A M I . Chem. I Q Q I , 63,2707-2795
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Ion Channel Sensors for Glutamic Acid Hirotsugu Minami, Masao Sugawara, Kazunori Odashima, and Yoshio Umezawa* Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060, J a p a n Masayuki Uto Department of Environmental Technology, Kitami Institute of Technology, Kitami 090,J a p a n Elias K. Michaelis Department of Pharmacology and Toxicology, and Center for Biomedical Research, University of Kansas, Lawrence, Kansas 66045 Theodore Kuwana Center for Bioanalytical Research, University of Kansas, Lawrence, Kansas 66046
Coulometric Moselwors using glutamate receptor (GluR) ion channel proteln as a signal-ampllfylng sensory element that expkn the gMamale4ggered Na' kn current through bllayer llpld membranes have been fabricated. The formatlon of stable planar bllayer llpld membranes was achieved by applying the foldlng method across a small circular aperture bored through a thin polylmlde film. The multichannel type sensing membranes, formed across an aperture of ca. 120 pm dlameier, contained more than 10 GluR proteins and showed L-glutamale-triggered response as a composlte of lndlvldual slnglethannel currents. The slngle-channel type senslng membranes, formed across an aperture d ca. 20 p n dlemeter, contalned a sufficiently small number of GluR protelns so that the response was observed as a serles of single-channel pulse currents. Dependence of the Integrated channel current on the glutamate concentration was examIned. A sharp concentration dependence of up to ca. 1.5 X lo-' M and 3 X lo4 M for the multichannel and slnglechannel type sensors, respectively, was observed. A high selectlvlty for L-giutamate compared wlth Dglutamate for Inducing the channel current was observed. A detectlon llmlt as low as ca. 3 X lo4 M was attained for the multichannel type sensor. W remarkable 8onsltlvky Is dkussed In term d the potmtMusedOMI Ion chsmd protein for a new type of rendng system.
INTRODUCTION A principle that holds promise in the development of highly sensitive and selective sensing systems is the use of sensory elements that have an intrinsic function of intense signal amplification. From this viewpoint, ligand-gated ion channels (receptor ion channels) that normally function in bilayer lipid membranes can be regarded as excellent candidates for the development of such sensors because of their intense signal amplification. Such amplification is based on the on/off switching of rapid ion flux through the membrane, triggered by ligand binding to the receptor site of the channel protein. Some recent studies on the physiological properties of receptor ion channel proteins (1-3) stimulated us to examine the possibility of creating highly sensitive and selective sensors that would be designated as "ion channel sensors" (4-6). Some preliminary studies toward the development of such signalamplifying sensors have been carried out using artificial (7-10) as well as biological (11) systems. The class of ion channel proteins that has been most widely studied so far is the nicotinic acetylcholine receptor (nAChR), 0003-2700/91/0363-2787$02.50/0
a representative neurotransmitter receptor ion channel protein (12-14). Its physiological properties have been extensively studied with the purified protein in reconstituted bilayer lipid membranes. In addition, some studies aimed at the development of biosensors using the nAChR protein have been reported recently (15-20). Although the prospect for a signal-amplifying sensor was demonstrated by Yager et al. (15), the receptor function rather than the ion channel function of the nAChR protein has been used in most cases, detecting the ligand binding as a change in potential (16),capacitance (17))impedance (18))or fluorescence (19,20). By use of the receptor function, an application to a much wider range of compounds including the antagonists that do not induce a channel current would be expected, though the advantage of the inherent function of the ion channel proteins, Le., intense signal amplification, is not utilized. Another class of proteins of interest for fundamental studies toward "ion channel sensors" is the glutamate receptor (GluR) ion channel protein (21-26). Increasing interest has been focused on the GluR proteins because L-glutamate (L-Glu) is considered to be the principal neurotransmitter that mediates fast excitatory synaptic transmission in the central nervous system of vertebrates. The physiological properties of GluR from several sources including synapses of mammalian neurons have been studied using, primarily, crude membrane patches due to the lack of well-established methods for GluR purification (27-30). After extensive studies in the isolation and purification of a glutamate-binding glycoprotein from synaptic membranes of rat brain (31-34),a reliable procedure was recently developed for obtaining an active preparation of GluR ion channel containing several essential components including the 71-kDa glutamate-binding glycoprotein (35-37). By application of this purification procedure with some modifications,a preliminary study aimed at the development of a novel signal-amplifying sensor (11) was carried out with two types of planar bilayer lipid membranes in which the purified GluR ion channel proteins were incorporated as a sensory element. In these sensors, the glutamate-triggered Na+ ion currents were detected. The bilayer sensing membranes were prepared by the folding method (38-42) and the tip-dip method (43). The membrane prepared by the folding method across an aperture of ca. 200 Mm diameter bored through a thin Teflon film (12.5 pm thickness) showed a multichannel response behavior. Since the membrane formed by this procedure contained a large number of GluR proteins, the observed current was a composite of a large number of single-channel currents (11). The channel current increased with the concentration of L-G~u, though the details of the concentration 0 1991 Amerlcan Chemical Society
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dependence remained for further investigation. At extremely low concentrations, however, it would be reasonable to m u m e that the output signals would change from a composite current to a series of single pulse currents, reflecting the discontinuous nature of the single-channel current. Considering that the analyte detection becomes more significant in a lower concentration range, it will be quite important to develop an understanding of the correlation between the analyte concentration and the single-channel response. In this context, a bilayer lipid membrane sensor was also fabricated with a planar bilayer lipid membrane formed across the tip of a patch pipet (ca. 1 pm diameter) by the tip-dip method (11). This membrane contained an extremely small number of GluR proteins, so that the observation of individual single-channel current signals was possible. As a result, a signal amplification of ca. 2.1 x lo5 could be estimated from the averages of the single-channel current and the channelopen time. However, a detailed analyis of the relationship between the concentration of L - G ~ uand single-channel responses was not performed because of insufficient stability of the reconstituted bilayer membrane formed by the tip-dip method. Such membrane instability caused difficulties in changing the L - G ~ uconcentration without disrupting the membrane. In order to understand in detail the relationship between L - G ~ uconcentration and single-channel responses, a more stable membrane must be used that contains a sufficiently small number of GluR to observe single-channel currents. In the present study, multichannel and single-channel type Coulometric biosensors using GluR ion