. The blocked nAchR shows similarly low binding properties to BgTx surfaces as nAchR does to nonfunctionalized streptavidin surfaces. From this comparison, the ratio between specific and nonspecific interactions of nAchR to the BgTx sensor surface was determined to be 20:1. Functionality of the Detergent-Solubilized nAchR. Since BgTx is known to also bind proteolytic fragments of the nAchR R-subunit,59 the experiments presented so far do not fully demonstrate the functionality of the receptor. Additional ligands that are known to require the integrity of the receptor were therefore tested by competitive assays: A bulk solution of the nAchR was first incubated with a particular ligand and subsequently reacted with the functionalized sensor surface. If the ligand under investigation binds to the same binding site as BgTx, the binding of nAchR to the BgTx sensor surface is reduced, depending on the amount of ligand in the bulk. Here incubation has been performed with conotoxin M1, conotoxin SI, WF6 antibody, decamethonium bromide, or carbamoylcholine, which are all known to bind to the same binding site on the nAchR. In all cases, binding of the nAchR to the functionalized sensor surface was reduced, indicating that the solubilized receptor retained its agonist and antagonist binding activity (results not shown).

Displacement of Surface-Bound nAchR. After binding nAchR to a BgTx-modified surface, high amounts of competing ligand were added to the washing buffer to induce receptor displacement. Even with 0.1 or 0.5 M solutions of carbamoylcholine or decamethonium bromide, no significant desorption could be monitored. This result is in good agreement with published data stating that the complex between nAchR and BgTx shows extremely low dissociation constants (KD ) 10-11 M30) with low rates of dissociation; sometimes, its binding is referred to as irreversible. Weber and Changeux25 reported an exchange of bound radiolabeled BgTx against BgTx with a half-time of 55 h. Moreover, due to the high surface concentration of BgTx, nAchR might bind to two toxin molecules,41 leading to an actual irreversible binding. Determination of Dissociation Constants to nAchR. For the evaluation of the apparent dissociation constants, a bivalent interaction is assumed between nAchR dimers and the ligand in bulk solution. As discussed above, the surface reaction between the receptor and the immobilized BgTx has to be mass transport limited. The initial binding rates of nAchR in the absence of competing ligand should depend linearly on the concentration of injected nAchR. We find a straight line with a correlation coefficient of 0.98, a slope of 9.6 × 106 RU M-1 s-1 and an intercept of 0.52 RU s-1 for the solubilized and a straight line with a correlation coefficient of 0.99, a slope of 5.82 × 105 RU M-1 s-1 and an intercept of 0.022 RU s-1 for the lipid reconstituted receptor. Therefore, we conclude that the conditions used are appropriate and the evaluation is valid. Figure 4 shows the concentration influence of carbamoylcholine and decamethonium bromide on nAchR binding to a BgTxfunctionalized sensor surface. The equilibrium dissociation constants were obtained by fitting the initial binding rate to eq 5: A KD ) 3.5 × 10-6 M for carbamoylcholine and KD ) 4.5 × 10-5 M for decamethonium bromide was found. Comparison of Determined Dissociation Constants with Published Data. The nAchR is the most intensively studied ligand-gated ion channel. Many authors have published dissociation constants of different ligands under diverse conditions, e.g., in vivo and in vitro, in the active and desensitized state, in the presence and absence of divalent ions, and for detergent-solubilized and reconstituted or native receptor, studied by electrophysiology, ELISA, radioligand binding, or fluorescence or evanescent wave techniques. Under these different conditions and techniques, KD values have been measured spanning several orders of magnitude. For instance, the KD values for decamethonium bromide range from 1.7 × 10-4 M 60 to 1.8 × 10-8 M 61 and those for carbamoylcholine from 4.5 × 10-3 M 31 to 5 × 10-7 M.22 To test the relevance of a particular bioanalytical assay it is mandatory to compare the obtained data with those determined under physiological conditions. Kasai and Changeux62 measured the affinities of both ligands and found them to be nearly identical for in vivo (measuring steady-state membrane potentials at a single isolated electroplax) and in vitro (22Na+ flux measurements on microsacs) systems. They reported in both cases a KD of 1.2 × 10-6 M for decamethonium bromide and a KD of (2.6-3.0) × 10-5 M for carbamoylcholine in vivo and a KD of (3.3-3) × 10-5 M in

(59) Wilson, P. T.; Gershoni, J. M.; Hawrot, E.; Lentz, T. L. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 2553-2557.

