Abstract
Seven cell lines expressing native and transfected nicotinic receptor subtypes were evaluated functionally by using fluorescent assays based on membrane potential and calcium dynamics with “no-wash” dye systems. Both assays provided the same rank orders of potency for (±)-epibatidine, 2S-(−)-nicotine, 7R,9S-(−)-cytisine, and 1,1-dimethyl-4-phenylpiperazinium in a cell line expressing rat α3β4 receptors. Nicotinic antagonists mecamylamine and dihydro-β-erythroidine inhibited responses in both assays. Both agonist and antagonist activity were assessed within the same experiment. Agonists seemed more potent in the membrane potential assay than in the calcium assay, whereas the converse was true for antagonists. The membrane potential assay afforded robust responses in K-177 cells expressing human α4β2 receptors, in IMR-32 and SH-SY5Y cells expressing human ganglionic receptors, and in TE-671 cells expressing human neuromuscular receptors. These lines gave weak to modest calcium responses. Moreover, membrane potential responses were obtained in cell lines expressing rat α4β2 and α4β4 receptors, which were devoid of calcium responses. Thus, membrane potential serves as a sensitive measure of nicotinic activity, and the resulting depolarization may be as important as calcium in cell signaling.
Nicotinic acetylcholine receptors are a class of pentameric ligand-gated ion channels present in the central and peripheral nervous systems (1–4). Different subtypes of these channels are involved in neuromuscular, autonomic, and CNS neurotransmission (2). Nicotinic receptors may be involved in number of diseases, including Alzheimer's and Parkinson's diseases, schizophrenia, Tourette's syndrome, and certain epilepsies, as well as nicotine addiction. Additionally, nicotinic receptor agonists have potential as analgetics, anxiolytics, and nootropics (3–7). Nicotinic agonists and antagonists used in the present study are shown in Fig. 1.
Figure 1.
Nicotinic agonists and antagonists used in this study.
Methods of pharmacologic analysis of nicotinic receptors have included both radioligand binding assays (8–11) and functional assays (12–16). Whereas binding assays are rapid and relatively simple to perform, they fail to distinguish agonists from antagonists. Functional assays for ion channels include classical electrophysiology (12, 17), radioisotopic ion flux (13–15), and fluorescent assays using ion- or voltage-sensitive dyes (16, 18–20). In the current study, nicotinic receptor activation was measured by using a membrane potential-sensitive fluorescent dye (21, 22) as an indicator of ionic flux in cell lines expressing various nicotinic receptor subtypes, both native (8, 23, 24) and transfected (25, 26). This result was compared with measurements using a calcium-sensitive dye. The membrane potential assay provides a rapid and sensitive method for the assessment of nicotinic responses. Indeed, readily quantifiable membrane potential responses were obtained in cell lines in which significant calcium responses could not be detected. Moreover, this method affords kinetic data, and allows measurement of agonism and antagonism within the same experiment. The paradigm used here may also be used to investigate loss of nicotinic response. This loss of response can reflect desensitization, in which the receptor becomes unresponsive to agonist (27, 28), or open channel block at high agonist concentrations (29, 30). Membrane depolarization is undoubtedly an important cell-signaling event for ligand-gated ion channels, as this provides a trigger for activation of voltage-sensitive ion channels. Kinetic fluorescence measurements thus provide a valuable approach for measuring and understanding drug actions at nicotinic receptors as well as for high-throughput screening.
Materials and Methods
Reagents.
All chemicals and reagents were obtained from commercial sources. ABT-418 hydrochloride was a gift of Abbott. It and (S)-nicotine di-d-tartrate, mecamylamine hydrochloride, (±)-epibatidine dihydrochloride, (7R,9S)-cytisine, 1,1-dimethyl-4-phenylpiperazinium iodide, A-85380 dihydrochloride, RJR-2429 dihydrochloride, (±)-epiboxidine hydrochloride, dihydro-β-erythroidine hydrobromide, and (1R,6R)-(+)-anatoxin-a fumarate were prepared as 1-mM or 10-mM stock solutions in methanol. Ionomycin-free acid and carbonyl cyanide, p-(trifluoromethoxy)phenylhydrazone (FCCP) were prepared as stock solutions in DMSO.
Buffers.
Hanks' balanced salt solution (HBSS) was prepared from commercial 10× stock (without sodium bicarbonate or phenol red) and supplemented with 20 mM 4-hydroxyethyl-1-piperazinesulfonic acid (Hepes) and adjusted to pH 7.4 with sodium hydroxide.