channel protein as a sensory element have been fabricated. The formation of stable planar bilayer lipid membranes was performed by applying the folding method across a small circular aperture bored through a thin polyimide film. The improved stability of the present bilayer membranes as compared to those in our previous study (11) enabled a detailed investigation of the L-G~uconcentration dependence of the channel response, especially in the case of the single-channel type sensor. The present article describes (i) the dependence of the multichannel response on the L-G~uconcentration, (ii) the LG l u / ~ - G l uselectivity of the multichannel response, and further (iii) the dependence of the single-channel response on the L-Glu concentration. EXPERIMENTAL SECTION Materials. L-a-Phosphatidylcholine (PC; from soybean; approximately 40%; Catalog No. P3644) was purchased from Sigma Chemical Co. (St. Louis, MO) and purified according to the reported procedure (44) with a slight modification as follows. Commercial PC was stirred at 4 “C overnight with dry acetone containing N-phenyl-p-phenylenediamineas antioxidant (2.5 mg/500 mL). After decantation of the acetone, the residue was dried under a nitrogen stream and used for the experiments. L-a-Phosphatidylethanolamine(PE; from dog brain; Catalog No. P 7514) was purchased from Sigma Chemical Co. and used without purification. Cholesterol (C 3137) was purchased from Sigma Chemical Co. and recrystallized twice from methanol. L-Glutamic acid (G 1251) was of the highest quality grade available from Sigma Chemical Co. Gramicidin (Gramicidin D; G 5002) was purchased from Sigma Chemical Co. and used without purification. HPLC grade hexane was used for the preparation of the lipid solutions. Concanavalin A was purchased either as a lyophilized water-soluble powder (Sigma Chemical Co.; C 2010) or as a 4 M NaCl solution (Boehringer Mannheim GmbH, Mannheim, Germany; 103594). The other chemicals were of analytical grade and used without further purification. The sample and buffer solutions were prepared with purified water obtained either by distillation over KMn04 of deionized water from a batch-type water still (Autostill Model WB-21, Yamato Scientific Co., Tokyo, Japan) or of deionized and charcoal-treated water from a Barnstead apparatus. Stock solutions of L-glutamate (0.1, 1, and 10 mM in water or 10 mM HCI) were
stored at 4 OC and used within 3 weeks. Isolation and Purification of Glutamate Receptor (GluR) Ion Channel Proteins. The procedures used for the isolation of synaptic plasma membranes from whole rat brain homogenates were similar to those previously described (34-36). Homogenates were obtained from eight rat brains of adult animals weighing 150 g or more. In the present work, the following protease inhibitors were used in all solutions to prevent proteolysis in all steps from the homogenization to the membrane protein solubilization: 2 pM pepstatin, 0.25 mM benzamide, 0.25 mM benzamidine.HC1, 25 mM c-amino-n-caproic acid (ACA), and 0.25 mM ethylene glycol bis(P-aminoethy1)ether N,N,”,”-tetraacetic acid (EGTA). For the solubilization and purification of the GluR proteins, the reported procedure (11) was used with some modifications described below to afford the following two kinds of preparations. Preparation A. The main modification points were that n-octyl 0-D-glucopyranoside was used in place of Triton X-100 for solubilization of the synaptic membranes and that, following the purification of GluR proteins, the concentrated liposome suspension of the protein was directly used for the fusion with bilayer membranes after adjusting the final NaCl concentration to 0.15 M. Preparation B. In addition to the modifications for obtaining preparation A, further purification was carried out by affinity column chromatography using ReactiGel (Pierce Chemical Co., Rockford, IL) to which L-glutamate was covalently bound (35). A mixture of two fractions containing the 71-, 42-, and 36-kDa proteins and the 58-kDa protein, respectively, as the major component(s) was used for the experiments. No substantial difference has been found between these two kinds of protein preparations in the liposome experiments (36, 37). Thus, in a typical purification process, 6.3 mL of lipid suspension of purified GluR protein containing 90g / m L of GluR and 5-10 mg/mL of a mixture of PE and cholesterol (4:l weight ratio) was finally obtained. The purity of the GluR protein was checked by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in order to confirm enrichment of the 71-kDa major band and the associated 54-59-, 42-, and 36-kDa bands observed in recent reconstitution studies (36,37). The GluR concentration in the PE/cholesterol liposome suspension was estimated by Peterson’s modification of the micro-Lowry method (45) and by the fluorescence method using benzoxanthene yellow (Hoechst 2495) (46). The GluR protein suspension was stored at 4 “C under argon. Formation of Planar Bilayer Lipid Membranes Containing GluR Ion Channel Protein(s). The folding method (Takagi-Montal method) (38-42) was used for the formation of planar bilayer lipid membranes as the sensing membranes. The bilayer membranes were formed across a small, smooth, circular aperture produced in a thin polyimide film (7.5 pm thickness; kindly supplied by Ube Industries, Kyoto, Japan) by passing an electrical spark generated by an automobile ignition coil (47). The shape of the aperture as well as the smoothness of its edge was checked under a microscope. The aperture diameters used were ca. 120 and 20 pm for the multichannel and single-channel type sensing membranes, respectively. The chamber was fabricated according to Montal(14) with a slight modification. The polyimide film containing a small aperture was cleaned with chloroform and fixed vertically in a specially fabricated Teflon chamber by tight sealing with thinly painted silicon grease, so that the two compartments, cis and trans, were separated (Figure 1). The Teflon chamber was placed on a magnetic stirrer enclosed in a Faraday cage that was mounted on a vibration-free table. Before each set of experiments, the polyimide film was precoated with a thin film of hexadecane painted by a clean cotton swab around the aperture on both sides. The whole procedure including the formation of bilayer membranes, the incorporation of GluR proteins, and the measurement of channel currents was carried out at 20 1 OC. Buffer solutions (1.45 mL), filtered just before use through a cellulose nitrate type membrane filter (pore size 0.2 rm; Advantec Toyo, Ltd., Tokyo, Japan; A020A013A), were added to each compartment with micropipets. In the general procedure, both of the cis and trans solutions contained citric acid (20 mM), NaCl (0.15 M), glycine (5.0 pM), and CaC12 (0.20 mM), and were adjusted to pH 4.0 with NaOH. The reference electrodes (Ag/AgCl)
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 23, DECEMBER 1, 1991 Amplifier
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Figure 1. Experimentalsetup for the measurement of the ion channel currents of the multichannel and single-channel type bilayer lipid membrane sensors using glutamate receptor (GIuR) ion channel protein as a sensory element. The polyimide film (a) was tightly sealed with thinly painted silicon grease in the middle of the Teflon chamber (b) placed on a magnetic stirrer (c), which was enclosed in a Faraday cage (d) mounted on a vibration-free table (e). Ag/AgCI electrodes (f) that were placed in both cis and trans compartments separated by the polyimide film were connected to the computer system through an amplifier and a low-pass filter. The cis electrode (left) was set to the command voltage relative to the trans electrode (right) connected to ground.
