Introduction

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Transmission at fast chemical synapses is a fundamental process in the central and peripheral nervous systems. The best-studied example is the neuromuscular junction. The nicotinic acetylcholine receptor (nAChR) mediates rapid synaptic transmission by increasing the conductance of the postsynaptic cell membrane in response to nerve-released ACh. High speed is achieved because both the receptor and the ion channel are parts of a single protein molecule.

One complicating feature of neuromuscular transmission is the presence of multiple binding sites for ACh. There are two nonidentical ACh binding sites on the receptor itself. The low affinity agonist blocking site within the pore of the channel is a third site. Acetylcholinesterase also recognizes ACh. Extrasynaptic muscle ACh receptors and presynaptic neuronal ACh receptors provide additional ACh binding sites. A neuronal subtype ACh receptor has been found on the postsynaptic membrane as well (Tsuneki et al., 1995). The presence of competitive inhibitors (muscle relaxants) to block movement during surgery introduces yet another molecule that can bind to the ACh receptors. Finally, because ACh is rapidly released and hydrolyzed, equilibrium conditions are never reached within the synapse.

A detailed description of the structure and function of the ACh receptor is emerging from several areas of investigation. Electron microscopy of receptors from Torpedo californica electroplax membranes has provided structural images at 4.6-Å resolution (Miyazawa et al., 1999) and has revealed structural differences between the open and closed states (Unwin, 1995). Site-directed mutagenesis studies have identified particular amino acids involved in agonist and antagonist binding (Arias, 2000) and ion selectivity and permeation (Corringer et al., 2000). High-resolution, single-channel measurements have determined the rate constants for transitions between states of the receptor (Colquhoun and Sakmann, 1981; Zhang et al., 1995; Maconochie and Steinbach, 1998) and lower limits on the speed of channel opening (Maconochie et al., 1995; Parzefall et al., 1998). Patch-clamp recording during rapid application of agonist has provided a controlled way of mimicking the physiological exposure of ACh receptors to ACh (Brett et al., 1986; Franke et al., 1987; Maconochie and Knight, 1989).

Measurements of the kinetics of agonist association suggest that they bind more slowly than predicted from simple diffusion (Gutfreund, 1972; Zhang et al., 1995). Structural studies of T. californica nAChR suggest the existence of ACh binding pockets accessible through narrow tunnels in the channel wall (Miyazawa et al., 1999). Diffusion through these tunnels may be the limiting factor for ACh association. It would be interesting to see if this is also true for antagonists. The binding affinity of the prototypical antagonist, (+)-tubocurarine, has been determined as 400 nM in frog muscle (Jenkinson, 1960) and 250 nM in rat muscle (Colquhoun and Rang, 1976). However, measurements of the kinetics of antagonist binding have produced inconsistent results. Estimates of the association rate of the prototypical antagonist, (+)-tubocurarine range from 10⁹ M¹ s¹ (Colquhoun and Sheridan, 1982; Le Dain et al., 1991) to 5 × 10⁵ M¹ s¹ (Bufler et al., 1996). Similarly, estimates of the dissociation rate range from 1000 to 4000/s (Colquhoun and Sheridan, 1982; Le Dain et al., 1991) to 0.1/s (Bufler et al., 1996). We undertook this study to try to resolve these discrepancies. Pancuronium was also studied as an example of a higher affinity competitive antagonist in current use as a neuromuscular blocking agent.

	Materials and Methods

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We studied the embryonic mouse muscle nicotinic ACh receptors expressed in clonal BC3H-1 cells. The cells were cultured as described previously (Sine and Steinbach, 1984). To prepare cells for patch-clamp recording, the culture medium was replaced with an "extracellular" solution (ECS) containing 150 mM NaCl, 5.6 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, and 10 mM HEPES, pH 7.3. Patch pipettes were fabricated from borosilicate glass and firepolished. They were filled with a solution consisting of 140 mM KCl, 5 mM EGTA, 5 MgCl₂, 10 (+)-glucose, and 10 mM HEPES, pH 7.3, and had resistances of 3 to 5 M. An outside-out patch (Hamill et al., 1981) with a seal resistance of 5 G or greater was obtained from a cell and moved into position at the outflow of a perfusion system. The perfusion system [modified from Liu and Dilger (1991)] consisted of solution reservoirs; computer-controlled, solenoid-driven pinch valves; and a three-tube device immersed in the culture dish. The three tubes were made of glass and were attached with epoxy so that they were coplanar and at 45° angles from each other. One tube was connected to a reservoir containing ECS without agonist (control), the second arm was connected to a reservoir containing ECS with 100 µM ACh (agonist solution), and the third arm was connected to a reservoir containing ECS with either (+)-tubocurarine or pancuronium (test solution) [(+)-tubocurarine chloride and pancuronium bromide; Sigma Chemical Co., St. Louis, MO]. In the resting position, control solution perfused the patch. The perfusion system allowed for a rapid solution exchange within 100 to 200 µs between each tube. When making onset- or recovery experiments (see below), we could reliably achieve a minimum duration of 10 ms for the second solution. We tested the timing systematically using an open electrode perfused with solutions of different ionic strength and confirmed the optimal position of the electrode frequently using open electrode tests.

