Anal. Chem. 1998, 70, 1331-1338
Screening Ligands for Membrane Protein Receptors by Total Internal Reflection Fluorescence: The 5-HT3 Serotonin Receptor Evelyne L. Schmid, Ana-Paula Tairi, Ruud Hovius, and Horst Vogel*
Laboratoire de Chimie Physique des Polyme` res et Membranes, Ecole Polytechnique Fe´ de´ rale de Lausanne, CH-1015 Lausanne, Switzerland
The screening of ligands for membrane receptor proteins is central to the discovery of new pharmaceutical drugs. We present a general method to reversibly attach receptor proteins via an affinity tag to a quartz surface and subsequently detect with high sensitivity the real-time binding of ligands by total internal reflection fluorescence. A serotonin-gated ion channel protein was immobilized, and the binding of a fluorescent ligand was investigated. The affinity and the kinetic parameters of binding were measured, and the effect of unlabeled compounds was determined by competition. The pharmacology of the immobilized receptor was identical to that of the native receptor. The affinity of unlabeled ligands was rapidly and effectively determined. The method described here is generally applicable for membrane proteins and opens new ways for the discovery of pharmacologically active compounds. The development of sensitive and rapid ligand-binding assays for receptors, which are targets for pharmaceutical effectors, is central to the screening and discovery of new drugs. In this context, surface-sensitive optical techniques have become increasingly important. The advantage of these techniques is that they provide real-time binding data. If one of the reacting partners is immobilized on the surface, the subsequent binding of the complementary analyte is detected by a change in the optical properties at the sensor surface, without any additional separation step. Among these techniques, total internal reflection fluorescence (TIRF) offers the combined advantages of surface specificity and high sensitivity of fluorescence. In addition, TIRF is suitable for microfluidic applications.1,2 Membrane proteins are at the center of communication between cells and, as such, play an important role in signal transduction processes. Any ligand that binds to and changes the properties of signal-regulating proteins, such as ion channels or G-protein-coupled receptors, is a potential drug. The serotoningated ion channel receptor is present in the central and peripheral nervous system, and dysfunctions in its regulation have been associated with mood and appetite disorders. Ligands that bind (1) Plowman, T. E.; Reichert, W. M.; Peters, C. R.; Wang, H. K.; Christensen, D. A.; Herron, J. N. Biosens. Bioelectron. 1996, 11, 149-160. (2) Proceedings of the 2nd International Symposium on Miniaturized Total Analysis Systems; Widmer, H. M, Verpoorte, E., Barnard S., Eds.; AMI: Basel, 1996. S0003-2700(97)01265-1 CCC: $15.00 Published on Web 02/28/1998
© 1998 American Chemical Society
to this receptor have use as anti-emetic drugs during cancer treatment and cures for depression.3 The attachment of functional membrane proteins to solid supports for ligand-binding assays is a major challenge. Simple receptors containing a single transmembrane segment have been successfully immobilized on solid supports by the fusion of vesicles containing the receptor for subsequent ligand-binding studies.4-6 More complex receptors, i.e., comprised of different subunits, each spanning the membrane several times, are proving more difficult to immobilize in a functional state. The most extensively studied membrane protein in this context is the nicotinic acetylcholine receptor, due to its availability from the electric organs of Torpedo. Different approaches have been applied; physisorption,7-9 where the protein is adsorbed to the surface at low pH without any detergent or lipid present, has given promising results. The pharmacology of the immobilized receptor is similar to that of native receptor, except for the far too low affinity of agonists. It was suggested that the immobilized receptor cannot undergo the necessary conformational changes. Other approaches involve the fusion of membranes containing the receptor with the solid substrate10,11 and of inclusion of the receptor in a polymer matrix.12 While these approaches lead to the subsequent binding of bungarotoxin, none have shown a pharmacology corresponding to functional receptor immobilization. We have previously demonstrated a generally applicable method for the functional immobilization of proteins on oxide surfaces using an affinity-tag approach.13 Combining this method with TIRF, we now create a real-time ligand-binding assay for the (3) Hamon, M. Central and peripheral 5-HT3 receptors; Academic Press: London, 1992. (4) Sui, S.; Urumow, T.; Sackmann, E. Biochemistry 1988, 27, 7463-7469. (5) Poglitsch, C. L.; Sumner, M. T.; L.Thompson, N. Biochemistry 1991, 30, 6662-6671. (6) Hsieh, H. V.; Thompson, N. L. Biochemistry 1995, 34, 12481-12488. (7) Rogers, K. R.; Valdes, J. J.; Eldefrawi, M. E. Anal. Biochem. 1989, 182, 353-359. (8) Rogers, K. R.; Valdes, J. J.; Eldefrawi, M. E. Biosens. Bioelectron. 1991, 6, 1-8. (9) Rogers, K. R.; Eldefrawi, M. E.; Menking, D. E.; Thompson, R. G.; Valdes, J. J. Biosens. Bioelectron. 1991, 6, 507-516. (10) Downer, N. W.; Li, J.; Penniman, E. M.; DeLuca, L. W.; Smith, H. G. Biosens. Bioelectron. 1992, 7, 429-440. (11) Puu, G.; Gustafson, I.; Artursson, E.; Ohlsson, P. Biosens. Bioelectron. 1995, 10, 463-476. (12) Taylor, R. F.; Marenchic, I. G.; Cook, E. J. Anal. Chim. Acta 1988, 213, 131-138.