and the two Teflon tubes connected to syringes were fixed in the holes bored in the chamber (see ref 14). The cis electrode was set to the command voltage relative to the trans electrode connected to ground. Initially, the water level in each compartment was set below the aperture of the polyimide film by sucking the solutions with the syringes. A solution of PC and cholesterol in hexane (5 pL), prepared by dissolving 16 mg PC and 4 mg cholesterol/mL of HPLC grade hexane, was spread on the buffer solutions in both compartments using a microsyringe. A 30-min period was allowed for complete evaporation of hexane so that the lipid monolayers were spontaneously formed at the air/water interface. The folding of the monolayers into a planar bilayer membrane, either before or after the incorporation of GluR proteins (vide infra), was conducted as follows. The water level in the two compartments was gradually and simultaneously raised by careful operation of the two syringes, so that both solutions were raised above the aperture. Using an EPC-7 patch-clamp amplifier (List-ElectronicCo., Darmstadt-Eberstadt, Germany), potentials of +lo0 and -100 mV were applied to the electrodes to monitor bilayer formation by the increase in resistance. The resistance of the bilayer membrane formed across the aperture was calculated from the monitored current. The electrical noise fluctuation was also checked. Regardless of the resistance of the membrane and the noise fluctuation at this stage, the solutions of both compartments were discarded so that the washable part of the painted hexadecane could be removed. Without further painting with hexadecane, the above procedure was repeated until a stable bilayer membrane could be obtained. Only bilayer membranes with resistances over 100 GQ and noise fluctuation amplitudes below 2 pA were regarded as sufficiently stable to use for further experimentation. Hereafter, the potential was maintained at 0 mV by the patch-clamp amplifier. Only when membrane resistance was measured were potentials of +lo0 and -100 mV applied successively. In preliminary experiments specifically designed to document the formation of a bilayer by the above procedure, the gramicidin method (48) was used. This is based on the characteristicproperty of gramicidin A that leads to the formation of a conducting channel only if present in a dimerized state in bilayer membranes (49). Gramicidin was injected into the cis buffer as a 100 ng/mL methanol solution of gramicidin D which is a mixture of the gramicidins A, B, and C. Single-channel current with an amplitude of about 0.9 f 0.1 pA (9.0 f 1.0 pS) was observed at applied potentials of +lo0 and -100 mV. Since this amplitude corresponds to previously reported values (9.18 f 0.08 pS) obtained under the same solution condition (100 mM NaCl) (50),the formation of a bilayer membrane was confirmed. Injection of the same volume of methanol did not induce any changes in the current. Gramicidin-containing membranes were not used for any of the GluR studies described below. Incorporation of the GluR proteins were performed by either of the following two methods.
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Method A. In this method, a bilayer membrane without GluR protein was formed first by the folding of the monolayers as described above. Then, the proteoliposomes containing GluR proteins were added to the cis solution and fused with the preformed bilayer to form the sensing membrane containing GluR ion channel. A typical procedure for the GluR incorporation was as follows. The bilayer membrane formed was allowed to stand for about 30 min for stabilization. The resistance and the noise fluctuation amplitude were checked again. Under gentle stirring, a 10-pL aliquot of proteoliposome suspension containing 0.9 pg of GluR protein in PE/cholesterol liposomes was added to the cis solution. The cis solution was stirred for an additional 5-min period and then allowed to stand for 15 min. Method B. After the formation of monolayers on the solutions of both cis and trans compartments, a 10-pL aliquot of proteoliposome suspension containing 2-2.5 pg GluR protein in PE/ cholesterol liposomes was carefully applied to the air/water interface of the cis solution. A period of ca. 30 min was allowed for incorporation of GluR protein into the preformed monolayer. Then, the folding was conducted as described above between the monolayer containing the channel proteins (cis side) and that containing only lipids (trans side). After the bilayer formation, the cis solution was stirred gently for 5 min and then allowed to stand for 15 min. In typical experiments with the GluR-containing bilayer membranes formed by both methods A and B, the pH of both solutions was adjusted to 7.6 by addition of 1.3 M aqueous solution of tris(hydroxymethy1)aminomethane (TRIS) under gentle stirring. Then, a solution of concanavalinA in 3 or 4 M aqueous NaCl was added into the trans solution to the final concentration of 25 pg/mL. The stirring was stopped after 60 s. The final composition of the trans solution was citric acid (20 mM), HEPES (5.0 mM), NaCl(O.15 M), glycine (5.0 pM), CaC12(0.20 mM), and concanavalinA (25 pg/mL), pH 7.6. The cis solution was identical except for the omission of concanavalin A. The resistance and the electrical noise fluctuation were checked before and after the stirring. The final volume of the solution was 1500 pL for both cis and trans compartments. Measurement of Multichannel and Single-ChannelCurrents and Data Treatment. To investigate the dependence of the channel responses on the L-G~uconcentration, the channel current at varying concentrations of L-G~uwas measured using a single preparation of GluR-containing bilayer membrane. For the multichannel type sensor, 22 concentrations of L-G~uin the M) were range from 30 nM to 35 pM (3.0 X 10-8-3.5 X examined. For the single-channeltype sensor, 18 concentrations of L-G~u in the range from 0.30 pM to 1.8 mM (3.0 X 10-'-1.8 X M) were examined. After the addition of concanavalin A, the solutions were first allowed to stand for 5-15 min at an applied potential of 0 mV. Then, a potential of +lo0 mV was applied, and after waiting for 20 s, the background current was recorded. Recordings were carried out successivelyat 0 and -100 mV in the same manner as described above. The potential was set back to 0 mV, and an aliquot of L-G~u stock solution was added under gentle stirring with a micro stirrer tip to the trans solution (initial volume 1500 pL) to generate the to be examined. The stirring was lowest concentration of L-G~u continued for an additional 1-min period. After a 1-min equilibration period without stirring, the current was recorded successively at +100, 0, and -100 mV in the same manner as described above. The above cycle was repeated with further additions of to increase the concentration appropriate stock solutions of G G ~ U of L-G~uin the trans solution. The channel current recording for the highest L-G~uconcentration was done 2.5-3 h after the formation of the GluR-containing bilayer membrane. Channel currents, though weaker than those in the recording period, were observed about 7 h after GluR incorporation into bilayer membranes. This shows much improved stability of the proteincontaining planar bilayer membranes prepared in the present study when compared with those prepared at the tips of patch pipets used in the previous study (11). The actual success probability of these experiments was such that the channel currents could be observed in five out of more than twenty membranes which retained high resistance and low noise fluctuation after incorporation of GluR protein was attempted. Failure to observe such signals with the rest of the