Dyes.
Calcium ion and membrane potential dyes were obtained from Molecular Devices. Calcium ion dye solution was prepared by dissolving the contents of one bottle of the dry reagent in HBSS/Hepes to a volume of 15 ml. Membrane potential dye solution was prepared in identical fashion, with dilution to 36 ml in HBSS/Hepes. In our hands, these were the maximum useful dilutions for the cells studied here. The dye solutions were kept in the dark and were stable at least 8 h at room temperature and 2 wk in a −20°C freezer.
The no-wash dyes are of proprietary composition (31). The responses are sensitive to dye concentration and loading time, as are other intensity dyes. The efficiency of dye loading was quite good in all lines reported here, allowing a reduction in dye usage of 5- to 12-fold relative to recommended loading described in package literature without substantial degradation of response characteristics. Further dilution, however, led to signal artifacts.
Cell Lines.
K-177 cells (26), expressing human α4β2 receptors, were a gift of Abbott. KXα4β2R2 and KXα4β4R1 cells, expressing rat α4β2 and α4β4 receptors, respectively, were transfected and selected as described for KXα3β4R2 cells expressing α3β4 receptors (25, 31). TE-671 rhabdomyosarcoma cells, expressing human neuromuscular receptors (8, 32), and IMR-32 (9, 23, 33–35) and SH-SY5Y (23, 24, 35–37) neuroblastoma (36) cells, expressing human ganglionic (9, 23) receptors, were obtained from the American Type Culture Collection.
Cell Culture.
All cell lines were maintained in a culture medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. KXα3β4R2, KXα4β2R2, and KXα4β4R1 cells were further supplemented with 0.6 mg/ml genetecin. K-177 cells were further supplemented with 0.6 mg/ml genetecin and 90 μg/ml hygromycin-B. Cells were maintained in an atmosphere of 5% CO2 in a humidified incubator at 37°C and were subcultured weekly at a subcultivation ratio of between 1:20 and 1:100. For assays, 96-well plates were coated by treatment with 50 μl per well of a sterile solution of 50 μg/ml poly-d-lysine (30–70 kDa) for 1 h followed by aspiration and washing with 75 μl of sterile water and allowing the plates to dry in a laminar flow hood. Cells were seeded into these plates at a subcultivation ratio of between 1:2 and 1:4 and grown to near confluence in 75 μl of culture medium. IMR-32 cells were seeded into 96-well plates precoated with collagen type I (Becton Dickinson) to improve cell attachment and reduce fibroblast populations (38).
Calcium Fluorescence Measurements.
The method used for analysis was an adaptation of the method for fluo-3 calcium measurements (18). Sample plates for dose-response analysis were prepared by evaporation of an appropriate volume of methanolic stock solution in 96-well round-bottomed plates and reconstitution in HBSS/Hepes, followed by serial dilution to produce 270 μl of sample volume, sufficient for 3 to 4 assays, and sealed with parafilm until use. All reagents and test compounds were stored at −20°C when not in use. Cell cultures in 96-well plates were removed from the incubator, allowed to reach room temperature over the course of 10 min, and then washed twice with 100 μl of HBSS/Hepes. Subsequently, the medium was replaced with 30 μl of calcium ion dye solution, and the cells were loaded for 1 h in the dark, after which plates were read on a Flexstation fluorescence plate reader (Molecular Devices). Excitation and emission wavelengths were set to 485 nm and 525 nm, respectively, with a cutoff of 515 nm. Measurements were made at 1- to 1.5-s intervals. Basal fluorescence was measured for 15–30 s, followed by addition of 30 μl of a 2× solution of test compound (to assess agonist activity), and measurement of fluorescence for 100–180 s. Subsequently, 30 μl of a solution of nicotine di-d-tartrate was added to a final concentration of 100 μM (to assess antagonism of nicotine response), and fluorescence was measured for 40–60 s. Finally, 30 μl of a 4× calibrant solution containing 20 μM ionomycin (a calcium ionophore), 400 μM carbamylcholine chloride (a muscarinic agonist), and 80 μM FCCP (a mitochondrial toxin) was added to maximize intracellular calcium levels (17). The resultant fluorescence was then measured for 30–40 s. Finally, responses were calculated as follows:
 |
 |
 |
 |
Absolute calcium concentrations were not determined, as these tend to be unreliable with intensity dyes.
Membrane Potential Measurements.