Analytical Chemistry, Vol. 70, No. 7, April 1, 1998 1331
Figure 1. Schematic representation of the binding of the serotonin receptor to the quartz glass surface. The chelator is covalently immobilized to the quartz surface and loaded with Ni2+, with which the His tag of the receptor interacts. TIRF creates an evanescent light wave at the surface, the intensity (I) of which decays exponentially with the distance (d) from the surface (indicated by the varying intensity of the background in the figure, as well as by the graph on the right-hand side. (Note, the drawing is not to scale; the receptor is 14 nm long, and the decay length of the evanescent field is about 100 nm.) The evanescent light wave makes TIRF a surface-sensitive technique, where only those fluorescent ligands which are located within the evanescent field, i.e., preferentially those bound to the surface-immobilized receptor, are excited (indicated by a bright circle). Ligands in the bulk buffer phase are not excited (indicated by a dark dot).
serotonin-gated ion channel membrane receptor 5-HT3R. Here, we show how to immobilize the membrane protein on an oxide surface for TIRF measurements (schematically outlined in Figure 1), that the protein retains its native pharmacology, and how drugs can be efficiently screened by this method. EXPERIMENTAL SECTION Chemicals. The fluorescent ligand GR-fluorescein (GR186741X, i.e., fluorescein-labeled GR119566X), ondansetron, and granisetron were obtained from Glaxo Wellcome (Geneva, Switzerland). The radioligand 3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)propanone ([3H]GR65630) was from NEN-DuPont (Boston, MA). Further receptor ligands were from RBI (Natick, MA) and Tocris Cookson (Langford, U.K.). Nickel(II) nitrilotriacetic acid (NiNTA) agarose was from Qiagen (Hilden, Germany). Aqueous solutions were made up in deionized water (Nanopure, >18 MΩ‚ cm). To avoid bubble formation in the cell during TIRF measurements, all buffers were degassed by sonication before use. All other products used were of the highest quality available and obtained from regular sources. 5-HT3R Expression and Purification. The expression and purification of the functional C-terminally histidine-tagged 5-HT3R are described elsewhere.14 In brief, the Semliki Forest virus system was used to express the homopentameric receptor to high (13) Schmid, E. L.; Keller, T. A.; Dienes, Z.; Vogel, H. Anal. Chem. 1997, 69, 1979-1985. (14) Hovius, R.; Tairi, A.-P.; Blasey, H.; Bernard, A.; Lundstro¨m, K.; Vogel, H. J. Neurochem. 1998, 70, 824-834.
1332 Analytical Chemistry, Vol. 70, No. 7, April 1, 1998
Figure 2. Elution profile of the 5-HT3R receptor. The His-tagged receptor was purified by IMAC, and the radioligand-binding activity of the recovered fractions was measured. After sample loading, the column was washed with 20 mM imidazole (a), and the receptor was eluted with 80 mM imidazole (b).
levels and on a large scale in mammalian cells. The membranes were solubilized with the detergent nonaethyleneglycol monododecyl ether (C12E9), and the receptor was purified in one step by immobilized metal ion affinity chromatography (IMAC). All manipulations were performed at 4 °C. The Ni-NTA agarose was equilibrated with 20 mM imidazole in 10 mM NaPi, 500 mM NaCl, and 0.4 mM C12E9, pH 7.4, and was incubated overnight with the solubilized receptor. After column packing, nonspecifically bound protein was removed by washing with buffer. The receptor elution was with 80 mM imidazole in the same buffer and was followed by measuring the absorption at 280 nm. Figure 2 shows the radioligand binding capacity of the collected fractions. Before further use of the eluted protein, imidazole was removed by gel filtration on a G-25 column (NAP10, Pharmacia, Uppsala, Sweden) equilibrated with 10 mM HEPES and 0.4 mM C12E9, pH 7.4. Purity was demonstrated by SDSPAGE.14 Purified receptor preparations were tested for ligandbinding activity by a radioactive ligand-binding assay and stored in aliquots at -80 °C until use. The receptor showed no loss of ligand-binding activity after thawing. TIRF on Modified Slides. The TIRF setup and NTA immobilization on quartz slides were as previously described.13 In brief, microscope slides were chemically modified by gas-phase silanization with (3-mercaptopropyl)trimethoxysilane. Thiol groups were then used to covalently immobilize a lysine derivative of nitrilotriacetic acid via a bifunctional maleimide-succinimide cross-linker. A modified quartz plate was mounted on a Teflon spacer joined to a glass window, creating a flow-through cell with a volume of either 1 mL or 30 µL. A three-way connection allowed punctual volume injections via a syringe or continuous buffer flow (5 mL/min) via a peristaltic pump (Minipuls 3, Gilson, Villierle-Bel, France). The excitation light beam (488-nm line of an argon ion laser at a power of 10 µW, 532-AP, Omnichrome, Chino, CA) was totally internally reflected at the quartz surface, creating a 2 × 103 µm2 evanescent field of approximately 100 nm in depth. Fluorescence excited by this field was collected through a water immersion objective (40×, 0.75 NA, Zeiss, Oberkochen, Germany), measured by a photomultiplier tube (R928, Hamamatsu, Bridgewater, NJ) in photon-counting mode run by a DM3000 system (SPEX, Middlesex, U.K.).
