Abstract
The α3/β4 rat neuronal nicotinic acetylcholine receptor, stably transfected in human embryonic kidney cells, was examined using the whole-cell-clamp technique and 2-dimensional confocal imaging. Application of agonists (nicotine, cytisine, epibatidine) activated a large (100–200 pA/pF) inwardly rectifying monovalent current, with little current at voltages between 0 and +40 mV. Rapid application of nicotine and cytisine indicated EC50 values of ≅22 and ≅64 μM, respectively, and suggested second order binding kinetics (Hill coefficient ∼2). The time constant of desensitization (decay) of nicotine-activated current was concentration-dependent (typically ∼10 s at 30 μM versus ∼1.0 s at 100–1000 μM), but not voltage-dependent and was significantly smaller than the ∼200 s reported for the α3/β4 receptor expressed in Xenopus oocytes. Nicotine-activated current was rapidly and reversibly blocked by coapplication of mecamylamine andd-tubocurarine. At −80 mV holding potentials, the current was also suppressed by ∼25% either upon complete removal or elevation of Ca2+ to 10 mM. Total replacement of Na+ by Ca2+ also completely blocked the current. On the other hand, evidence for permeation of Ca2+was indicated by increased inward current at −40 mV upon elevation of Ca2+ from 2 to 10 mM, as well as a rise in the cytosolic Ca2+ proportional to the current carried by the receptor. These findings are consistent with the idea that Ca2+, in addition to its channel-permeating properties, may also regulate the receptor from an extracellular site. Our results suggest that the α3/β4 neuronal nicotinic acetylcholine receptor, when stably expressed in human embryonic kidney 293 cells, has desensitization kinetics and Ca2+ regulatory mechanisms somewhat different from those described for the receptor expressed inXenopus oocytes.
Neuronal nicotinic acetylcholine receptors (nAChRs) composed of α and β subunits are differentially expressed in the nervous system and constitute a family of ligand-gated cationic channels. Eight α (α2–α9) and three β (β2–β4) neuronal subunit genes have been cloned in chicks, rodents, and humans, consistent with the idea that neurons may express nAChR with a variety of subunit combinations (see McGehee and Role, 1995; Lindstrom, 1996, for recent reviews).
The central nervous system expresses mRNA for all of the nAChR subunits. The receptors that contain the α3 subunit, although lower in abundance than α4, may mediate important effects of acetylcholine and nicotine in the brain (Connolly et al., 1995) and possibly in the spinal cord. Compared with α4/β2 receptors, nAChR containing α3 subunits appear to have much lower affinity for most nicotinic agonists in the central nervous system (Albuquerque et al., 1997).
The nAChR comprised of α3 subunits in combination with either β2 or β4 subunits may be the predominant nicotinic receptor in the mammalian sympathetic, parasympathetic, and trigeminal ganglia, and in adrenal chromaffin cells (Nooney and Feltz, 1995). Rat and chicken superior cervical ganglion, chick ciliary ganglion, and PC12 cells also express mRNA for designated α3, α5, β2, and β4 subunits (McGehee and Role, 1995).
Expression studies in Xenopus oocytes have shown that functional neuronal nAChRs can be formed by pairwise coinjection of one kind of α and one kind of β subunit (Boulter et al., 1987). The receptor forms with an apparent subunit stoichiometry of two α and three β (Anand et al., 1991) or, in some cases (α7 and α8), as homomeric assemblies (Séguéla et al., 1993). Both α and β subunits contribute to the pharmacological and biophysical properties of these receptors, giving each subunit combination unique characteristics (Wada et al., 1988; Luetje and Patrick, 1991).
In this report we explore the biophysical and pharmacological characteristics of the α3/β4 nAChR subtype in a stably transfected mammalian human embryonic kidney (HEK) 293 cell line recently developed in our laboratory (Xiao et al., 1998). Our results suggest that α3/β4-transfected cells generate a rapidly activating, voltage-dependent monovalent current on exposure to nicotinic receptor agonists. The current shows characteristic inward rectification, desensitization, kinetics, and pharmacology similar but not identical with the neuronal nAChR in chromaffin cells. The electrophysiological and confocal Ca2+ imaging data suggest that Ca2+ may both permeate and allosterically regulate the α3/β4 receptor (Lena and Changeux, 1993).
Experimental Procedures
Materials.