(60) Mochly-Rosen, D.; Fuchs, S. Biochemistry 1981, 20, 5920-5924. (61) Gu, Y.; Lee, H.; Hudson, R. A. J. Med. Chem. 1994, 37, 417-420. (62) Kasai, M.; Changeux, J. P. J. Membr. Biol. 1971, 6, 1-23.

Figure 3. Surface binding of nAchR before and after functionalization with biot-BgTx. At (1), OG-solubilized receptor is applied to a streptavidin-modified surface, leading to nonspecific binding. Upon washing with buffer (2), nearly no nonspecifically adsorbed receptor is left. At (3), biot-BgTx is bound to the sensor surface; subsequent injection of nAchR (4) induces a pronounced increase in the SPR response, which remained after washing (last negative jump).

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a

Table 1. Equilibrium Dissociation Constants for Decamethonium Bromide and Carbamoylcholine authors Franklin and Pottera,66 Weber and Changeuxb,26 c Kasai and Changeuxd,62 O’Brien et al.e,67 Weiland and Taylorf,22

b

Figure 4. Binding of detergent-solubilized nAchR to a BgTxmodified surface after preincubation with the indicated concentrations of either carbamoylcholine (a) or decamethonium bromide (b). Points are experimental data, the continuous lines correspond to particular fits according to eq 5.

vitro. These data have been measured without competing toxin. Weber and Changeux26 reported for membrane-bound nAchR preincubated with either decamethonium bromide or carbamoylcholine with subsequent binding of R-najatoxin a KD of 0.8 × 10-6 M for decamethonium bromide and a KD of 4 × 10-5 M for carbamoylcholine. These nearly identical results show that competition ligand-binding assays deliver reasonable KD values. The dissociation constants for carbamoylcholine measured in this paper (KD ) 3.5 × 10-6 M) and for decamethonium bromide (KD ) 4.5 × 10-5 M) are in good agreement with the constants measured in vivo, at natural or homogenized membranes (Table 1). The deviations might not be surprising since our assay has some notable differences from the published ones (to our knowledge, no completely comparable data are available). We worked with solubilized receptor, a procedure that is reported to alter the binding properties of ligands.19,63 A loss of interconversion of affinity states induced by agonists upon dissolution in neutral detergents or cholate has been described,64 but no complete characterization of the effect of OG upon nAchR solubilization has been published yet. Therefore, some important aspects or possible influences are not clear, especially the influence of the solubilization with OG on the low- and high-affinity binding site during desensitization of the receptor or the interconversion between (63) Chang, H. W.; Bock, E. Biochemistry 1979, 18, 172-179. (64) Sugiyama, H.; Popot, J. L.; Changeux, J. P. J. Mol. Biol. 1976, 106, 485496.

carbamoylcholine KD (M) 5 × 10-6 5 × 10-7 4 × 10-5 (3.3-5) × 10-5 7 × 10-6

decamethonium bromide KD (M) 8 × 10-7 0.8 × 10-6 1.2 × 10-6 0.7 × 10-7-2 × 10-5

a Protection constant66 on Torpedo membrane fragments. b Protection constants against 3H acetylcholine measured on Torpedo membrane fragments. c Protection constants against 3H BgTx measured on Electrophorus membrane fragments at 22 °C. d Efflux of 22Na+ from microsacs at 22 °C. e Protection constants on Torpedo membrane fragments, depending on the receptor concentration. f Protection constant against [3H]BgTx measured on Torpedo membrane fragments.