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membranes was probably due to failure to incorporate the GluR into the bilayer membrane or due to inappropriate orientation of the protein in the bilayer membrane; Le., the receptor site was facing the cis solution that did not contain cGlu. Another possible reason is that it might be intrinsically difficult for the separated subunits to spontaneously reassemble to form the active channel complex, especially when the channel subunits are separated from each other during the purification process, as in the case of preparation B (vide supra). In addition to the low probability for observing the channel currents, the repeatability of the magnitudes of the integrated channel currents with different membranes was insufficient so that the calculation of standard deviation would not make sense. Possible reasons for the low repeatability would be varying numbers of the CluR ion channel proteins incorporated into the membranes, as well as varying activities of the incorporated ion channel proteins. However, in the present study, the discussions on the concentration dependence of the integrated channel current were made on the basis of the representative results shown in Figures 6 and 8, because in two sets of experiment carried out with the multichannel and single-channel type membranes, respectively, the orders of magnitude were the same for the amplitudes of the integrated channel current at fiied concentrations, as well as for the concentration showing a steep rise of the curve and for the concentration range showing a sharp concentration dependence. Data acquisition and analysis were performed as follows. Data acquisition for the background current and the glutamate-triggered channel current at each of the glutamate concentrations examined was continued for 30-60 s. Digitization of the analog data was made with amplitude resolutions of 0.2 and 0.1 pA for the multichannel and single-channel type sensors, respectively. The time resolution (sampling interval) was 200 ps for both type of sensors. The data acquired in the first 10 s were analyzed. Noise filtration was made at 1 kHz by an eight-pole low-pass filter of Bessel type (NF Electronic Instruments, Kanagawa, Japan). The digitization was made with a COMPAQ DESKPRO 3868 personal computer (Compaq Computer Corp., Houston, TX) in which a pCLAMP software (Axon Instruments Inc., Burlingame, CA) was installed. In the data treatment, a histogram of the current amplitude was made first. The integrated channel current was calculated from the histogram by integrating the event number with a weight of amplitude over the whole amplitude range. Correction was made at each point for the background current at +lo0 or -100 mV in the absence of L-G~u,which could be ascribed to the leak of the bilayer membrane without relation to a channel function of the incorporated GluR protein. The acquired signals, temporarily stored in a fixed disk, were transferred for permanent storage to an ordinary tape using a tape drive or to a VCR tape using Auto Fax Imager software (Auto Fax Corp., Ben Lomond, CAI.
RESULTS AND DISCUSSION Formation of Stable Bilayer Lipid Membranes Containing GluR Ion Channel Protein. As described earlier, stability of the GluR-containing bilayer membranes is essential for a detailed investigation of the dependence of channel responses on t G l u concentrations. In addition, the ease with which L-G~uconcentrations can be changed is another point to be considered. Especially in the case of singlechannel type sensor, rather than in the case of the multichannel type sensor, these two requirements are of crucial importance because membranes of smaller size, hence with less stability, are required. These points were satisfied by the use of the Takagi-Montal planar bilayer lipid membrane system (38-42) in which a thin polymide film (7.5 pm thickness) having a small aperture of appropriate diameter was used (Figure 1). In the present study, multichannel and single-channel type Coulometric biosensors were fabricated. The formation of remarkably stable planar bilayer membranes was performed by applying the folding method (38-42) across apertures of ca.120 and 20 pm diameters. A thin polyimide film was used as support material because it was easier to form bilayer lipid
membranes with polyimide as compared with Teflon. The number of GluR proteins incorporated into the bilayer membrane could be controlled by the area of the membrane without substantially changing the amount of the protein added as proteoliposome. By using a polyimide film with a 120-pm aperture, a multichannel type sensing membrane containing more than 10 ion channels was formed. On the other hand, by using a polyimide film with a 20-pm aperture, a singlechannel type sensing membrane containing one or two ion channel(s) could be formed (vide infra). With respect to the single-channel type sensor, the 20 pm diameter aperture enabled the application of the folding method to the formation of a stable bilayer membrane that was 100 times smaller by area than that used in our previous study (Teflon film of 12.5 pm thickness with an aperture of ca. 200 pm diameter) (11). Even though the area of the membrane formed across the 20-pm aperture was still about 400 times larger than the membrane area at the tip of patch pipets (ca. 1 pm diameter) used in the previous work (111, the GluR proteins actually incorporated could be restricted to a sufficiently small number so that single-channel currents were observed. The GluR-containing bilayer membranes were formed by fusing the GluR-containing proteoliposomes either with the preformed bilayer membrane (method A) or with the monolayer on the cis solution before the bilayer formation (method B), as described in Experimental Section. Fusion was facilitated by maintaining a Jow pH (pH 4.0) in the cis solution (51). Since ion channel proteins normally reside in bilayer membranes, fusion to a bilayer (method A) would be milder than fusion to a monolayer (method B), although the probability of protein incorporation was somewhat higher in the latter method. After the incorporation of the channel protein into the membrane, the pH of both solutions was adjusted to 7.6,so that further fusion of the GluR-containing liposomes with the bilayer membrane will be diminished. This is an important requirement for measuring the channel current without the number of incorporated protein complexes increasing throughout the experiment. pH 7.6 was chosen on the basis of a recent report by Traynelis and Cull-Candy (52) showing that the ion channel activity of a related GluR protein in neuronal membranes decreases at a lower pH. Advantage of the Experimental Section. In the experimental system used in the present study (Figure l), possible contact between the added glutamate and the GluR proteins that are not incorporated in the