Stably transfected HEK 293 (ATCC CRL 1573)-expressing α3/β4 neuronal nAChRs (cell line designation: KXα3β4R2 cells) were prepared as described previously (Xiao et al., 1998). Cells were plated in tissue culture medium (Gibco-BRL, Gaithersburg, MD) containing bovine serum and antibiotics. ACh-, nicotine, andd-tubocuraine (d-tc) were obtained from Sigma Chemical Company (St. Louis, MO). Mecamylamine and epibatidine were purchased from Research Biochemicals (Natick, MA). A similar stable transfection of the rat α3/β4 receptor in HEK 293 cell line has been reported previously (Stetzer et al., 1996).
Cell Transfection and Culture.
HEK 293 cells were maintained at 37°C with 5% CO2 in the incubator. Growth medium for HEK 293 cells was minimum essential medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin. Stably transfected cell lines were raised in selective growth medium containing 0.7 mg/ml of Geneticin (G418). Transfection was conducted by the calcium phosphate method (Chen and Okayama, 1987). Briefly, exponentially growing HEK 293 cells were plated in 100-mm dishes containing 10 ml of the growth medium 24 h before transfection. For transfection 1.0 ml of solution containing 10 μg of linearized DNA, 125 mM CaCl2, 25 mM HEPES, 140 mM KCl, 6 mM glucose, 0.75 mM Na2HPO4(pH 7.05) was added to the cell-containing dish in drop-wise fashion. The cells were incubated with the transfection mixture for 16 h in the incubator and then were grown in fresh growth medium for 24 h. They were collected and plated at a range of densities. The selection process was continued for 3 to 4 weeks. G418-resistant clones were picked up by cloning cylinders and have been maintained in continuous culture for about 1 year.
Electrophysiological Recording.
Functional expression of nicotinic receptors was evaluated in the whole-cell configuration of the patch clamp technique using a DAGAN 8900 amplifier (Dagan Corp., Minneapolis, MN). The patch electrodes, pulled from borosilicate glass capillaries, had a resistance of 3 to 5 MΩ when filled with the internal solution containing (in mM): CsCl, 110; tetraethylammonium chloride, 20; NaCl, 0 to 20; MgATP, 5; EGTA, 14; HEPES, 20 and titrated to pH 7.2 with CsOH. About 90% of the electrode resistance was compensated electronically, so that effective series resistance in the cell-attached configuration was always less than 1 MΩ. Stably transfected HEK cells were studied 2 to 4 days after plating the cells on coverslips. Generation of voltage-clamp protocols and acquisition of data were carried out using pCLAMP software (Axon Instruments, Inc., Burlingame, CA). Sampling frequency was 0.5 to 2.0 kHz and current signals were filtered at 10 kHz before digitization and storage. All experiments were performed at room temperature (23–25°C). Currents were normalized relative to the membrane capacitance (10–30 pF) and then calculated as the mean ± S.E.M. for the number of cells examined (n).
Perfusion System.
Cells plated on plastic coverslips (15 mm round thermanox, Nunc, Inc., Napierville, IL) were transferred to an experimental chamber mounted on the stage of an inverted microscope (Diaphot, Nikon, Nagano, Japan) and were bathed in a solution containing (in mM): NaCl, 137; CaCl2, 2; HEPES, 10; glucose, 10; MgCl2, 1 (pH 7.4 with NaOH). The experimental chamber was constantly perfused at a rate of about 1 ml/min with the control bathing solution. The amplitude and time course of the nicotinic current was highly dependent on the speed of application of nicotine. A reduction in flow rate significantly slowed the activation, decreased the amplitude, and slowed the desensitization of the nicotinic current (Callewaert et al., 1991). We therefore used servo-controlled miniature solenoid valves for rapid switching between control and test solutions (Cleemann and Morad, 1991). The effective switching time was determined in rat ventricular cells by measuring the peak Na+ currents at various times after triggering a change in [Na+]o or by measuring the holding current at the tip of an open patch pipette subjected to a solution of low Cl- (Davies et al., 1988). Under optimal conditions, such changes in solution had a delay of 6 to 8 ms (corresponding mainly to the pull and release of the solenoid valves and replacement of fluid in the common outlet of the perfusion manifold) followed by a transition period where the measured current changed with a time constant of 5 to 10 ms. In repeated applications the delay was fairly reproducible, but it did show some variation in different experiments with different hydrostatic pressures and perfusion manifolds. The transition time was typically about 20 ms.