This assay was conducted in a similar fashion to the calcium assay, with modifications. Briefly, cells were equilibrated and washed as before, followed by loading for 45 min with 30 μl of membrane potential dye solution. Fluorescence measurements were taken as before, but with excitation and emission wavelengths set to 535 nm and 560 nm, respectively, and with a cutoff of 550 nm. Finally, the ionomycin/carbachol/FCCP calibrant solution was replaced by a 4× calibrant solution containing 160 mM KCl. Responses were calculated as before with KCl serving as the calibrant, clamping the membrane potential to ≈−35 mV. In a few cases, especially when the dye concentration is low, a slowly dropping baseline was observed. In such cases, the baseline is taken as that immediately before the nicotine addition.
Data Analysis.
Responses were normalized to the maximum response of (±)-epibatidine and fitted to a standard four parameter logistic equation. Data were either fitted within the flexstation control software or exported to prism (V. 3.0, GraphPad, San Diego). In cases where bell shaped curves were observed, data points on the ascending side were used for determination of potency and efficacy. Hill coefficients were typically one or greater; however, the number of concentration points did not allow for reliable measurements of this parameter.
Results
The general protocol of this method involved acquisition of a basal signal, followed by addition of the test compound to assess agonist potency (EC50) and efficacy. Subsequently, a reference agonist (nicotine) was added to assess antagonism of the nicotine reference response (IC50). Such antagonism may be either the result of open channel blockade or desensitization (27–30). In the cell lines tested, however, time-dependent desensitization did not seem to contribute significantly within the time frame of the experiment (see below). Finally, a calibrant was added to normalize for dye loading and cell count. This protocol resulted in a signal having three distinct phases as illustrated for epibatidine and nicotine in Fig. 2.
Figure 2.
Signals acquired for calcium fluorescence dynamics and membrane potential fluorescence by using KXα3β4R2 cells. Agents are added sequentially as indicated by arrows. Note that agonist signals are not entirely decayed before addition of nicotine reference.
After subtraction of basal fluorescence, the response was calculated as the ratio of the fluorescence increase produced by the test compound to that produced by the calibrant. The nicotine reference response was calculated in a similar fashion. Response values were then normalized to the percentage of the maximum (±)-epibatidine response for activation, or the nicotine reference response for inhibition, resulting in concentration-response curves as shown in Fig. 3. In KXα3β4R2 cells, EC50 values for activation were similar to IC50 values for inhibition of the nicotine reference response as shown in Table 1. This result is consistent with a simple saturation mechanism, because the subsequent response of an already stimulated system should be reduced relative to an unstimulated one. The timescale of the experiment is short relative to the desensitization periods for the receptor subtypes assessed in this study (27). Indeed, the IC50 for nicotine did not change from 30 s up to 1 h of preexposure in KXα3β4R2 cells (data not shown). However, during the time course of the usual assays, the agonist-evoked signal did not decay entirely in the calcium dynamics experiments. Often in the case of membrane potential, the agonist signal did not decay at all (see Fig. 2). Thus, the derived IC50 values based on apparent reduction of the subsequent nicotine signal should be viewed with caution.
Figure 3.
Activation/Inhibition curves for (±)-epibatidine in KXα3β4R2 cells. Filled symbols represent percentage activation relative to a maximally efficacious concentration of (±)-epibatidine. Open symbols represent normalized fluorescence for subsequent 100 μM (s)-nicotine addition. Error bars represent SEM for three experiments conducted in duplicate.
Table 1.
Comparison of agonist activation and inhibition parameters using KXα3β4R2 cells
| Agonist | Calcium dynamics
|
Membrane potential
|
||
|---|---|---|---|---|
| EC50 | IC50 | EC50 | IC50 | |
| (±)-Epibatidine | 0.035 ± 0.012 | 0.059 ± 0.024 | 0.018 ± 0.011 | 0.019 ± 0.015 |
| S-Nicotine | 14 ± 4 | 31 ± 7 | 8.0 ± 4.6 | 5.7 ± 4.3 |
| (−)-Cytisine | 35 ± 18 | 76 ± 43 | 11 ± 5 | 8.2 ± 6.3 |
| DMPP | 11 ± 5 | 69 ± 33 | 9.4 ± 3.5 | 9.6 ± 7.7 |
EC50, Response produced by the agonist alone. IC50, Inhibition of subsequent 100 μM nicotine stimulation. All values are in μM and represent the average ± SEM for three experiments conducted in duplicate.