bilayer was eliminated by making all additions of L-G~uto the solution of the trans compartment. By such a way, the added glutamate would not bind with the excess GluR proteins existing in the monolayer or in the liposomes in the cis compartment (Figure 2a). On the other hand, in the patch pipet, tip-dip experimental system, glutamate had to be introduced in such a way that possible contact with the GluR proteins remaining in the monolayer or in the liposomes in the outer solution could be eliminated. Accordingly, in order to obtain ion channel responses of a single preparation of membrane to varying glutamate concentrations, glutamate has to be introduced into the outer solution after exchanging the initial outer solution that contained GluR proteins or into the inner solution of the patch pipet (ca.1 pm diameter) without exchanging the outer solution (Figure 2b). The former approach was not feasible because of the high probability of membrane rupture due to instability of the reconstituted bilayer membranes as compared with the more stable crude membrane patches from cell membranes. The alternative approach, i.e., that of changing the inner solution of the pipet, was practically impossible because of the extremely small volume within the pipets. For all these reasons, the Takagi-Montal cell with planar bilayer
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Figure 3. Typical example of multichannel current observed as a composite of a large number of singlechannel currents. Conditions: [L-GIu] = 0, 50, and 700 nM; applied potential = -100 mV. The sensing membrane was prepared by method B with preparation B of the purified GluR protein. This membrane was used for the investigation of the L-GIu concentration dependence of the channel response (Figure 6). The values indicating the amplitude of the composite current are corrected for the background current at -100 mV in the absence of L-Glu. OPEN CLOSED 40 ms
Figure 2. Schematic representations of the planar bilayer lipid mew branes containing GIuR ion channel proteins, formed by the folding method and the tipdip method. (a) The experimental system of the folding method. (b) The experimental system of the tipdip method.
membranes was selected as the most suitable system for a detailed investigation of the concentration dependence of channel responses, especially for the single-channel type sensor.
Conditions for Measurement of Channel Currents Triggered by L-Glutamate. An important point that should be considered in the characterization of receptor-induced ion channel currents is desensitization of the receptor, i.e., inactivation of the channel during prolonged exposure of the receptor to an agonist at concentrations optimal for channel opening. Desensitization usually accompanies repeated cycles of a burst current with abnormally high amplitude, followed by inactivation that lasts for several seconds. Most of the receptor ion channel proteins are subject to desensitization, which may cause a delay of response and a decrease in sensitivity. Studies by several groups (53-57) have shown that concanavalin A and other lectins with similar sugar specificities reduce desensitization of the GluR in membranes from mammalian neurons (mouse (57)) and invertebrate muscle cells (locust (53-55) and crayfish (56)). In the present study, concanavalin A was added to the trans solution at the final concentration of 25 pg/mL after the formation of the GluRcontaining bilayer membrane. Accordingly, the observation of channel current was performed in the presence of concanavalin A bound to GluR protein. In previous studies by Michaelis et al. (e.g., see refs 33,34), it was demonstrated that concanavalin A binds to the proteins in the isolated receptor complex. Burst currents that are characteristic of desensitization were not observed at the L - G ~ uconcentrations employed in the present study. Ion flux through incorporated GluR ion channels observed following addition of L-G~u was driven by an applied potential rather than by an established concentration gradient of channel-permeable cations between the cis and trans solutions. Under the present conditions with NaCl as the major electrolyte, the ion channel current composed mainly of Na+ ion current would be detected. An applied potential under conditions of equal concentrations of Na+ ions in the cis and trans
1 6 p A
Figure 4. Typical example of single-channel current observed as a series of pulse currents showing an amplitude of 5.6 pA during the channel open times with varying durations. Conditions: [L-GIu]= 5.0 pM; applied potential = +75 mV. The sensing membrane was prepared by method B with preparation B of the purified GluR protein. solutions is optimal for detecting glutamate-triggered current, first because the background current due to ion leak through the bilayer membrane is small and second because the electrochemical potential can be kept constant by the patch-clamp amplifier during the measurement. The applied potential was initially set to 0 mV to minimize the possibility of membrane rupture.
Observation of Multichannel and Single-Channel Responses. Figure 3 shows a typical example of current vs time curves for multichannel type responses at 0,50, and 700 nM L-G~uconcentrations under an applied potential of -100 mV. The glutamate-triggered multichannel responses appear to be a composite of a large number of single-channelcurrents due to simultaneous opening of the channels. The amplitude of the composite current increased from -9.9 to -38.4 pA (negative values because of the negative applied potential) as glutamate concentration was increased from 50 to 700 nM. Both of these two curves have the characteristics of pulse-like responses reflecting the component single-channel currents. The number of GluR ion channel proteins incorporated in this membrane was more than 10 as estimated from the conductance level of the channel current induced a t the highest concentration of L-G~uexamined (vide infra). In the case of the single-channel type sensor, the channel current had an amplitude much smaller than that of the multichannel type sensor and was observed as a series of pulse signals with a rectangular shape and of varying duration that reflected varying channel-open times (Figure 4). The proportion of channel-open time increased with increasing concentration of L-Glu as observed in our previous study (11). The amplitude of the single-channel current was linearly related to the applied potential within both the positive and negative potential regions, as shown in Figure 5. However, the channel currents induced by negative potentials were somewhat larger than those induced by the corresponding
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Positive Applied Potential
Figure 5. Dependence of the ampliude of the single-channel current on the applied potential in the range +lo0 to -100 mV for a singlechannel type sensor. Condition: [L-Glu] = 5.0 pM. The sensing membrane was prepared by method B with preparation B of the purified GluR protein. A schematic representation of the glutamate-bcund channel protein with the direction of the cation permeation is shown for the posklve and negative applied potentials. The cis electrode was set to the command voltage relative to the trans electrode connected to ground.