Confocal Microscopy.
Nicotine-induced Ca2+ signals in single voltage-clamped HEK cells were measured by adding 1 mM of the fluorescent Ca2+ indicator dye fluo-3 to the dialyzing pipette solution and scanning the cell at a rate of 30 frames per second with a confocal microscope (Odyssey XL, Noran Inc., Madison, WI; microscope: Zeiss, Axiovert 135, 40× Zeiss 440052 c-apochromat objective, NA. 1.2). At this rate, the microscope collects full two-dimensional frames of 640 × 480 pixels on a 0.207 μm grid by collecting data at 10 MHz and sweeping the rapidly scanned direction with an acousto-optical deflector at 17 kHz. Detected fluorescent light passes through a variable slit extending in the rapidly scanned direction so that the instrument is confocal only in the slowly scanned direction. Ca2+ signals were measured as the change in fluorescence intensity over the entire cell, or they were normalized relative to the fluorescence intensity before application of nicotine (ΔF/F). Cells examined with confocal microscopy were cultured on 25-mm glass coverslips and subjected to reduced flow rates.
Results
Agonist-Induced Current.
Stably transfected HEK 293 cells expressing α3/β4 nAChR generated rapidly activating inward current at −60 mV holding potentials when exposed to small concentrations of nACh receptor agonists. Figure 1 compares representative traces of current activated by application of 10 μM ACh (A), 10 μM nicotine (B), and 10 nM epibatidine (C). One micromolar concentrations of atropine were used to block any endogenous muscarinic receptors when ACh was used as an agonist. Because the time courses of activation and deactivation of the current on application and withdrawal of nicotinic agonists (Fig. 1) are significantly slower than the step change in agonist concentration (∼20 ms; see Fig. 1B of Davies et al., 1988; Tang et al., 1989), it is possible that the time courses of rise and fall of the current reflect the binding or gating kinetics of the nAChR channel. Comparison of activation and deactivation of the current shows clearly that epibatidine-evoked currents decayed significantly slower on removal of the drug than those induced by acetylcholine, nicotine, and cytisine (Figs. 1 and 2, A and B), consistent with the higher binding affinity of the drug to the receptor (Xiao et al., 1998). Comparison of the peak currents generated by epibatidine and nicotine at −60 mV holding potentials showed that 10 nM epibatidine generated currents (88.3 ± 24.8 S.E.M. pA/pF, n = 5) equivalent to those activated by 200 μM ACh (93.0 ± 34.5 pA/pF, n = 4).
Dose-Response Relation.
Figure 2 compares the dose dependence and kinetics of activation of α3/β4 nAChR to rapid application of various concentrations of nicotine and cytisine. The dose-response (D) was measured by exposing each cell to at least three concentrations of either nicotine or cytisine. Nicotine and cytisine at concentrations of ∼1 μM failed to activate a detectable current in α3/β4-expressing cells. At higher concentrations (10–1000 μM) nicotine and cytisine evoked a rapidly activating inward current that varied in its magnitude and gating kinetics with concentration. The peak amplitude of nicotine- and cytisine-activated currents at −80 mV, when plotted as a function of agonist concentrations, showed that nicotine and cytisine at concentrations of 100 to 200 μM generally produced their maximal responses. The dose-response relationships were normalized and fitted with the empirical Hill equationy = 1/(1+((EC50/[agonist])n)) yielding a cooperativity factor (n), which was close to 2 for both agonists (nicotine: 1.96 ± 0.30; cytisine: 2.05 ± 0.17). The data indicate an EC50 of 22 ± 2 μM for nicotine and 64 ± 7 μM for cytisine. The magnitude of the fully activated current (corresponding to unity in Fig. 2D) was 121 ± 16 pA/pF (n = 14) in the group of cells activated by nicotine and 182 ± 20 pA/pF (n = 11) in the cells activated by cytisine, suggesting about equal efficacy for the two compounds.
The differential potency was tested by measuring the responses to 50 μM of both compounds in each of eight cells (Fig. 2, A-C). The results showed that the peak current activated by nicotine was about twice as large as that activated by cytisine. Consulting the dose-response curves (Fig. 2D), this is consistent with the notion of equal efficacy. Notice, however, that the rate of desensitization was consistently larger for nicotine than for cytisine, so that the currents after 500 ms of drug exposure were almost identical (Fig. 2C). This suggests that the relative potency and efficacy of nicotine and cytisine may depend on the speed of drug application and the timing of current or flux measurements.