Binding of GR-Fluorescein to Receptor Immobilized on the Surface. Binding of 5-HT3R to the Surface. The NTA-modified quartz glass surface was loaded with Ni2+ by incubating for 5 min with a 50 mM NiCl2 solution, rinsing with 50 mL of water followed by 50 mL of the appropriate buffer.13 Subsequent surface immobilization of 5-HT3R was done from a 10-30 nM receptor solution for 90 min in high salt buffer (20 mM NaPi, 250 mM NaCl, 0.4 mM C12E9, pH 7.4). A high concentration of salt was used to minimize possible nonspecific electrostatic interactions between the protein and the surface. The cell was subsequently rinsed with 50 mL of high salt buffer. Determination of the Amount of Bound 5-HT3R. A 30 nM solution of receptor in detergent was incubated with the chelating quartz glass surface in high salt buffer. After 90 min, the cell was rinsed with 50 mL of buffer. The cell was rapidly emptied and immobilized receptor was released by incubation for 1 h with 1 mL of 500 mM imidazole in high salt buffer. This volume was recovered and, after removal of the imidazole by gel filtration on a G-25 column, the receptor content was determined by a radioligand-binding assay with [3H]GR65630. Binding of GR-Fluorescein to Immobilized 5-HT3R. Total ligandbinding (specific plus nonspecific) at increasing concentrations of GR-fluorescein to immobilized 5-HT3R was measured by TIRF in a low salt buffer (20 mM NaPi, 0.4 mM C12E9, pH 7.4) since a high salt concentration diminishes ligand-binding. Nonspecific binding of the fluorescent ligand was determined by binding identical concentrations of GR-fluorescein in the presence of an at least 100-fold excess of the nonfluorescent competitor quipazine. Competition Experiments. To study the pharmacology of the immobilized 5-HT3R, various competitors with different potencies were added to 3 nM GR-fluorescein before ligand-binding was measured. For each experiment, receptor was immobilized at the surface from a 10 nM receptor solution in detergent. Regeneration. The NTA quartz glass slides can be regenerated after protein immobilization. Rinsing with 500 mM imidazole removes all specifically bound receptor from the surface. Surfaces exposed to nonpurified receptor preparations (e.g., raw detergent extracts of cell membranes) or to samples in which nonspecific binding of protein is important are regenerated by adding 0.5% SDS to the imidazole buffer. The cell is then rinsed with water, and the surface is finally reactivated by Ni2+. Binding of GR-Fluorescein to Receptor in Solution. The affinity of GR-fluorescein for the solubilized 5-HT3R in solution was measured by fluorescence spectroscopy with a Fluorolog 2 fluorometer (SPEX). GR-fluorescein (2 nM in 10 mM HEPES, 0.4 mM C12E9, pH 7.4) was placed in a stirred cuvette (Hellma GmbH, Mu¨llheim, Germany) and was titrated with increasing concentrations of 5-HT3R. The sample was excited at 488 nm and emission collected at 520 nm. Excitation and emission bandpasses were 1 nm. The change in fluorescence of the ligand upon binding to the receptor was recorded until equilibrium was reached. To determine the affinity of unlabeled ligands, varying concentrations of these ligands were mixed with GR-fluorescein before the addition of the solubilized receptor. Radioligand Binding Assays. The amount of solubilized receptor was determined by radioligand-binding assays with the antagonist [3H]GR65630.14 The incubation was terminated by
Figure 3. Reversible binding of 5-HT3R to the chelating quartz glass surface. Receptor (30 nM) was first incubated with GR-fluorescein (5 nM), thus forming a 5-HT3R/GR-fluorescein complex (more than 99% of the GR-fluorescein is bound to receptor). This was then injected into the TIRF cell. The fluorescence increased (a), indicating that the ligand-receptor complex bound to the quartz glass surface (functionalized by NTA and charged with Ni2+). The protein was removed from the surface by rinsing with 500 mM imidazole (starting at dashed line at b).