positive potentials. For example, the channel current at -100 mV was 32% larger than that at +lo0 mV. This might reflect more feasible permeation of ions through the channel under a "physiologically matched" condition. Although the direction of the channel proteins incorporated into the bilayer membrane would be random (i.e., the receptor sites would be facing either the cis or trans solution), only those facing the trans solution containing L - G ~ should u be "effective" for induction of channel current (see Figures 2a and 5). Considering that the extracellular side of the membrane is the positive side of the membrane potential under normal physiological conditions, it would be reasonable to expect that the ion permeation through the channel is more feasible when the direction of the applied potential is such that the side of the bilayer membrane facing the trans solution is the positive side of membrane potential. This corresponds to a "negative" potential applied to the cis side in the present experimental system (Figure 5, left inset). Analysis Based on Integrated Channel Current. In the present study, the dependence of the multichannel and single-channel responses on glutamate concentration was analyzed as the integrated current (number of coulombs detected). It corresponds to the number of ions passing through the channel during the duration of the open channel condition following the binding of glutamate to the receptor site. Compared with analyses based on parameters such as current amplitude (in the case of the multichannel type sensor) or open channel probability (in the case of the single-channel type sensor), the present analysis based on integrated current is characteristic in that this is a direct measure of signal amplification by this sensing system. Another advantage of this measure is that this analysis is not complicated by very fast channel open/close behavior occurring within the time resolution of measurement (200 ps), or by multiple levels of conductance usually observed with receptor ion channels (vide infra). To our knowledge, this is the first example in which the analysis of ion channel responses was made on the basis of integrated channel current. As mentioned above, the channel currents induced by negative potentials were larger than those induced by the
50
100
150
200
Concentration of Glutamate (nM) Flgure 6. Relationship between the concentration of L-glutamate and the integrated channel current (number of coulombs detected) for the multichannel type sensor. The sensing membrane was prepared by method B using preparation B of the purlfi GIUR proidin. The applled potential was -100 mV. A concentration range from 30 nM to 35 pM (3.0 X 10-*-3.5 X lo-' M) was investigated. Onty the region of sharp concentration dependence (up to 2.0 X lo-' M) is shown. corresponding positive potentials (Figure 5). This means that the signal amplification by GluR ion channel protein is more effective with a negative applied potential. Therefore, it would be more reasonable from the analytical viewpoint to measure the channel currents at negative applied potentials taking advantage of the greater signal amplification. Accordingly, the dependence of the channel response on the glutamate concentration was investigated at a negative applied potential, as described below. L - G ~ Concentration u Dependence of t h e Multichannel Type Sensor. The relationship between the concentration of L - G ~ uin the trans solution and the response of the multichannel type sensor as integrated channel current was examined at an applied potential of -100 mV in the L-G~u concentration range from 30 nM to 35 pM (3.0 X 104-3.5 X 10" M).Figure 6 shows the concentration-response curve in the region of sharp concentration dependence. A steep rise of the concentration-response curve was seen at a glutamate concentration on the order of M. A sharp concentration dependence was observed up to ca. 150 nM (1.5 X lo-' M) L-G~u,followed by a more gradual increase of integrated current at higher L - G ~ concentrations u (up to 3.5 X M) (figure not shown). The dependence of ion channel activation on L-G~uconcentration had the characteristics of a positively cooperative process. A high sensitivity was attained with a detection limit that could be regarded as being lower than the lowest concentration tested, Le., 30 nM (3 X M). The concentration-response curve at an applied potential of +100 mV (figure not shown) was obtained as a mirror image of the curve at -100 mV (Figure 6)except that the amplitude of the curve was somewhat smaller for the reason mentioned earlier. L - G ~ u / D - G Selectivity. ~u A preliminary examination was made on the L-G~u/D-G~u selectivity of the multichannel response. Figure 7 shows the changes in the integrated channel current upon successive addition of L- or D - G ~ to u the trans solution. A stepwise increase in L-G~u concentration to 50 nM resulted in an expected increase in the integrated current. The addition of 1pM D-G~u caused almost complete inhibition of the channel current. An increase in D-G~uconcentration to 5 pM did not produce a further inhibition of the current but rather a slight increase in the response. On the basis of these preliminary results, a possible interpretation may be that the present GluR ion channel protein exhibits a high selectivity for the L isomer of glutamate as compared with the D isomer for the channel activation and that the D isomer of glutamate acts not as a competitive antagonist but as an agonist with a weaker potency. The observed selectivity for the channel activation presumably reflects a combined effect of both the
ANALYTICAL CHEMISTRY, VOL. 63, NO. 23, DECEMBER 1, 1991
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Figure 7. Changes in the integratedchannel current upon successive addition of L- or BGiu for the muitlchannel type sensor, showing the ~-Glu/ffiluselectivity. The sensing membrane was prepared by method B using preparation B of the purified GluR protein. The applied potential was -100 mV. The integrated currents are shown with the concentrationsof L- or ~ G l in u the trans solution after each addition. About 50% greater cunent observed for this membrane as compared with the current observed at the same L-GIu concentration for the membrane in Figure 6 Is probably due to the larger number of active GluR proteins contained in this membrane.