Voltage Dependence of Agonist-Induced Current.
Figure3 compares the voltage dependence of three nAChR agonists. Comparison of superimposed tracings of the current activated with different agonist concentrations and test potentials shows that: 1) agonist-activated currents decay little at low concentrations and moderate holding potentials (∼−40 mV) during a 1.5-s drug exposure period; 2) at higher concentrations, the current decays more rapidly after its activation; 3) little or no current is activated at 0 to +40 mV even when [Na+]i was increased from 0 to 20 mM (data not shown); 4) the deactivation times increase at high agonist concentrations; and 5) the voltage dependence of the agonist-induced current displayed prominent inward rectification (Fig.3, A-C, a characteristic of the whole-cell neuronal nAChR current measured in sympathetic neurons, Mathie et al., 1990; adrenal chromaffin cells, Callewaert et al., 1991; Nooney et al., 1992; and cultured hippocampal neurons, Bonfante-Cabarcas et al., 1996; or in HEK cells transiently transfected with α3/β4 nAChR, Wong et al., 1995).
Significant outward current was activated by nicotine or cytisine at potentials positive to +80 mV especially when using 200 to 1000 μM agonist concentrations (Fig. 3B). The voltage dependence of the epibatidine- and cytisine-induced current was similar to that of nicotine (Fig. 3A), i.e., large inward currents were measured at potentials negative to 0 mV with little or no current activated between 0 and 40 mV (Fig. 3, B and C). The most obvious difference between the epibatidine and nicotine or cytisine, in addition to the range of effective concentrations, was the off-rate of epibatidine current upon removal of the drug. Often at higher concentrations of epibatidine (1 μM), the activated current was significantly smaller than those generated by 100 nM epibatidine.
Intracellular magnesium has been proposed to regulate the rectification of the nicotinic current (Mathie et al., 1990; Albuquerque et al., 1997; Forster and Bertrand, 1995; Bonfante-Cabarcas et al., 1996). We also examined the effect of [Mg2+]i on the voltage dependence of the nicotine-activated current, but found no significant change in the rectification or the voltage dependence of the current even when cells were dialyzed for 10 to 15 min with Mg2+-free intracellular solutions containing 2 mM of the chelator EDTA (data not shown). Similarly, removal of extracellular Mg2+ had no measurable effects on the magnitude or the inward rectifying properties of the current. A similar insensitivity to Mg2+ was reported in α4/β2-expressing HEK cells (Buisson et al., 1996).
Decay of Agonist-Activated Current.
Nicotine-activated current decayed significantly in the presence of the drug, resulting in part from desensitization of the α3/β4 receptor. Time constants of the decay of the current in the presence of nicotine typically ranged between 1.0 and 10.0 s, depending on the agonist concentration (Fig. 4A and B). After activation of the receptor by 30 μM, 100 μM, and 1 mM nicotine, the time constant of the decay of the current at −80 mV was found to be respectively 11 ± 6 (n = 3), 1.4 ± 0.3 (n = 5), and 2.0 ± 0.4 (n = 4) s (○, Fig. 4B). The rate constant of decay of the current was mostly independent of membrane potential (■, −120 mV; ○, −80 mV; ▵, −40 mV), but increased with increasing concentration (Fig. 4B).
At high nicotine concentrations, the decay kinetics of current may also reflect, in part, the block of the open channel by the agonist (Sine and Steinbach, 1984). Consistent with this idea, the rapid removal of 1 mM nicotine or cytisine were often accompanied by the activation of a “rebound” current, which may represent the rapid unblock of the channel by the agonist (Fig. 4A, top right; see also Fig. 7A). The rebound current was most prominent at voltages negative to −60 mV, was absent at voltages between −40 to +80 mV, was highly concentration-dependent, and appeared to be somewhat enhanced at higher concentrations of Ca2+ (Fig. 7A). A detailed analysis of the rebound current in muscle end-plate acetylcholine receptor has been recently reported by Maconochie and Steinbach (1998)using 1.0 to 10 mM ACh.