them. In addition, it is not clear how the two ligands interact with the solubilized receptor. They do not necessarily have to behave identically, since carbamoylcholine and decamethonium bromide are known to have different properties: decamethonium bromide acts as an agonist (like carbamoylcholine) but in some aspects as a partial antagonist.22 In competition experiments against acetylcholine, carbamoylcholine shows one and decamethonium bromide two dissociation constants.65 This difference in behavior might be stressed upon OG solubilization and account for the difference in dissociation constants compared to literature values. Nonspecific Binding of nAchR Vesicles. The nAchR has been reconstituted into lipid vesicles in order to study its ligandbinding activity in a membrane environment. A lipid mixture (80% DOPC, 10% Chol, 10% DOPG) was used for reconstitution by dialysis. To be able to investigate the nAchR-ligand interaction, the NSB of the vesicle-reconstituted receptor to the BgTxmodified surface had to be minimized. Several parameters have been tested including lipid composition, charge of either the vesicle or the sensor surface, presence of detergents, and ionic strength of the buffer. Although all of these factors are suited to reduce the NSB, it still remained too high in our experiments. In a further attempt, PEG lipids, which are reported to reduce surface adsorption of proteins,38 were incorporated into the vesicles. The polymer forms a steric barrier outside the membrane, strongly reducing the NSB of the vesicles. PEG lipids with different polymer chain lengths (MT PEG ) 750, 2000, and 5000 Da) and different concentrations in the vesicles have been tested (Figure 5). According to theory,40 increasing the polymer chain length and concentration reduces the NSB. Unfortunately we found that the nAchR, reconstituted in these vesicles, showed reduced initial rates for specific binding to the BgTx surface. For further optimization, a lipid composition resulting in high specific nAchRBgTx interaction compared to nonspecific binding was chosen (1.75% POPE-PEG2000). Table 2 summarizes the results of some representative experiments performed to find optimal conditions to reduce the NSB of these SSVs to the sensor surface. It turned out that a major part of the unspecific binding of lipid vesicles was suppressed by increasing the ionic strength of the bulk buffer (65) Neubig, R. R.; Cohen, J. B. Biochemistry 1979, 18, 5464-5475. (66) Franklin, G. I.; Potter, L. T. FEBS Lett. 1972, 28, 101-106. (67) O’Brien, R. D.; Gilmour, L. P.; Eldefrawi, M. E. Proc. Natl. Acad. Sci. U.S.A. 1970, 65, 438-445.

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Figure 5. Total and nonspecific binding of nAchR in SSVs at different concentrations (mole %) and chain lengths of the PEG lipids to BgTx-modified surfaces. The filled symbols give the signals of nonblocked nAchR for DMPA-PEG750 ([), POPE-PEG2000 (b), and POPE-PEG5000 (9). The corresponding open symbols give the signals for BgTx-blocked nAchR SSVs. Table 2. NSB of NAchR Reconstituted in Lipid Vesicles type of vesicles, buffer, surface treatment standard vesicles, 500 mM NaCl SSVs, 150 mM NaCl SSVs, 500 mM NaCl SSVs, 500 mM NaCl, 1:10 cmc CHAPS SSVs, 500 mM NaCl, surface lipid-presaturated SSVs, 500 mM NaCl, 1:10 cmc CHAPS, surface lipid-presaturated

apparent response (RU) 395 135 100 70 40 40

Figure 6. Apparent binding of free (1) and blocked (2-4) nAchR in SSVs to BgTx-modified surfaces. The ligands used for blocking ligand receptor binding sites are acetylcholine (2), decamethonium bromide (3), and carbamoylcholine (4).

solution to 500 mM NaCl. Furthermore, by flushing the sensor surface with a lipid mixture prior to measurement, the NSB could almost completely be suppressed. Compared with the nAchR “standard vesicles,” we were able to reduce the NSB by 90%. Functionality of the Lipid Vesicle-Reconstituted nAchR. To test the functionality of the nAchR incorporated into SSVs, competition ligand-binding assays have been performed. A defined amount of nAchR in SSVs is incubated with an excess of carbamoylcholine, decamethonium bromide, or acetylcholine, respectively. Figure 6 shows the binding of nAchR-containing vesicles to the BgTx sensor surfaces. As the nAchR-binding affinity to the sensor surface is almost suppressed by the three ligands 3164 Analytical Chemistry, Vol. 71, No. 15, August 1, 1999