relative strength of L-and DGlu in binding to the receptor site of the ion chennel protein and the relative potency of the bound isomers to induce the ion channel current. For a quantitative estimation of the L-G~u/DG~u selectivity of the present ion channel sensor, it would be necessary to measure the channel currents triggered by each of the isomers under the same conditions. That is, the measurements of the channel current should be made for a pair of solutions of Land DGlu containing a fixed concentration of L-G~u(primary ion) or for a pair of separate solutions of L- and D-G~u. Quantitative determination of the L-G~u/D-G~u selectivity is under way by seeking an appropriate method applicable to the present bilayer membranes that are not stable enough to physically tolerate the exchange of solutions during the experiment. L - G ~ uConcentration Dependence of the Single-Cham ne1 T y p e Sensor. As described above, the observation of single-channel currents was made possible by the use of a thin polyimide film with a small aperture of ca. 20 pm diameter. Across this aperture, a planar bilayer sensing membrane with improved stability could be formed by the folding method. The single-channel currents observed with this sensor consisted of a series of pulse currents with several conductance states, although only a single conductance level is shown in Figure 4. Observation of several distinct conductance levels triggered by L-G~uand other GluR agonists has been reported for neuronal membranes from hippocampal and cerebellar neurons (27-30). Since the single-channel current signals were treated as integrated currents in the present study, a possible complication due to multiple conductance states could be eliminated. The relationship between the concentration of L-G~uin the trans solution and the response of two preparations of single-channel type sensors was investigated a t an applied POtentid of -100 mV and L-G~uconcentrations ranging from 0.30 pM to 1.8 mM (3.0 X 10-'-1.8 X lo9 M).Figure 8 shows the concentration-response curves for these two preparations of single-channel type sensors in the region of sharp concentration dependence. A steep rise of the concentration-response curve was seen a t a glutamate concentration on the order of 10-'-104 M. A sharp concentration dependence was observed up to ca. 3 pM (3 X lo4 M) L-G~u, followed by a more gradual increase of integrated current a t higher L-G~uconcentrations
the integrated channel current (number of coulombs detected) for two preparations of single-channeitype sensor, most likely Containing one and two GluR ion channel proteln(s) as represented by curves 1 (0) and 2 (O),respectively. The sensing membranes were prepared by method A using preparation A of the purified GluR protein. The applied potential was -100 mV. A concentrationrange from 0.30 pM to 1.8 mM (3.0 X 10-'-1.8 X M) was investigated. Only the region of sharp concentration dependence (up to 5.0 X M) is shown.
(up to 1.8 X M) (figure not shown). The concentrationresponse curve at an applied potential of +lo0 mV (figure not shown) was obtained as a mirror image of the curve at -100 mV (Figure 8) except that the amplitude of the curve was somewhat smaller for the reason mentioned earlier. Curves 1 and 2 in Figure 8 most likely represent the responses of the membranes containing one and two GluR ion channel protein(s), respectively, for the following reasons. (i) The channel currents of the membrane corresponding to curve 1showed three major conductance levels, with the highest level being 74 pS. These observations have been reproduced over many preparations (58). (ii) The channel currents of the membrane corresponding to curve 2 sometimes showed larger conductance levels up to 150 pS in addition to those observed with the membrane corresponding to curve 1. (iii) The integrated current in the constant region (>3 pM) in curve 2 is double of that in curve 1. (iv) The steep rise of the curve begins at a lower L - G concentration ~ in curve 2 as compared with curve 1. This is consistent with the assumption that a larger number of GluR proteins are contained in the former membrane, resulting in a higher probability of the glutamate activation of channels and hence a larger integrated current at a fixed glutamate concentration. This is further supported by the observation in Figure 6 that the steep rise of the curve begins at an even lower concentration of L-G~u(-lo* M) for the multichannel type sensor containing a much larger number of GluR proteins. Hence, if curve 1 represented the response of the membrane containing a single GluR ion channel protein, the detection limit of the very single-channel sensor could be regarded as being lower than 0.3 pM (3 X lo-' M). Comparison of Sensitivity w i t h O t h e r Glutamate Sensors. Different biosensors for L-glutamate have been described in the literature. These include microorganism electrodes using Escherichia coli (59) or Bacillus subtilis (60) and enzyme electrodes using L-glutamate dehydrogenase (61) or L-glutamate oxidase (62,63). Remarkable among these is the enzyme electrode developed by Yao et al. (63) using a coimmobilized enzyme system of L-glutamate oxidase and L-glutamate-pyruvate transaminase. In this enzyme electrode, an extremely high sensitivity was attained by signal amplification based on substrate recycling in the presence of excess L-alanine (source of amino transfer) and oxygen. Detection of L - G ~ uas an oxidation current of hydrogen peroxide generated by the action of L-glutamate oxidase was performed at a concentration on the order of lo* M. The detection limit (signal-to-noise ratio = 3) was estimated to be as low as 2 x
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ANALYTICAL CHEMISTRY, VOL.
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10-lo M. By substrate recycling, the estimated sensitivity was improved by 3 orders of magnitude as compared with the corresponding nonamplifying system (62).The sensitivity of the other related electrodes is on the order of 10-3-10-4 M (59-61).
In the present study, a detection limit at least on the order of M was attained by the multichannel type sensor. Although it is difficult to compare the sensitivity of this ion channel sensor with that of the substrate-recycling sensor, the present sensitivity of the ion channel sensor on the order of M may be regarded as remarkably high, since the detection of L-G~u by the present sensor is based on the response by a much smaller number of sensory elements (110 ion channel proteins).
CONCLUSION Multichannel and single-channel type Coulometric biosensors using the GluR ion channel as a sensory element have been fabricated on the basis of planar bilayer lipid membranes with improved stability. Analysis of the concentration dependence of the multichannel and single-channel responses was made on the basis of the integrated channel current, which would be a direct measure of the signal amplification by the ion channel protein. The detection of L - G ~ was u performed at a concentration as low as 10-8M (by the multichannel type sensor) despite the fact that the conditions were not optimized for highest sensitivity. This high sensitivity is evidently based on the intense signal amplification function of the GluR ion channel protein, as supported by the signal amplification factor of ca. 2.1 X lo6 estimated in our previous study (11). Even higher sensitivity could be attained by optimizing the conditions of protein incorporation and by the use of a larger bilayer membrane containing a greater number of channel proteins. Although an improvement of the stability of the bilayer membranes is a prerequisite for practical analytical use of the ion channel sensors, the results obtained in the present study with the GluR/bilayer membrane system are a demonstration of the potential use of ion channel proteins as sensory elements for a new type of sensing system.
ACKNOWLEDGMENT We gratefully acknowledge Ana Maria Ly, Keshava N. Kumar, and Nancy Marioli, Center for Biomedical Research, University of Kansas, for the purification of glutamate receptor protein, Richard L. Schowen and Gary L. Aistrup, Department of Chemistry, University of Kansas, for helpful discussion on the analysis of ion channel currents, Hajime Hirata, Department of Biochemistry, Jichi Medical School, Tochigi, Japan, for instruction of the aperture fabrication, and Ube Industries, Kyoto, Japan, for supplying the polyimide film.