In some cells we observed a 10-fold faster rate of desensitization (Fig. 4C), which was also accelerated by increasing nicotine concentrations (● Fig. 4B). The cells on a given day of experimentation would all have either slow or fast desensitization. During an experiment lasting 5 to 20 min the time constant of desensitization would often decrease by a factor of 2, but not enough to alter a slowly desensitizing cell to a fast one.
Nicotine-Activated Current Was Blocked by Mecamylamine andd-tc.
To further characterize the properties of the nicotine-induced current in α3/β4-expressing cells, we examined the kinetics, voltage, and concentration dependence of two well known nAChR blockers. To determine the relative speed of the antagonist action vis-à-vis the agonist-activated current, nicotine and the antagonist were coapplied to the cell. Rapid application of mecamylamine (1 μM) simultaneous with 10 μM nicotine suppressed the nicotine-induced current by ≅80% (n = 4; Fig.5A and C). The suppressive effect of the drug was voltage-dependent such that at −120 mV,mecamylamine blocked the current by ∼80% versus ∼60% at −80 mV (Fig. 5B). Mecamylamine also significantly enhanced the decay kinetics of the nicotine-activated current, consistent with the open channel-blocking property of the drug (Fig. 5A). Nicotine-activated current recovered rapidly upon washout of the blocker. Thus, mecamylamine appears to access the channel pore in a voltage- and time-dependent manner. The kinetics of the block at the 1 μM concentration were sufficiently fast as to prevent most of the nicotine-activated current when the agonist and antagonist were coapplied.
Similarly, d-tc (50 μM) when coapplied with 30 μM nicotine blocked the current rapidly (Fig.6). Concentrations of d-tc smaller than 10 μM, when coapplied with nicotine, had little or no effect on the current activated by nicotine (n = 3, data not shown). The onset of d-tc block, similar to mecamylamine, was fast and consistent with channel-blocking properties of these drugs (Fig. 6A).
Calcium Permeation and Block of Rat α3/β4 nAChR.
Reliable measurements of reversal potential are required to assess directly the ionic selectivity of agonist-induced current. Because little or no outward current could be measured on activation of the nicotinic receptor between 0 and +40 mV, the measurements of reversal potentials were less reliable. Thus, we attempted to evaluate the permeation of Ca2+ based on the larger current measured at very negative and positive potentials.
Figure 7A shows how the current through the α3/β4 nAChR was modified when the external Ca2+ concentrations were increased from 2.0 to 10 mM during application of 50 μM nicotine. At −120 mV and −80 mV the higher [Ca2+]o suppressed the current and the washout of the drug was accompanied by a noticeable rebound current, as if the blocking effect of Ca2+ washed out before the deactivation of the channel. In addition, the current activated by 50 μM nicotine, at −80 mV, was slightly suppressed when [Ca2+]o was reduced to 0.2 mM (○, Fig. 7B). This trend became significant in completely Ca2+-free solution over a wide range of nicotine concentrations (20 (▪), 200 (●) μM nicotine). Significant suppressions were also observed at 5 and 10 mM [Ca2+]o. Complete replacement of all the external NaCl with 90 mM CaCl2, completely blocked the inward current through the receptors during the drug exposure period (Fig. 7B) and suppressed the outward current at +120 mV to 18 ± 11% (n = 5) of values measured with 2 mM [Ca2+]o. Thus, if Ca2+ in addition to its blocking effect were also to permeate the channel, it would most likely generate significant current at the moderate Ca2+ concentrations. Accordingly, at 10 mM Ca2+ (Fig. 7C), the nicotine-induced currents appeared to decrease at −120, −80, and +120 mV, increase at −40 mV and remain undetectable at 0 and +40 mV. These changes were significant when measured relative to the current recorded in control solution with 2 mM Ca2+. The enhanced inward current at −40 mV suggest that Ca2+ may permeate through the nAChR channel and shift the reversal potential toward more negative potentials.