To evaluate the utility of membrane potential fluorescence for evaluation of nicotinic pharmacology, data obtained for several nicotinic agonists and antagonists were compared with parallel data from calcium fluorescence and published data for 86Rb efflux (30) for KXα3β4R2 cells (Table 2). As shown in Fig. 4, compounds show a similar rank order of potency although agonists seem more potent in the membrane potential assay than either calcium fluorescence or 86Rb efflux assays. Two nicotinic antagonists, mecamylamine and dihydro-β-erythroidine, were also assessed, and were less potent in the membrane potential assay than in calcium or 86Rb efflux assays. Calcium fluorescence and 86Rb efflux yielded very similar values for potency as observed previously (16, 30).
Table 2.
Comparison of functional assays for nicotinic receptors
| Membrane potential | Calcium dynamics | 86Rb efflux | |
|---|---|---|---|
| Agonist | EC50, μM | ||
| (±)-Epibatidine | 0.018 ± 0.011 (100) | 0.035 ± 0.012 (100) | 0.06 ± 0.01 (100) |
| (±)-Epiboxidine | 0.17 ± 0.11 (99) | 3.4 ± 1.8 (110) | — |
| RR-Anatoxin-a | 0.21 ± 0.04 (100) | 1.9 ± 3 (93) | — |
| RJR-2429 | 0.23 ± 0.08 (100) | 0.64 ± 0.08 (98) | — |
| A-85380 | 1.1 ± 0.2 (100) | 35 ± 13 (94) | 5.7 ± 0.3 (90) |
| S-Nicotine | 8.0 ± 4.6 (120) | 14 ± 4 (67) | 31 ± 1.5 (89) |
| DMPP | 9.4 ± 3.5 (120) | 11 ± 5 (58) | 28 ± 3.9 (60) |
| (−)-Cytisine | 11 ± 5 (110) | 35 ± 18 (91) | 24 ± 7.4 (80) |
| ABT-418 | 17 ± 1 (88) | >500 (79) | — |
| Antagonist | IC50, μM | ||
| (±)-Mecamylamine | 25 ± 3 | 0.95 ± 0.29 | 1.2 ± 0.4 |
| DHβE | >500 | ∼500 | 100 ± 6 |
KXα3β4R2 cells were assayed using membrane potential fluorescence and calcium fluorescence dynamics as described in Materials and Methods. EC50 values represent the average ± SEM for three experiments conducted in duplicate. Efficacies are in parentheses and are expressed relative to a maximally efficacious concentration of (±)-epibatidine. 86Rb efflux data are adapted from refs. 25 and 31.
Figure 4.
Comparison of EC50 values for calcium fluorescence, membrane potential fluorescence, and 86Rb efflux in KXα3β4R2 cells. The line of identity is presented for reference. Data are taken from Table 2.
A number of cell lines expressing a variety of nicotinic receptor subtypes were next evaluated. The K-177, IMR-32, SH-SY5Y, and TE-671 cells gave only weak signals by using calcium fluorescence, whereas KXα4β2R2, and KXα4β4R1 cells gave no detectable signal (data not shown). In contrast, quantifiable signals were obtained with all cell lines by using membrane potential (Table 3). Nicotine responses in both assays were blocked by mecamylamine in all cell lines tested. IC50 values are reported for both antagonists and agonists in Table 3, although IC50 values for agonists should be viewed with caution (see above). Fluorescent membrane potential assays thus provide results consistent with nicotinic activation in a variety of cell types, and are clearly more sensitive than the corresponding calcium assays.
Table 3.