The currents flowing during exposure of the patch to ACh were measured with a patch clamp amplifier (EPC-9; List Medical, Darmstadt, Germany), filtered at 3 kHz (3 db frequency, eight-pole Bessel filter), digitized and stored on the hard disk of a laboratory computer. Data analysis was performed off-line as described previously (Dilger et al., 1997). Experiments were performed at room temperature (20-23°C.). We recorded current responses (at 50 mV) during 100- to 200-ms applications (at 5-s intervals and sampled at 100-200 µs per point) of ECS containing 100 µM ACh, a concentration that opens about 93% of the nAChR channels from BC3H-1 cells (Dilger and Brett, 1990). We subsequently used this test solution to quantify loss of channel activity.

To determine antagonist onset kinetics, we equilibrated the patch with ECS, perfused with antagonist for a variable time interval (10-1500 ms) and then measured the current response to 100- to 200-ms applications of 100 µM ACh to assess the fraction of antagonist-free channels. To determine recovery kinetics, we equilibrated the patch with antagonist, removed antagonist for variable time intervals (10-1500 ms), and then applied ACh. We repeated this with various concentrations of antagonist. The association and dissociation rate constants were calculated from the assumption of a single-site binding model; this corresponds to the higher affinity antagonist binding site (Fletcher and Steinbach, 1996). Responses of the patch to 100 µM ACh applications were measured before and after each onset/recovery protocol to test for loss of channel activity. Data were accepted when these two differed by less than 10%. The ensemble mean current was calculated from 10 to 20 individual current traces. Under most circumstances, mean currents were fit to single exponential functions to obtain peak and steady-state current values and a time constant of the decay caused by desensitization. At high concentrations of (+)-tubocurarine, the currents had a biphasic time course (see Fig. 2). In these cases, the currents were fit to a biexponential function to determine the current at the end of the initial activation phase. The initial mean current was defined as the current after the initial activation phase. We calculated the ratio of this current in the presence and absence of the antagonists.

Data are expressed and graphed as mean ± S.D. To facilitate statistical comparison, we performed curve fits using all data points rather than the mean values. Parameters derived from curve fitting are expressed as best-fit values ± S.D. Statistical comparisons are made using an unpaired two-tailed t test. To compare association rates, we performed the t test based on the linear regression analysis (Igor Pro software; Wavemetrics, Lake Oswego, OR) of the reciprocal of the onset time constant versus antagonist concentration (see eq. 3). This results in a mean value and S.D. of the slope.

	Results

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Competitive Antagonism under Equilibrium Conditions. In these experiments, we equilibrated the patch with various concentrations of the antagonist for >1 min before applying a mixture of 100 µM ACh and the antagonist. Figure 1a shows examples of currents seen in a single patch with 0, 3, and 30 nM pancuronium. The control trace exhibits a rapid activation phase whereas 100 µM ACh activates about 93% of the receptors and a desensitization phase with a time constant of 53 ms. The main effect of pancuronium is to reduce the peak current. The decay phase is still described by a single exponential function, but the decay rate is slowed at high concentrations of pancuronium (the decay time constant was 200 ms at 30 nM.). Similar effects are seen with low concentrations of (+)-tubocurarine (Fig. 1b). However, at high concentrations of (+)-tubocurarine, the current exhibits a biphasic time course after the initial activation phase. Figure 2 shows an example of a current in the presence of 200 nM (+)-tubocurarine along with a 2-exponential fitting function that describes the biphasic time course.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   A, the effects of 3 and 30 nM pancuronium on currents evoked by rapid perfusion of 100 µM ACh to an outside out patch. V = 50 mV. Two control curves are shown. The traces for 30 nM and the corresponding control were scaled by a factor of 1.072 to compensate for loss of channel activity during this experiment. B, the effects of 25 and 75 nM (+)-tubocurarine on currents evoked by rapid perfusion of 100 µM ACh to an outside out patch. V = 50 mV. Two control curves are shown. The traces for 25 nM and the corresponding control were scaled by a factor of 1.094 to compensate for loss of channel activity during this experiment (there were 55 min between the two determinations).