To determine more directly whether activation of nicotine-induced current results in rise of [Ca2+]i, we measured confocal images of Ca2+ in Fluo 3-dialyzed voltage clamped cells. Figure 8A shows simultaneous traces of nicotine-activated current and intracellular Ca2+ in response to rapid application of 100 μM nicotine recorded at indicated locations in the cell. Cytosolic Ca2+ appears to rise more rapidly at peripheral and flatter segments of the cell (Fig. 8B, open symbols; see also Fig.9) than at the thicker midsection of the cell (Fig. 9B, closed symbols) even though the thicker segments seem to achieve higher “apparent” Ca2+concentrations. Intracellular Ca2+ (ΔF/F) rises continuously during the application of nicotine and decays slowly upon removal of nicotine. Comparison of total charge carried by the nicotinic channel in five different cells (represented by different symbols in Fig. 8C) and change in fluorescence (ΔF/F) showed direct correspondence of current and ΔF/F (Fig. 8C), suggesting that the rise in Ca2+ is directly correlated to the ionic charge transported by the α3/β4 receptor. Mecamylamine strongly suppressed the nicotine-activated current and simultaneously inhibited the rise in [Ca2+]i (Fig.8C, open symbols), suggesting that the nicotinic channel was responsible for the rise in Ca2+.
The experiment illustrated in Fig. 9 was performed to test the possibility that the influx and intracellular accumulation of Na+ might mediate the rise in cytosolic Ca2+ via transport of Ca2+on the Na+-Ca2+ exchanger. Such a mechanism might be accentuated in flatter and narrower parts of the cell where Na+ as well as Ca2+ could readily build up as a result of large agonist-activated current. We therefore compared the Ca2+ signals measured at −80-mV holding potentials, when K+ was substituted as extracellular charge carrier for Na+. In a set of three experiments, we found that nicotine-activated currents and the rise in cytosolic Ca2+ were similar whether Na+ (Control) or K+ (KCl) was the charge carrier through the channel (Fig. 9). The inset frames of Fig. 9 show that the ratiometric Ca2+ signals (ΔF/F) increase in strength toward the distal ends of the cellular protrusions more rapidly than the center of the cell and confirm that K+ substitution alters neither the magnitude nor the cellular distribution of the Ca2+ signals. Because K+ is known not to substitute for Na+ on the Na+-Ca2+ exchanger, this finding strongly suggests that the Ca2+ signals are not produced secondary to influx of Na+, but are directly related to the permeation of Ca2+via the nAChR channel.
Discussion
In response to application of nicotinic agonists (ACh, nicotine, cytisine, and epibatidine), HEK cells stably transfected with rat α3/β4 nAChR generate a large rapidly activating and slowly decaying current. This current displayed inwardly rectifying properties (generating little or no current between 0 and +40 mV, Fig. 3) and was rapidly and reversibly blocked by mecamylamine and d-tc (Figs. 5 and 6). The nicotine-activated channel, at physiological Ca2+ concentrations, appears to transport sufficient Ca2+ as to induce significant rise in cytosolic Ca2+ concentrations (Figs. 8 and 9). At higher concentrations, Ca2+ appears to suppress the Na+ current through the receptor, consistent with Ca2+ serving as a permanent blocker, but the complete block of the channel on isoosmolar replacement of Na+ by Ca2+ may suggest an additional regulatory role for extracellular Ca2+.
Dose-Response of Nicotine and Cytisine.
Nicotine and cytisine appeared to activate the α3/β4 recombinant receptor with EC50 values of about 22 and 64 μM, respectively, and with a cooperativity factor of ∼2.0 for both agonists. Hill coefficients ranging between 1.5 and 2.0 have been reported previously for both the recombinant and wild types of nAChR (Lindstrom, 1995; Wong et al., 1995). Significantly lower Hill coefficients of 0.6 and 1.4 were recently reported for the human α3/β4 recombinant receptor stably expressed in HEK cells (Stauderman et al., 1998). However, in those experiments, the coefficients were derived from measurements of intracellular Ca2+ in large populations of cells in response to slow application of nicotinic receptor agonists and could reflect both desensitization of the receptor and indirect functional coupling between activation of nicotinic receptor and rise in [Ca2+]i. Our electrophysiological measurements, based on rapid application of agonists, on the other hand, appear to fit a Hill coefficient close to 2.0 for both cytisine and nicotine (Fig. 2) as found also with human α3/β4 expressed in oocytes (Chavez-Noriega et al., 1997). Cytisine is generally reported to be at least as potent as nicotine in activating the α3/β4 receptor to generate inward current in oocytes (Chavez-Noriega et al., 1997; Gerzanich et al., 1998) or Ca2+ influx in HEK cells (Stauderman et al., 1998). In our experiments, we found a somewhat higher EC50 value for cytisine (Fig. 2D). However, this may be in part due to measuring the current shortly after the drug application and before the development of agonist-induced block or desensitization (Fig. 2, A-C). Because such rapid measurements are not possible in whole oocytes or dense populations of eukaryotic cells where average activation times of the receptor are significantly slower (>5 s), the previously reported dose-response relations may reflect the steady-state component of the agonist-activated current.