Pharmacological parameters for membrane potential response to nicotinic agonists for seven cell lines
| EC50, μM | Efficacy | IC50, μM | |
|---|---|---|---|
| IMR-32 (Human α3β4*) | |||
| (±)-Epibatidine | 0.011 ± 0.003 | 100 ± 1 | 0.028 ± 0.001 |
| S-Nicotine | 7.2 ± 1.9 | 71 ± 1 | 6.7 ± 0.7 |
| (−)-Cytisine | 6.9 ± 1.0 | 77 ± 4 | 130 ± 50 |
| DMPP | 5.0 ± 0.1 | 68 ± 1 | 9.2 ± 2.8 |
| Mecamylamine | — | — | 0.69 ± 0.19 |
| DHβE | — | — | 240 ± 60 |
| SH-SY5Y (Human α3*) | |||
| (±)-Epibatidine | 0.0080 ± 0.0005 | 100 ± 3 | 0.014 ± 0.001 |
| S-Nicotine | 6.2 ± 0.2 | 78 ± 7 | 11 ± 4 |
| (−)-Cytisine | 8.3 ± 2.2 | 91 ± 2 | 43 ± 5 |
| DMPP | 4.6 ± 0.6 | 67 ± 7 | 32 ± 17 |
| Mecamylamine | — | — | 0.35 ± 0.10 |
| DHβE | — | — | 310 ± 80 |
| TE-671 (Human α1β1γδ) | |||
| (±)-Epibatidine | 0.27 ± 0.03 | 100 ± 1 | 0.52 ± 0.01 |
| S-Nicotine | 26 ± 1 | 84 ± 5 | 41 ± 17 |
| (−)-Cytisine | 77 ± 17 | 67 ± 7 | 130 ± 10 |
| DMPP | 1.1 ± 0.4 | 110 ± 10 | 1.5 ± 0.3 |
| Mecamylamine | — | — | 62 ± 12 |
| DHβE | — | — | 87 ± 14 |
| K-177 (Human α4β2) | |||
| (±)-Epibatidine | 0.0053 ± 0.0026 | 100 ± 1 | 0.010 ± 0.002 |
| S-Nicotine | 0.86 ± 0.09 | 91 ± 21 | 0.71 ± 0.04 |
| (−)-Cytisine | 1.7 ± 0.4 | 44 ± 16 | 13 ± 1 |
| DMPP | 2.2 ± 0.1 | 79 ± 15 | 7.8 ± 1.5 |
| Mecamylamine | — | — | 16 ± 3 |
| DHβE | — | — | 25 ± 6 |
| KXα3β4R2 (Rat α3β4) | |||
| (±)-Epibatidine | 0.018 ± 0.011 | 100 ± 4 | 0.018 ± 0.015 |
| S-Nicotine | 8.0 ± 4.6 | 120 ± 10 | 5.7 ± 4.3 |
| (−)-Cytisine | 11 ± 5 | 110 ± 10 | 8.2 ± 6.3 |
| DMPP | 9.4 ± 3.5 | 120 ± 10 | 9.6 ± 7.7 |
| Mecamylamine | — | — | 19 ± 5 |
| DHβE | — | — | >500 |
| KXα4β2R2 (Rat α4β2) | |||
| (±)-Epibatidine | 0.034 ± 0.012 | 100 ± 10 | 0.0035 ± 0.0017 |
| S-Nicotine | 3.4 ± 1.3 | 84 ± 5 | 0.21 ± 0.05 |
| (−)-Cytisine | 15 ± 8 | 28 ± 5 | 0.37 ± 0.11 |
| DMPP | 7.6 ± 4.2 | 43 ± 4 | 1.2 ± 0.14 |
| Mecamylamine | — | — | 2.4 ± 1.1 |
| DHβE | — | — | 20 ± 7 |
| KXα4β4R1 (Rat α4β4) | |||
| (±)-Epibatidine | 0.0057 ± 0.0010 | 100 ± 5 | 0.0052 ± 0.0021 |
| S-Nicotine | 0.95 ± 0.29 | 110 ± 10 | 0.67 ± 0.25 |
| (−)-Cytisine | 0.085 ± 0.016 | 89 ± 5 | 0.23 ± 0.18 |
| DMPP | 11 ± 1 | 49 ± 5 | 22 ± 8 |
| Mecamylamine | — | — | 1.0 ± 0.2 |
| DHβE | — | — | >500 |
Values represent the average ± SEM for three experiments conducted in duplicate. Efficacies are expressed relative to a maximally efficacious concentration of (±)-epibatidine. IC50 values represent inhibition of subsequent 100 μM nicotine stimulation.
, Subunit combination not fully defined.
Discussion
High-throughput screening has led to miniaturization of assays and adaptation for screening large numbers of compounds in drug discovery and development efforts. Among the functional assay techniques for such screening, fluorescence has attracted much attention of late (16, 18–22). The most widely used fluorescent probes for nicotinic receptor functional analysis have been the calcium chelating Fluo- and Fura-dyes developed by Tsien (39–41). The calcium kit dye used in this study is similar in nature to the Fluo-3,4 intensity dyes, except that a secondary dye is incorporated to mask background fluorescence due to unloaded and extruded dye (31). This masking dye eliminates the need for washing out extracellular dye, thus simplifying the assay and enhancing reproducibility.