LITERATURE CITED Hille, B. Ionic Channels of Excitable Membranes; Slnauer: Sunderland, MA, 1984. Miller, C., Ed. Ion Channel Reconstitution;Plenum Press: New York, NY, 1986. Hoffman, J. F., Gieblsch, 0. Eds. hWecular Bbbgy of Ionic Channels; Current Topics in Membranes and Transport; Academic Press: Sen Diego. CA, 1988; Voi. 33. Umezawa. Y.; Sugawara. M.; Kataoka, M.; Odashima, K. I n Ion-Sel8CtlVe Electrodes; Pungor. E., Ed.; Akademlai Kiadd (Pergamon Press): Budapest (Oxford), 1989; Vol. 5, pp 211-234. Odashima, K.; Umezawa, Y. I n Biosensor Technology; Buck, R. P., Hatfleld, W. E., Umafia, M.. Bowden, E. F., Eds.; Marcel Dekker: New York. 1990; Chapter 6. Odashlma, K.; Sugawara, M.; Umezawa. Y. Trends Anal. Chem. 1981. 10, 207-215. Sugawara. M.; KoJima, K.; Sazawa, H.; Umezawa, Y. Anal. Chem. 1887, 5 9 , 2842-2846. Sugawara, M.; Kataoka, M.; Odashima, K.; Umezawa. Y. Thin Solid Fllms 1989, 180, 129-133. Nagase. S.; Kataoka, M.; Naganawa, R.; Komatsu, R.; Odashima. K.; Umezawa, Y. Anal. Chem. 1990, 62, 1252-1259. SUgaWara. M.; Sazawa. H.; Umezawa, Y. Submitted fcf publication. WO, M.; Michaelis, E. K.; Hu, I.F.; Umezawa, Y.; Kuwana, T. Anal. SCi. 1890, 6 , 221-225.
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Research (Y.U.) from the Ministry of Education, Science, and Culture, Japan, by the Japan-U.S. Cooperative'sciente'program (Y.U., T.K.) sponsored by the Japan Society for the Promotion of Science and the United States Nation2 Science Foundation, and by the United States Army Research Office Grant DAAL 03-88-K-0017 (E.K.M.).
T.;.Yaimto,
RECEIVEDfor review June 14,1991. Accepted September 12, 1991. This work is supported by a Grant-in-Aid for Scientific
TECHNICAL NOTES Improved Separation of Nucleic Acids with Analyte Velocity Modulation Capillary Electrophoresis Tshenge Demana, Maureen Lanan, and Michael D. Morris* University of Michigan, Department of Chemistry, Ann Arbor, Michigan 48109-1055 INTRODUCTION Capillary gel electrophoresis (CGE) is increasingly applied to the separation and sequencing of nucleic acids. The separation of polydeoxyoligonucleotides (l),single-stranded DNA sequence reaction products (2), as well as double-stranded DNA fragments (3, 4 ) , have been reported. As with other capillary electrophoresis techniques, CGE provides faster separations with higher resolution than slab gel versions (3). In high electric fields, nucleic acids become roughly oriented along the field direction and move at a velocity which is approximately size-independent (5-7). Changing the field direction disrupts the alignment and recovers size-dependent mobility. Because the time scale of nucleic acid motions is size-dependent, different field change intervals are necessary to optimize a separation for a given size range. These pulsed-field techniques have been most extensively developed for fragments larger than 10 kbp (thousands of base pairs) (8).
At the fields used in CGE (>lo0 V/cm) even short fragments align in gels, and resolution can suffer. Heiger, Cohen, and Karger have recently demonstrated that pulsed field operation improves the resolution of short restriction fragments in linear polyacrylamide-filled capillaries (4). For resolving the 4363- and 7253-bp fragments in 6% linear polyacrylamide, best results were obtained with 50% duty cycle and a frequency of about 100 Hz. They were unable to achieve resolution in this size range in conventional cross-linked gels. In the present communication, we report on the use of analyte velocity modulation capillary electrophoresis (9,lO) as a form of pulsed-field CGE. Sinusoidal changes in field strength and direction replace the customary step changes of pulsed-field electrophoresis. Analyte velocity modulation was developed to improve the performance of capillary zone electrophoresis (CZE) detectors that are excess noise limited. In CGE, an ac electric field of the proper frequency superimposed on the driving dc field would be expected to shorten nucleic acid fragment transit times and improve resolution in gel-filled capillaries. Although analyte velocity modulation does not change migration times or band shapes in CZE (9), it does increase the heat dissipation in an electrophoresis capillary, as a simple argument shows. With a superimposed ac field, the RMS voltage in the capillary is Vdc(l vdc/2vac)2)'/2,where Vd, and V,, are the dc and peak ac voltages, respectively. Thus, the RMS power is proportional to Vd:(1 + (vac/2vdc)2). Under the usual modulation conditions, V,, I0.5Vdc,and the excess power never exceeds 12.5%. Even when v,, = Vd,, the
+
excess power is increased by 50%
I
EXPERIMENTAL SECTION Electrophoresis was performed in 75 pm i.d. quartz capillaries (Polymicro Technologies, TSP/075/375). The total capillary length was 25 cm, with an entrance to detector length of 20 cm. The capillaries were filled with 3.5% T, 3.3% C polyacrylamide gel (Sigma), using the protocol of Paulus, Gassmann, and Field (11). Urea was not used in our gel preparations. The samples were 4X 174 RF DNA HaeIII fragments (Bethesda Research Laboratories). Separations were performed in 1X (90mM Tris) TBE buffer (Sigma) containing 0.5 rg/mL ethidium bromide. Electrophoretic separations were performed in the analyte velocity modulation apparatus previously described (9)using 180 V/cm dc and 0-120'70 depth of modulation (ratio of ac to dc voltage). A dc high voltage was applied between the capillary entrance and ground. A bucking voltage was applied between the capillary exit end and ground by an 88:l step-up transformer driven by a signal generator and power operational amplifier (Apex PA85). Detection of the ethidium bromide complexed nucleic acids was by laser-inducedfluorescence, using 325-nm He-Cd laser illumination and observation at 580 nm with a sharp-cut filter (Wratten gelatin filter No. 23A) and a photomultiplier tube. The dc signal was amplified, low-pass-filtered,(