Decay of Nicotine-Activated Current.
The rate of decay of nicotine-induced current in the presence of nicotine was strongly concentration-dependent, such that at low concentrations of agonists (10–30 μM) time constants in excess of 10 s were required for full decay of the current, whereas at 100 to 1000 μM drug concentrations the current decayed within 1 to 2 s (Fig. 4).
Although it is tempting to attribute the decay of the current entirely to the desensitization of the receptor, the decay of current in the presence of the agonist may also reflect, in part, agonist-induced block of the channel. This might be particularly the case at higher concentrations of agonists, as demonstrated for the muscle endplate nicotinic receptors (Sine and Steinbach, 1984; Maconochie and Steinbach, 1998). The bell-shaped dose-response relation was also seen in the α3/β4-expressing HEK cells in our laboratory using Rb+ efflux to measure channel function (Xiao et al., 1998). That agonist may serve as a channel blocker at high concentrations is also consistent with significant acceleration in the decay kinetics of α3/β4-generated current (Fig. 3) and the enhancement of “rebound” current at negative potentials (Figs. 3B,4A, and 7A). Our data are, therefore, consistent with the idea that at higher concentrations and negative potentials ACh, nicotine, and cytisine may gain access to the channel permeation sites and serve as open channel blockers (see scheme proposed by Colquhoun et al., 1987;Maconochie and Steinbach, 1998).
Comparing the kinetics of decay of nicotine-activated current in α3/β4-expressing HEK cells with those of primary cultures of adult rat chromaffin cells exposed to the same set of experimental conditions and techniques suggests significantly slower decay kinetics of ACh- or nicotine-activated current in recombinant versus the native receptor. The time constant of decay of nicotinic current in primary cultures of rat chromaffin cells was 324 ± 58 ms (n = 5) when activated with 30 μM and 128 ± 26 ms (n = 5) with 1 mM nicotine compared with 10.9 ± 5.9 s (n = 3) and 2.1 ± 0.3 s (n = 4), respectively, in transfected HEK cells expressing the α3/β4 recombinant receptor.
In some populations of cells, the rate of decay of the current was much faster, by as much as an order of magnitude (cf. Fig. 4, A and C), approaching the decay kinetics of the current recorded in primary cultures of rat chromaffin cells (Callewaert et al., 1991). Such cell populations were relatively rare, and their occurrence could not be attributed to any known culture or experimental condition.
The kinetics of decay of α3/β4 current reported here are significantly faster than those recently reported for this receptor expressed in Xenopus oocytes. At 50 μM nicotine concentrations, time constants of about 200 s were measured in oocytes (Fenster et al., 1997), compared with about 1.0 s in HEK cells (Fig. 4). Such differences may reflect the role of host cells in determining the regulatory properties of neuronal nicotinic receptors (Lester and Dani, 1995).
Ca2+ Permeability of α3/β4 Receptor.
Permeation of Ca2+ through the native and the recombinant nicotinic receptor has been the subject of considerable interest and investigation. From the shift in the reversal potential, on fractional increases of extracellular Ca2+, a PCa/PNa of 0.9 for α3/β4 and 0.1 for the muscle α1/β1/γ/δ expressed in oocyte have been estimated (Costa et al., 1994). Direct comparison of charge transported by the channel and the rise of intracellular Ca2+ measured in chromaffin cells and in oocytes expressing α3/β4 suggest that approximately 2 to 4% of the total charge may be transported by Ca2+ at physiological Ca2+ concentrations (Vernino et al., 1992; Decker and Dani, 1990). Unexpectedly, however, Vernino et al. (1992) also found that a 10-fold increase in [Ca2+]o also enhanced the outward current through the α3/β4 receptor at positive (+30 mV) membrane potentials, where enhancement of inward Ca2+ current was expected (see also Amador and Dani, 1995). These findings support the idea that Ca2+ may both permeate and allosterically regulate the nAChR from an extracellular site (Lena and Changeux, 1993). Consistent with a possible regulatory role of Ca2+ on nAChR, Buisson et al. (1996) reported a 50% suppression of the current on a 10-fold increase of [Ca2+]o in α4/β2-transfected HEK cells. The decrease in the whole-cell current appears to be carried by suppression of the single channel conductance on elevation of [Ca2+]oin both the native and recombinant receptors (Vernino et al., 1992;Mulle et al., 1992; Amador and Dani, 1995; Buisson et al., 1996).