The present methods allow for rapid screening of compounds for evaluation of structure-activity and subtype specificity relationships for nicotinic receptors. The 96-well instrument used in the current study has an integrated fluidics module that dispenses reagents during acquisition of signal. Up to three additions can be programmed, which allows acquisition of responses from the compound to be tested, a reference agonist, and the calibrant. This protocol allows agonist and antagonist activity to be assessed within the same experiment, because an agonist will produce an initial fluorescence response, whereas an antagonist will not. However, both will produce an inhibition of subsequent nicotine stimulation. The inhibition observed for nicotinic agonists could result from desensitization or open channel blockade (27–30). EC50 and IC50 values for the agonists were similar in magnitude in KXα3β4R2, KXα4β4R1, and TE-671 cells. In IMR-32, SH-SY5Y, and K-177 cells, IC50 values for inhibition of the nicotine response by cytisine were significantly higher than EC50 values for cytisine activation. Curiously, KXα4β2R2 cells required lower concentrations for inhibition of nicotine response than for activation by all four agonists (Table 3). Further kinetic studies are required to reveal the reasons for such results, because the agonist responses had often not fully decayed before subsequent addition of the reference agonist, nicotine. Whereas the pharmacological interpretation of inhibition curves for agonists is not clear, they may serve as a diagnostic tool for nicotinic activity. In the case of antagonists, inhibition parameters will depended on the mechanism and time course of blockade.
The current paradigm provides kinetic data and allows for assessment of agonism and antagonism within the same experiment. The use of dyes that do not require washing reduces variability due to cell detachment. Finally, the use of an automated plate reader/liquid handler increases reproducibility of additions. The membrane potential assay seems much more sensitive than the calcium fluorescence assay, but provides data consonant with either calcium fluorescence or 86Rb efflux assays (Table 2 and Fig. 4).
Although fluorescence measurement of nicotinic receptor activity through calcium dynamics has been documented extensively in recent years (16, 18–20, 31), the use of membrane potential for nicotinic receptors is novel and quite sensitive. The use of membrane potential is especially important for cell lines that pass little or no calcium. TE-671 cells, expressing the neuromuscular subtype of nicotinic receptor (8), have a low calcium conductance and expression. Indeed, we were unable to obtain a significant calcium response until well past cell confluence, and then only weakly. Weak calcium signals were also observed with K-177 cells expressing human α4β2 receptors, and with SH-SY5Y, and IMR-32 cells, expressing human ganglionic receptor subtypes. No detectable calcium signals could be observed with KXα4β2R2 and KXα4β4R1 cells (data not shown). Membrane potential, however, affords acceptable signals in all cell lines used here and very strong signals in several.
Membrane potential fluorescence, while providing complementary data to calcium fluorescence and 86Rb efflux, did not provide identical data. Agonists were consistently and significantly more potent in the membrane potential assay, whereas antagonists were less potent. Moreover, the potency of mecamylamine in KXα3β4R2 cells was ≈20- to 40-fold less than in IMR-32 or SH-SY5Y cells, although these cell lines are thought to express similar ganglionic-type nicotinic receptors. Functional responses in IMR-32 cells have been attributed mainly to α3β4 receptors based on electrophysiological measurements (35). IMR-32 and SH-SY5Y cells express receptor at <100 fmol/mg protein (23, 37), whereas KXα3β4R2 cells express receptors at ≈8,000 fmol/mg protein (25). It is possible that the differences in potency are due to differences in fractional receptor activation requirements for membrane depolarization. This mechanism could also explain the differences in potency between the calcium and membrane potential assays for antagonists. In the case of agonists, it is possible that only a small fraction of receptors need be activated to depolarize the membrane (the fraction depending on receptor number), resulting in increased apparent potency. Conversely, antagonists would need to inhibit a much greater fraction of receptors to prevent depolarization, resulting in reduced apparent potency. This is a potentially important observation, as such depolarizations act as the triggering event for opening voltage-gated ion channels and other membrane potential-dependent processes.
Conclusions
Membrane potential fluorescence is a sensitive measure of nicotinic receptor functional response, giving data that is complementary to calcium fluorescence and radioisotopic ion flux methods. Further, membrane potential signals can be obtained in cell lines in which it has been difficult to measure responses with other functional assays. It affords robust signals and reproducible pharmacology for a variety of nicotinic receptor subtypes. Membrane potential fluorescence should prove a valuable tool for characterizing nicotinic receptor function and for discovering novel nicotinic agents.