Figure 7. Binding of nAchR in SSVs to BgTx-modified surfaces after preincubation with indicated concentrations of carbamoylcholine. The continuous line corresponds to a fit according to eq 5.

tested, we conclude that the agonist and antagonist binding affinity is conserved. A KD ) 1.4 × 10-5 M is calculated for carbamoylcholinereceptor interaction fitting the initial binding to eq 5 (Figure 7). Taking the differences in preparation and evaluation into account, the results are in reasonable agreement with the KD values of the detergent-solubilized receptor determined in this paper (3.5 × 10-6 M) and in the literature. The initial binding rate is significantly reduced, but the general binding properties are not affected. We conclude that the PEG chains interfere with the receptor only by steric shielding. From simple geometric considerations, this is consistent with the size of the receptor and the PEG. Typically, PEG chains in the fully extended conformation have a length of 0.5 nm/monomer,40 giving lengths from 8 to 55 nm for the polymers used; the extracellular domain of the receptor molecule extends 6-7 nm from the surface of the lipid bilayer.16 Thus, the PEG could form a steric barrier that completely covers the receptor binding sites. Due to the high conformational flexibility of the polymer, the receptor binding sites are on time average still accessible for ligand binding, though to a lower extent than in the case without PEG lipids; this accounts for the reduced initial binding rates. CONCLUSIONS We have presented a competition assay to study the interaction of ligands with their membrane receptor by a label-free optical technique. Two different approaches have been successfully realized by using either detergent-solubilized or lipid vesiclereconstituted receptors. Further we have developed a new method for reducing NSB in membrane receptor assays. The specific binding properties of the receptor molecules are fully preserved and nonspecific binding is strongly reduced. This methodology could be applied to other membrane receptors and is not limited to the SPR transducer described here. It is easily possible and highly attractive to transfer this approach to other types of sensing surfaces. With respect to sensitivity, the measurement of receptor molecules reconstituted into lipid vesicles offers an additional advantage in evanescent wave sensing devices compared to detergent solubilization. Upon binding of the same number of receptor molecules to the sensing surface, the mass increase of receptor vesicles is much higher than that of detergent-solubilized receptors, consequently the sensitivity is enhanced.

Glossary. BgTx, R-bungarotoxin; biot-BgTx, biotinylated R-bungarotoxin; Chol, cholesterol; cmc, critical micellar concentration; DMPA-PEG750, 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid [poly(ethylene glycol)] ester (Mr PEG ) 750); DOPC, 1,2-dioleoyl-snglycero-3-phosphatidylcholine; DOPG, 1,2-dioleoyl-sn-glycero-3phosphatidylglycerol; EDC, N-ethyl-N′-dimethylaminopropylcarbodiimide; HBS, HEPES-buffered saline; nAchR, nicotinic acetylcholine receptor; NHS, N-hydroxysuccinimide; NSB, nonspecific binding; OG, n-octyl-β-D-glucopyranoside; PEG, poly(ethylene glycol); POPE-PEG2000/5000, 1-palmitoyl-2-oleoyl-sn-glycerophosphoethanolamine-N-[poly(ethylene glycol)] (Mr PEG ) 2000/ 5000); RU, resonance units; SAM, self-assembled monolayer; SPR, surface plasmon resonance; SSVs, sterically stabilized vesicles.

ACKNOWLEDGMENT We thank Prof. Alfred Maelicke for the anti nAchR antibody WF6. We are especially grateful to Ruud Hovius, Andreas Brecht, and Peter Ulrich for their extensive help and to Andreas Heusler for synthesising the 16-mercaptohexadecanoic acid, the 11mercaptoundecanol, and the 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid [poly(ethyleneglycol)] ester. This work was supported by the Swiss National Science Foundation Priority Program on Biotechnology, Grant 5002-045017 (H.V.) Received for review December 29, 1998. Accepted March 11, 1999. AC9814391

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