Our electrophysiological data on α3/β4-transfected HEK cells showed a bell shaped response for the magnitude of the current versus the extracellular Ca2+ concentration (Fig. 7B). The reduction of the current at low Ca2+concentrations (Fig. 7B) may in part reflect a surface charge effect that would shift the current-voltage relations to the left when the divalent cation concentrations were lowered. The elevations of Ca2+ from 2 to 10 to 90 mM partially or completely (Fig. 7B) blocked the nicotine-activated current, consistent with the data in α4/β2-expressing HEK cells (Bouisson et al., 1996). However, detailed examination of the current-voltage relations (Fig. 7C) showed that elevation of [Ca2+]o increased the inward current at −40 mV. This finding is consistent with the measurements in oocytes where the currents are large enough to reveal the effects of Ca2+ at and near the reversal potential (Gerzanich et al., 1998). Our findings are in part consistent with the idea that Ca2+ permeates through the nAChR channel, but in the process briefly occludes the channel, thereby lowering its single channel conductance. However, such a mechanism would require that the blocking effect depend on the electrical potential driving Ca2+ into the channel, somewhat inconsistent with Fig. 7D where the degree of block appears to be equivalent at −120 mV and +120 mV.
Direct measurement of intracellular Ca2+ (Figs. 8and 9) shows that the rise in intracellular Ca2+was directly related to the magnitude of the charge transferred through the receptor (Fig. 8, a 10–30% rise of ΔF/F for currents ranging from 1.5–4 nA). Because these cells were dialyzed with 1 mM fluo-3 (K d ≅ 300 nM) and assuming resting Ca2+ activity of ≅100 nM, the predicted rise in [Ca2+]i is about 33 to 100 μM, producing PCa/PNa≈ 1, consistent with the PCa/PNa values measured in native cells or α3/β4 receptor expressed in oocytes (Costa et al., 1994; Vernino et al., 1992), but is not easily reconciled with the complete suppression of inward current after replacement of all Na+ with Ca2+ (Fig. 7B). We also considered whether the rise in cytosolic Ca2+ occurs indirectly through a voltage-gated Ca2+ channel or via the Na+-Ca2+ exchanger. The persistence of the Ca2+ signals at −80 mV and when Na+ was replaced by K+as the charge carrier (Fig. 9), however, suggests that Ca2+ permeates directly through the nAChR channel. Thus, fairly small and difficult to detect Ca2+ currents through the α3/β4 receptor may cause significant increases in cytosolic Ca2+ in transfected HEK cells (average cell volume and capacitance, 4 pl and 20 pF; Figs. 8 and 9). Although our Ca2+ imaging data are consistent with the findings of others on native and recombinant α3/β4 receptor measured in neurons, oocytes, and eukaryotic cells, the suppressive effect of Ca2+on the α3/β4 receptor appears to be more consistent with that observed in HEK cells (Bouisson et al., 1996) rather than oocytes (Vernino et al., 1992). It is intriguing to speculate whether the regulation of Ca2+ permeability of nAChR may, in part, depend on endogenous presence of other Ca2+-sensitive ion transporters or subunit assembly of the receptor in the host cells (Lewis et al., 1997; Cooper and Miller, 1997).
Footnotes
- Received November 4, 1998.
- Accepted March 15, 1999.
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Send reprint requests to: Dr. Martin Morad, Georgetown University Medical Center, Department of Pharmacology, 3900 Reservoir Road N.W., Washington, DC 20007. E-mail: moradm{at}gunet.georgetown.edu
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Supported by National Institutes of Health Grants HL16152 and DA06486.
Abbreviations
- nAChR
- nicotinic acetylcholine receptor
- Ach
- acetylcholine
- HEK
- human embryonic kidney
- d-tc
- d-tubocuraine
- The American Society for Pharmacology and Experimental Therapeutics