Abbreviations
- FCCP
carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone
- DMPP
1,1-dimethyl-4-phenylpiperazinium
- DHβE
dihydro-β-erythroidine
- HBSS
Hanks' balanced salt solution
References
- 1.Lukas R J, Changeux J-P, LeNovère N, Albuquerque E X, Balfour D J K, Berg D K, Bertrand D, Chiappinelli V A, Clarke P B S, Collins A C, et al. Pharmacol Rev. 1999;51:397–401. [PubMed] [Google Scholar]
- 2.Lindstrom J. Handb Exp Pharmacol. 2000;144:101–162. [Google Scholar]
- 3.Dani J A. Biol Psychiatry. 2001;49:166–174. doi: 10.1016/s0006-3223(00)01011-8. [DOI] [PubMed] [Google Scholar]
- 4.Holladay M W, Dart J, Lynch J K. J Med Chem. 1997;40:4169–4194. doi: 10.1021/jm970377o. [DOI] [PubMed] [Google Scholar]
- 5.Decker M W, Meyer M D, Sullivan J P. Exp Opin Invest Drugs. 2001;10:1819–1830. doi: 10.1517/13543784.10.10.1819. [DOI] [PubMed] [Google Scholar]
- 6.Lloyd G K, Williams M. J Pharmacol Exp Ther. 2000;292:461–467. [PubMed] [Google Scholar]
- 7.Schmidt J D, Bencherif M. Annu Rep Med Chem. 2000;35:41–51. [Google Scholar]
- 8.Lukas R J. J Neurochem. 1986;46:1936–1941. doi: 10.1111/j.1471-4159.1986.tb08516.x. [DOI] [PubMed] [Google Scholar]
- 9.Gotti C, Wanke E, Sher E, Fornasari D, Cabrini D, Clementi F. Biochem Biophys Res Commun. 1986;137:1141–1147. doi: 10.1016/0006-291x(86)90344-x. [DOI] [PubMed] [Google Scholar]
- 10.Houghtling R A, Davila-Garcia M I, Kellar K J. Mol Pharmacol. 1995;48:280–287. [PubMed] [Google Scholar]
- 11.Davies A R L, Hardick D J, Blagbrough I S, Potter B V L, Wolstenholme A J, Wonnacott S. Neuropharmacology. 1999;38:679–690. doi: 10.1016/s0028-3908(98)00221-4. [DOI] [PubMed] [Google Scholar]
- 12.Connolly J C, Kennedy C. Drugs Pharm Sci. 1998;89:107–133. [Google Scholar]
- 13.Bertrand D, Buissand B, Krause R M, Hu H Y, Bertrand S. J Recept Sig Transd Res. 1997;17:227–242. doi: 10.3109/10799899709036606. [DOI] [PubMed] [Google Scholar]
- 14.Catterall W A. J Biol Chem. 1974;250:1776–1781. [PubMed] [Google Scholar]
- 15.Lukas R J, Cullen M J. Anal Biochem. 1988;175:212–218. doi: 10.1016/0003-2697(88)90380-6. [DOI] [PubMed] [Google Scholar]
- 16.Villarroya M, López M G, Cano-Abad M F, Garcia A G. Methods Mol Biol. 1999;114:137–147. doi: 10.1385/1-59259-250-3:137. [DOI] [PubMed] [Google Scholar]
- 17.Veliçelebi G, Stauderman K A, Varney M A, Akong M, Hess S D, Johnson E C. Methods Enzymol. 1999;279:20–47. doi: 10.1016/s0076-6879(99)94005-3. [DOI] [PubMed] [Google Scholar]
- 18.Chavez-Noriega L E, Gillespie A, Stauderman K A, Crona J H, O'Neil Claeps B, Elliott K J, Reid R T, Rao T S, Veliçelebi G, Harpold M M, et al. Neuropharmacology. 2000;39:2543–2560. doi: 10.1016/s0028-3908(00)00134-9. [DOI] [PubMed] [Google Scholar]
- 19.Manning T J, Jr, Sontheimer H. J Neurosci Methods. 1999;91:73–81. doi: 10.1016/s0165-0270(99)00083-7. [DOI] [PubMed] [Google Scholar]
- 20.Kuntzweiler T A, Arneric S P, Donnelly-Roberts D L. Drug Dev Res. 1998;44:14–20. [Google Scholar]