rapid filtration through GF/B filters (Whatman, Springfield Mill, U.K.), presoaked for 15 min in 0.5% (w/v) polyethylenimine. Filters were transferred into scintillation vials, and scintillation liquid was added (Ultimagold, Packard, Meriden, CT). Radioactivity was measured in a Tri-Carb 2200CA liquid scintillation counter (Packard). Nonspecific binding was determined in the presence of 1 µM quipazine. The affinity of nonradioactive compounds was determined by inclusion of increasing concentrations of these substances in the standard binding assay. RESULTS AND DISCUSSION Binding of 5-HT3R to the Surface. To monitor the specific binding of the His-tagged receptor to the chelating surface, a 60 nM solution of 5-HT3R was first incubated with 10 nM GRfluorescein in low salt buffer (more than 99% of the ligand is then receptor-bound). After the ligand had bound to the receptor (20 min), the solution was diluted to yield 30 nM 5-HT3R in high salt buffer. The binding of the fluorescent complex to the surface was measured by TIRF. Figure 3 shows the time course of the immobilization of the ligand-protein complex to the quartz glass surface. After 30 min, 75% of the binding was achieved, and equilibrium was reached after 90 min. In this manner, the binding of the receptor to the surface could be followed. However, due to the change of buffer necessary to avoid electrostatic interactions with the charged surface, the affinity of the receptor for the ligand diminishes. Therefore, it is impossible to compare fluorescence signals obtained in these conditions with the following experiments. In addition, flowing buffer at this stage will gradually remove the ligand from the receptor, and receptor stability at the surface cannot be determined in this manner. However, a flow of 500 mM imidazole (in high salt buffer), which competes with the His tag for the coordination of the Ni2+ ions, very rapidly reduced the fluorescence to its initial level by effectively removing the protein from the surface. Thus, since Analytical Chemistry, Vol. 70, No. 7, April 1, 1998
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the chelating groups are covalently immobilized on the slide, a single surface can be used repetitively after treatment with imidazole, washing with water, and recharging with Ni2+. Binding of the ligand/receptor complex to the surface was reproducible for three cycles ((9%). Quantification of Surface-Bound 5-HT3R. Receptor was bound to the quartz glass surface and later detached by competition with imidazole. The amount of receptor recovered from the surface was determined by a radioactive ligand-binding assay, and quantities of 0.5 pmol ((0.1 pmol) of receptor were repeatedly retrieved in 1 mL of imidazole buffer. The reproducibility of the process demonstrated that the receptor remained tightly bound to the surface upon a flow of buffer and that imidazole was necessary to displace it specifically. In addition, it showed that receptor which had been immobilized could be recovered from the surface and still showed ligand-binding properties. The total surface accessible to the protein was 660 × 1012 nm2 (1-mL TIRF cell), and, from the amount of receptor recovered, the surface per binding site was determined to be 2.2 × 103 nm2. As the receptor can be described as a cylindrical protein 14 nm high with a diameter of 8 nm,15 the surface coverage by the receptor protein was on the order of 0.5%. When working with surface-immobilized receptor proteins, one needs a surface receptor density high enough to detect the desired molecular events, while taking care to keep it low enough to avoid steric hindrance. Additionally, high receptor coverage often leads to mass-transportlimited binding over much of the measurement, until the density of free receptors at the surface drops sufficiently. If kinetic parameters are to be measured, high receptor coverage must be avoided. As will be seen further, the surface coverage we obtain under our present experimental conditions fulfils both requirements. The affinity of the His-tagged 5-HT3R for the surface can be calculated by taking into account the number of chelating groups at the surface. In a previous publication,13 we reported a density of 0.27 molecules/nm2 of NTA on the quartz slide. Since 4.5 × 104 molecules/nm2 bind to the surface from a 30 nM solution of 5-HT3R, the Kd of the protein for the surface is around 18 µM. From these measurements at one concentration, the affinity of this His-tagged membrane protein for the surface appears to be approximately 50 times lower than that of the water-soluble Histagged Green Fluorescent Protein (GFP), which was investigated elsewhere.13 Independently of factors such as the presence of detergent in the buffer to solubilize the membrane protein, it is a well-known fact that His tags presented by different proteins exhibit varying affinities for the immobilized metal ions.16 Binding and Displacement of GR-Fluorescein to Immobilized 5-HT3R. Figure 4 shows a typical time course for the binding of GR-fluorescein to the receptor, which was immobilized to the quartz glass surface before 3 nM of the fluorescent ligand was injected into the cell and its binding followed by TIRF. At this concentration, equilibrium was reached after 90 min. To see if this binding was specific to the receptor or nonspecific, either to the quartz glass surface or to other sites on/around the receptor, a high concentration of nonfluorescent competitor (15) Boess, F. G.; Beroukhim, R.; Martin, I. L. J. Neurochem. 1995, 64, 14011405. (16) Schmidt, A. M.; Mu ¨ ller, H. N.; Skerra, A. Chem. Biol. 1996, 3, 645-653.
1334 Analytical Chemistry, Vol. 70, No. 7, April 1, 1998
Figure 4. Reversible binding of GR-fluorescein to immobilized 5-HT3R. Receptor was immobilized to the quartz glass surface from a 30 nM solution in high salt buffer. After washing the cell thoroughly, 3 nM GR-fluorescein in low salt buffer was injected (a). Binding equilibrium was reached after 90 min. Injecting a 10 µM concentration of the nonfluorescent competitor quipazine (b) led to a gradual displacement of the fluorescent ligand from the receptor, as indicated by the decrease of the fluorescence signal. From the remaining fluorescence, nonspecific binding of GR-fluorescein to the surface was estimated to be below 15% (relative to the saturation signal of (a)).