- 21.Whiteaker K L, Gopalakrishnan S M, Groebe D, Sheih C C, Warrior U, Burns D J, Coghlan M J, Scott V E, Gopalakrishnan M. J Biomol Screen. 2001;6:305–312. doi: 10.1177/108705710100600504. [DOI] [PubMed] [Google Scholar]
- 22.Baxter D F, Kirk M, Garcia A F, Raimondi A, Holmqvist M H, Flint K K, Bojanic D, Distefano P S, Curtis R, Xie Y. J Biomol Screen. 2002;7:79–85. doi: 10.1177/108705710200700110. [DOI] [PubMed] [Google Scholar]
- 23.Lukas R J. J Pharmacol Exp Ther. 1986;265:294–302. [PubMed] [Google Scholar]
- 24.Lukas R, Norman S, Lucero L. Mol Cell Neurosci. 1993;4:1–12. doi: 10.1006/mcne.1993.1001. [DOI] [PubMed] [Google Scholar]
- 25.Xiao Y, Meyer E L, Thompson J M, Surin A, Wroblewski J, Kellar K J. Mol Pharmacol. 1996;54:322–333. doi: 10.1124/mol.54.2.322. [DOI] [PubMed] [Google Scholar]
- 26.Gopalakrishnan M, Monteggia L M, Anderson D J, Molinari E J, Piattoni-Kaplan M, Donnelly-Roberts D, Arneric S P, Sullivan J P. J Pharmacol Exp Ther. 1996;276:289–297. [PubMed] [Google Scholar]
- 27.Fenster C P, Rains M F, Noerager B, Quick M W, Lester R A J. J Neurosci. 1997;17:5747–5759. doi: 10.1523/JNEUROSCI.17-15-05747.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Marks M J, Robinson S F, Collins A C. J Pharmacol Exp Ther. 1996;277:1383–1396. [PubMed] [Google Scholar]
- 29.Zhang J, Xiao Y, Abrakhamanova G, Wang W, Cleeman L, Kellar K J, Morad M. Mol Pharmacol. 1999;55:970–981. doi: 10.1124/mol.55.6.970. [DOI] [PubMed] [Google Scholar]
- 30.Meyer E L, Xiao Y, Kellar K J. Mol Pharmacol. 2001;60:568–576. [PubMed] [Google Scholar]
- 31.Krahn T, Paffhausen W, Schade A, Bechem M, Schmidt D. U.S. Patent 6,420,183; (2002) Chem Abstr. 2002;128:59162. [Google Scholar]
- 32.Teicher B A. In: Human Cell Culture. Masters J R W, Palsson B, editors. Vol. 1. New York: Kluwer; 1999. pp. 1–19. [Google Scholar]
- 33.Tumilowicz J J, Nichols W W, Cholon J J, Greene A E. Cancer Res. 1970;30:2110–2118. [PubMed] [Google Scholar]
- 34.Clementi F, Cabrini D, Gotti C, Sher E. J Neurochem. 1986;47:291–297. doi: 10.1111/j.1471-4159.1986.tb02861.x. [DOI] [PubMed] [Google Scholar]
- 35.Nelson M E, Wang F, Kuryatov A, Choi D H, Gersanich V, Lindstrom J. J Gen Physiol. 2001;118:563–582. doi: 10.1085/jgp.118.5.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Ross R A, Spengler B A, Biedler J L. J Natl Cancer Inst. 1983;71:741–749. [PubMed] [Google Scholar]
- 37.Warpman U, Friberg L, Gillespie A, Hellström-Lindahl E, Zhang X, Nordberg A. J Neurochem. 1998;70:2028–2036. doi: 10.1046/j.1471-4159.1998.70052028.x. [DOI] [PubMed] [Google Scholar]
- 38.Theile C J. In: Human Cell Culture. Masters J R W, Palsson B, editors. Vol. 1. New York: Kluwer; 1999. pp. 21–53. [Google Scholar]
- 39.Minta A, Kao J P, Tsien R Y. J Biol Chem. 1989;264:8171–8178. [PubMed] [Google Scholar]
- 40.Grynkiewicz G, Poenie M, Tsien R Y. J Biol Chem. 1985;260:3440–3450. [PubMed] [Google Scholar]
- 41.Gee K R, Brown K A, Chen W N U, Bishop-Stewart J, Gray D, Johnson I. Cell Calcium. 2000;27:97–106. doi: 10.1054/ceca.1999.0095. [DOI] [PubMed] [Google Scholar]