Figure 5. Binding of various concentrations of GR-fluoresein to immobilized 5-HT3R. Receptor was immobilized on the quartz glass surface, and the binding of increasing concentrations of GRfluorescein was measured. The lower concentrations used are shown: 3 ([), 30 (b), and 100 pM (9).
(10 µM quipazine) was injected. As quipazine competed with GRfluorescein,17 the latter was gradually displaced from the receptor and the measured fluorescence decreased (Figure 4b). The displacement of GR-fluorescein was slow, and, after 4 h, equilibrium still was not reached. However, it is already apparent that, at this concentration, the nonspecific binding of GR-fluorescein was lower than 15%. Affinity of GR-Fluorescein for the Immobilized 5-HT3R. Figure 5 shows typical time courses of the binding of increasing concentrations of GR-fluorescein to surface-immobilized receptor. Binding (total and nonspecific) was measured until equilibrium was reached, and Figure 6 shows the resulting specific binding (17) Tairi, A.; Hovius, R.; Vogel, H., manuscript in preparation.
Table 1. Pharmacology of 5-HT3R Immobilized on the Surface Compared with Receptor in Solution and in Native Membranesa pKd versus GR-fluorescein pKd versus pKd versus surface [3H]GR65630, [3H]zacopride, equil 15 min solution solution membranes serotonin* MDL72222 ondansetron mCPBG* granisetron
Figure 6. Affinity of GR-fluorescein for immobilized 5-HT3R. Specific binding of GR-fluorescein to the receptor is plotted as a function of the concentration of ligand applied. The Langmuir isotherm (continuous line) resulted in a Kd of 0.3 nM. Total and nonspecific binding were measured twice, the error bars show the resulting deviation.
of the ligand to the immobilized receptor (measured by fluorescence intensity amplitudes) as a function of injected ligand concentration. Considering a simple equilibrium between the receptor R and the ligand L of the type R + L / RL, the dissociation constant, Kd, of the ligand from the receptor was determined by fitting the data to a Langmuir isotherm, taking into account ligand depletion in the bulk.
RL* )
[
]
(Lt + Rt + Kd) - ((Lt + Rt + Kd)2 - 4RtLt)1/2 f (1) 2
RL* (counts s-1) is the measured fluorescence intensity from ligand bound to surface-immobilized receptor, Lt (M) is the total concentration of ligand injected, and Kd (M) is the dissociation constant between the ligand and receptor. In this expression, an additional parameter, f (counts s-1 M-1), is introduced to convert molar concentrations to counts per second. The amount of receptor immobilized at the surface, Rt, is normally expressed in molecules per unit area; however, to use eq 1, Rt must be expressed in molar concentrations and was thus related to the overhead bulk volume (1 mL). To fit the binding data to eq 1, Kd, Rt, and f were optimized with the Levenberg-Marquardt algorithm to minimize χ2. In this manner, the dissociation constant of GR-fluorescein for immobilized 5-HT3R was 0.3 nM (Table 1). The number of surface-immobilized receptors was obtained from fitting the data to eq 1, giving a formal total concentration of 0.55 nM in 1 mL (0.55 pmol). This is in agreement with the amount of protein recovered by imidazole from the surface. Therefore, all the receptors immobilized specifically by the His tag at the surface and recovered in solution with functional ligandbinding sites are regarded to be functional when immobilized at the surface. Additionally, the ligand-binding isotherm can serve to quantify directly the amount of receptor immobilized at the surface, without the need for an additional calibration procedure. It is interesting, at this stage, to determine the sensitivity of the method. The surface density of receptors has already been estimated (454 molecules/µm2), and, since the actual spot size is
6.4 7.4 8.2 8.5 9.0
7.8 8.5 8.8 8.8 9.1
7.3 ndb 8.3 7.7-9.0 8.7
7.3 ndb 8.8 8.3 9.4
6.4-6.5 7.4-7.7 8.1-8.2 8.5 8.6-9.0
a Affinity of various agonists (*) and antagonists for the 5-HT R 3 immobilized on the surface (at equilibrium and after 15 min) or in solution is determined by competition against GR-fluorescein or against the radioligand [3H]GR65630. Comparison is made with the affinity determined against [3H]zacopride in native membranes of the rat cerebral cortex.26,29 b Not determined.
2 × 103 µm2, there is 1.5 amol of immobilized receptor in the illuminated area. The affinity of the ligand for the receptor was measured (Kd ) 0.3 nM), and Figures 5 (total binding) and 6 (specific binding) show that binding of ligand from a 30 pM solution is detected easily. Therefore, the binding of as little as 0.2 amol of ligand can be readily measured. This is a great improvement compared to the maximum sensitivity of 25 fmol reported in the nicotinic acetylcholine receptor-based fluorosensor.7,18 Kinetics of GR-Fluorescein Binding. The kinetics of GRfluorescein binding to immobilized receptor were analyzed according to ref 13. In brief, the fluorescence intensity, I, resulting from the binding of increasing concentrations of GR-fluorescein (30 pM-1 nM) was measured as a function of time. As previously discussed,13 whereas the initial part of the process is limited by diffusion, the major part is limited by the binding reaction itself, allowing the determination of the kinetic parameters of the ligand-receptor interaction. For this part of the measurement, plotting (dI/dt)/I versus each concentration of GR-fluorescein applied (Figure 7) yielded a slope equal to the on-rate, kon, while the intercept gave the off-rate, koff. Thus, we obtained kon ) 8.6 × 105 M-1 s-1 ((2 × 105) and koff ) 7.5 × 10-4 s-1 ((1 × 10-4). The off-rate can, in addition, be determined directly by the competition experiment shown in Figure 4. Fitting the desorption curve with a single exponential results in a time constant of 3 × 10-4 s-1, which is coherent with the off-rate estimated in Figure 7. The curve is, however, better fitted with a double exponential (time constants 1.3 × 10-3 and 2 × 10-4 s-1), possibly indicating the presence of binding sites with different affinities, and pointing to a complex mechanism for receptor-ligand interactions.20-22 (18) The precision and reproducibility of the measurements can be improved, first by choosing appropriate sensor surfaces with reduced nonspecific ligand binding, and second by using an automated fluid-handling system connected to a flow-through cell. Work in progress, applying the recently described optical waveguide sensor19 to the present ligand-receptor system, confirmed the ligand-binding isotherms quantitatively. (19) Duveneck, G.; Pawlak, M.; Neuscha¨fer, D.; Budach, W.; Ehrat, M. SPIE Proc. 1996, 2928, 98-109. (20) Yakel, J. L.; Lagurtta, A.; Adelman, J. P.; North, R. A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5030-5033. (21) Hope, A. G.; Downie, D. L.; Sutherland, L.; Lambert, J. J.; Peters, J. A.; Burchell, B. Eur. J. Pharmacol. 1993, 245, 187-192.
Analytical Chemistry, Vol. 70, No. 7, April 1, 1998
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Figure 7. Kinetic parameters of GR-fluorescein binding to immobilized 5-HT3R. (dI/dt)/I is plotted versus the ligand concentration in solution. The on-rate is determined from the slope of the straight line fitted to the experimental points, the off-rate from the intercept.
Likewise, the rates obtained from Figure 7 must be compared to the measured affinity of the ligand for the receptor. Indeed, koff/kon should be identical to the Kd of Figure 6. The dissociation constant obtained from the kinetic parameters is 0.9 nM ((0.2), which is comparable to the one resulting from the Langmuir isotherm. Although the binding is originally limited by diffusion, most of the process is limited by the binding reaction itself. Therefore, increasing the amount of receptor bound to the surface would only create a longer mass-transport-limited regime. Imposing a continuous flow of ligand during the experiments would further reduce the diffusion limitation and allow quicker rate determinations. The presented results demonstrate the feasibility of determining kinetics of ligand-receptor interactions by our experimental approach. As discussed elsewhere,23,24 a detailed analysis of the measured binding and desorption kinetics should take into consideration that the ligand-receptor interaction at the sensor surface is influenced by mass transport effects of the ligand to and from this surface under the experimental conditions. This analysis is, however, only reasonable if kinetic data of improved precision are available. Measurements in this direction are in progress by using an automatic fluid handling system connected to a flow-through cell. Competition Experiments. The characterization of the binding properties of a single labeled ligand is sufficient to determine the affinity of unlabeled ligands by comparison. Figure 8 shows how increasing concentrations of the antagonist granisetron prevented binding of GR-fluorescein. Having deduced the concentration of competitor causing half-maximal binding of the fluorescent ligand (IC50), and knowing both the dissociation constant of the fluorescent ligand, Kd, and its total concentration, Lt, the dissociation constant, Kdc, of the competitor was determined using the Cheng-Prusoff equation.25 (22) Bonhaus, D. W.; Stefanich, E.; Loury, D. N.; Hsu, S. A. O.; Eglen, R. M.; Wong, E. H. F. J. Neurochem. 1995, 65, 104-110. (23) Duschl, C.; Se´vin-landais, A.-F.; Vogel, H. Biophys. J. 1996, 70, 1985-1995. (24) Shuck, P. Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 541-566. (25) Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099-3108.
1336 Analytical Chemistry, Vol. 70, No. 7, April 1, 1998
Figure 8. Inhibition dose-response curve for the competitor granisetron. Increasing concentrations of granisetron were mixed with 3 nM GR-fluorescein before addition to immobilized receptor. The fraction of binding relative to that in the absence of competitor is plotted as a function of granisetron concentration. Nonspecific binding has been subtracted. By comparison with the affinity of GRfluorescein, the IC50 allows the determination of the affinity of granisetron. The affinity of granisetron for immobilized receptor is identical to its affinity for wild-type receptor in detergent-solubilized membranes and to receptor in detergent solution.
Kdc )
[IC50] (1 + Lt/Kd)
(2)
The dissociation constant of granisetron from the immobilized receptor was 1.4 × 10-9 M (Table 1). This is in agreement with the affinity of this substance measured for native receptor in cortical membranes by radioactive binding assays.26 This is an important result indicating that the receptor showed the same affinity for this antagonist upon immobilization to the surface as in native membranes. In addition, using a single fluorescent ligand and low concentrations of immobilized receptor protein, our system is perfectly suited to accurately determine the affinity of unlabeled ligands by competition. To further characterize the pharmacology of the immobilized receptor, a series of agonists and antagonists were studied. In each case, a 100 nM concentration of a particular unlabeled competitor (C in eq 3) was incubated with 3 nM GR-fluorescein (Lt in eq 3), and the binding of the fluorescent ligand to the surface-immobilized receptor was measured by TIRF (Figure 9). From such a single experiment on each competitor, the dissociation constant, Kdc, was determined from the amount of binding measured at equilibrium in the presence of the competitor (I), compared to the binding without competitor (I0) using eq 3. The
1 + Lt/Kd I ) I0 1 + Lt/Kd + C/Kdc
(3)
results are summarized in Table 1. Comparison with the values found in the literature for native receptor in its original membrane environment show that the pharmacology of the receptor was identical between surface-bound receptor and receptor in its native membrane. (26) Bolanos, F. J.; Schechter, L. E.; Miquel, M. C.; Emerit, M. B.; Rumigny, J. F.; Hamon, M.; Gozlan, H. Biochem. Pharmacol. 1990, 40, 1541-1550.
Figure 9. Binding of various agonists (*) and antagonists to immobilized 5-HT3R. Different competitors (100 nM) were mixed with GR-fluorescein (3 nM), and the resulting binding to the surfaceimmobilized receptor was measured by TIRF. Key: no competitor (b), serotonin* (4), MDL72222 (2), ondansetron (9), mCPBG* (0), and granisetron ([).
Figure 10. Binding of GR-fluorescein to receptor immobilized from solubilized raw extract. A nonpurified extract containing 15 nM of receptor was incubated with the surface. After rinsing, 3 nM GRfluorescein was injected and left to bind (a). An excess of quipazine (10 µM) displaced the fraction of GR-fluorescein specifically bound to the receptor (b).
For the agonist serotonin, the binding measured in the presence of the competitor was close to the binding measured in absence of the competitor; this is due to its low affinity for the receptor. To obtain a more visible effect on the binding of the fluorescent ligand, a higher concentration of serotonin could be used. However, as far as determining affinities of the different competitors, this approach is highly valuable. For the other, more potent compounds, the absolute values of the measured Kdc were also in close agreement. Therefore, this approach is valuable in the screening of potential new drugs of unknown potencies using a single concentration of the compound. Once the high-affinity molecules are identified, a precise determination of their Kdc can be obtained by measuring a full doseresponse curve. Ligand Binding to the 5-HT3R in Solution. The affinity of GR-fluorescein for the solubilized receptor in solution Kd was 0.2 nM. This value was identical to the affinity determined at the surface between this ligand and the receptor. In addition, the pharmacology of solubilized receptor in solution, by radioactive binding assay and by competition against the fluorescent ligand, was in good agreement with those measured on immobilized receptor and on native receptor. Again, the receptor binding properties were not affected by the immobilization process. Response Time. To shorten the response time of such a screening procedure on the surface, the affinities of the different compounds were estimated after 15 min of incubation, well before equilibrium was reached (Figure 9). To do so, the binding measured after 15 min was used in eq 3 to determine Kdc(15 min) (Table 1). As expected, these values were less precise than the values at equilibrium. However, the order of potencies for the compounds was already correct; thus, an unknown compound could be compared to known drugs already after 15 min. In this experiment, 100 nM concentrations of all the compounds were used to show the full range of potencies measurable, but, when searching for a highly potent drug (such as granisetron), it is clear that a high inhibition must be obtained from low concentrations of the compound. In a sense, the desired Kdc
is known, and a concentration C in that range should be used, leading to highly effective screening between the compounds. Another way to shorten the measurement would be to avoid a new protein deposition step before each ligand/competitor binding measurement and to always use the same immobilized protein. However, the off-rate of ligand is so slow that it is impossible to rapidly remove all the fluorescent ligand from the receptor. If the same immobilized receptor was to be used again to determine the affinity of a competitor, it would be necessary to remove the GR-fluorescein solely with buffer (to avoid saturating it with quipazine). For that matter, having a novel fluorescent ligand with a faster off-rate would be a great improvement. Raw Extract. To explore the full potential of this novel ligandbinding assay for membrane receptor proteins, experiments with nonpurified receptor in solubilized raw cell extract were undertaken. Working with such a preparation dramatically reduces the amount of material necessary, eliminates the losses during purification, and both simplifies and quickens the procedure. Solubilized cell membranes containing 15 nM receptor were incubated with the chelating surface in the same way as was the purified preparation. Figure 10 shows the subsequent binding of 3 nM GR-fluorescein, followed by the displacement of the bound ligand by an excess of quipazine (10 µM). From the difference in intensity before and after the addition of quipazine, we can clearly distinguish between total and nonspecific binding. As discussed previously, having measured the binding in the absence of a competitor, the same immobilized receptor could be used to determine the affinity of unlabeled competitors if a fluorescent ligand with a fast off-rate was obtained. In another experiment, raw extract containing the receptor was freshly immobilized on the surface before each ligand-binding experiment. Contrary to immobilization from a pure protein sample, the reproducibility of the immobilization was not immediate. In fact, similarly to the IMAC procedure, we found that saturating the chelating groups at the surface with 20 mM imidazole before incubating the raw extract in a 5 mM imidazole solution dramatically increased the reproducibility of the binding measurement. To keep the lengthened procedure as short as Analytical Chemistry, Vol. 70, No. 7, April 1, 1998
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possible, ligand-binding was measured for no more than 33 min (2 × 103 s). In this manner, the total binding of 3 nM GRfluorescein was also clearly distinguishable from the nonspecific binding, while providing fresh receptor for each ligand-binding measurement. Another publication will address how this procedure can be optimized to allow for the quantitative determination of competitors. CONCLUSIONS We presented here a highly effective method for the investigation of ligand-receptor interactions. Because the receptor was immobilized on a quartz surface, the real-time binding of ligands could be measured by TIRF with a direct discrimination between bound and unbound ligands. No additional perturbing separation steps were necessary, such as is the case for classical radioligandbinding assays. In contrast to established surface-sensitive techniques such as SPR, in which the signal is proportional to the deposited mass,27 our fluorescence technique functions without the limitation in ligand size. Binding of molecules of any size can be detected, either directly by fluorescence, or by competition against a fluorescent ligand. In addition to real-time and mass-independent detection, the system has a very high sensitivity because the surface immobilization is combined with effective fluorescence detection. In this case, where fluorescein was used as the reporter group, the sensitivity was below 0.2 amol. The sensitivity of the system is such that the collected signal comes from a surface comprising no more than 1.6 amol of receptor. In other words, the 5-HT3 receptors expressed by one of the engineered mammalian cells (with a typical receptor expression of about 5 × 106 copies/cell) would be sufficient for ligand-binding assays. In parallel, we aim to produce a fluorescent ligand for the 5-HT3R with a faster off-rate. Assays could then include a single receptor immobilization step, followed by ligand-binding, removal by buffer, and finally incubation with the competitor of interest. This approach could open the way to optimized, quantitative measurements with nonpurified cell extract. Combining this novel method for ligand-receptor interaction measurement with the microfluidic systems would lead to a very efficient, high-throughput assay.2 The presence of a continuous flow would enhance reproducibility and allow ligand-binding kinetics to be studied in more detail. The overall speed of the (27) Keller, T. A.; Duschl, C.; Kro ¨ger, D.; Se´vin-Landais, A.-F.; Vogel, H.; Cervigni, S. E.; Dummy, P. Supramol. Sci. 1995, 2, 155-160.
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assay could be greatly augmented; in particular, a variation in the kinetics of ligand-binding could be used as a means of detecting the presence of competitors. The protein immobilization procedure presented here is applicable to all proteins. We have previously demonstrated this approach for the soluble His-tagged GFP. Here, a membrane receptor is immobilized in the same manner in a functionally active form. In principle, any His-tagged protein or macromolecule can now be immobilized on oxide surfaces for ligand-receptor, protein-protein, and DNA-protein interaction or DNA hybridization measurements. The flexibility of the His tag approach would also allow the immobilization of receptor proteins in a bilayer system. Choosing to express the tag on the cytoplasmic side of the receptor would present the ligand-binding site away from the surface. The bilayer can be formed either directly by fusion of vesicles containing the receptor4,28 or, alternatively, after the solubilized receptor has been immobilized. We have now shown for two cases that the proteins are immobilized in a functional state. In a previous publication, the GFP remained fluorescent, i.e., correctly folded at the surface. Here, the 5-HT3R exhibited identical binding properties for the different ligands investigated, either immobilized on the surface, in solution, or in its native membrane environment. This means that not only can ligand-receptor interaction measurements be carried out, but also they are of pharmacological value. In fact, with a single fluorescent ligand at hand, the presence of competitors was quantitatively detected, making this approach valuable in the screening of potential new drugs. ACKNOWLEDGMENT We are grateful to Dr. Kenneth Lundstro¨m (Hoffmann-LaRoche, Basel), who developed the His-tagged 5-HT3R expression vector, and to Dr. Horst Blasey (Geneva Biomedical Research Institute), who was responsible for the cell cultures. This work was supported by the Swiss National Science Foundation Priority Program (SPP) on Biotechnology, Grant 5002-35180.
Received for review November 18, 1997. January 27, 1998.
Accepted
AC9712658 (28) Heyse, S.; Ernst, O. P.; Dienes, Z.; Hofmann, K.-P.; Vogel, H. Biochemistry 1998, 37, 507-522. (29) Laporte, A. M.; Koscielniak, T.; Ponchant, M.; Verge´, D.; Hamon, M.; Gozlan, H. Synapse 1992, 10, 271-281.