View larger version (36K): [in this window] [in a new window]   Fig. 2.   An example of the biphasic current response to 100 µM ACh in the presence of 200 nM (+)-tubocurarine. The inset shows the two exponential fit used to characterize such responses. The fit functions were (currents in pA, time in ms; control fits not shown): control 1, I(t) = 16 245*exp(t/47.1); 200 nM (+)-tubocurarine, I(t) = 2.61 65.1*exp(t/71.3) + 28.8*exp(t/17.8); control 2, I(t) = 13.6 227*exp(t/45.8)

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View larger version (20K): [in this window] [in a new window]   Fig. 3.   Equilibrium concentration response curves for pancuronium and (+)-tubocurarine on nAChR from BC3H-1 cells. The solid lines are fits of eq. 1 to the data. The fitting parameters are given in Table 1.

View this table: [in this window] [in a new window]   TABLE 1 Equilibrium and kinetic parameters for pancuronium and (+)-tubocurarine obtained from fitting the data in Figs. 3 and 8 The two binding site model (eq. 2) did not converge for the (+)-tubocurarine data. Residency time and off/on are calculated using the value of off determined from recovery experiments. The number of original data points (before averaging by concentration) is given in parentheses.

The Kinetics of Competitive Antagonism. We speculated that the biphasic current response seen with (+)-tubocurarine but not with pancuronium might be related to the rate at which the drug dissociates from the receptor. If (+)-tubocurarine were to dissociate on the 100-ms time scale and pancuronium were to dissociate more slowly, the slow rise in current might be caused by ACh binding to receptors previously bound by (+)-tubocurarine. Such behavior has been predicted (Rang, 1966; Aoshima et al., 1992). Our first experimental approach to determining the kinetics of (+)-tubocurarine is illustrated in Fig. 4. Channels were activated by 3 µM ACh, a concentration that induces only a small amount of desensitization within 200 ms. Equilibrium application of 500 nM (+)-tubocurarine inhibited the current to 15% of control. Simultaneous application of ACh and (+)-tubocurarine produced a time-dependent current that decayed from the control level to the equilibrium level with a time constant of 16 ms (Fig. 4A.). Equilibration with (+)-tubocurarine followed by perfusion of ACh without (+)-tubocurarine produced a relaxation from the equilibrium to the control level with a time constant of 75 ms (Fig. 4B.). These two numbers provided a first estimate of the kinetics of (+)-tubocurarine association and dissociation. This experimental approach is limited in that fast relaxation times are distorted by the onset of the ACh-activated currents and slow relaxation times are distorted by desensitization.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   The kinetics of inhibition by 400 nM curare revealed by different perfusion protocols with a nonsaturating concentration of ACh. 3 µM ACh, V = 50 mV. The gray traces are control and equilibrium exposure to curare. Black traces were fit to a single exponential decay; the time constants are indicated. A, the black trace was obtained by simultaneous perfusion of ACh and (+)-tubocurarine. B, the black trace was obtained by simultaneous perfusion of ACh and removal of (+)-tubocurarine.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Examples of onset kinetics obtained using the 3-tube perfusion system. A, family of 20 traces for 20 nM pancuronium. The three black traces are for 0-, 151-, and 428-ms exposure to pancuronium before application of 100 µM ACh. B, family of 20 traces for 150 nM (+)-tubocurarine. The three black traces are for 0-, 60-, and 428-ms exposure to (+)-tubocurarine before application of 100 µM ACh.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Examples of recovery kinetics obtained using the 3-tube perfusion system. Data are from the same patches as shown in Fig. 5. A, family of 20 traces for 20 nM pancuronium. The three black traces are for 20, 341, and 1430 ms after removal of pancuronium before application of 100 µM ACh. B, family of 20 traces for 150 nM (+)-tubocurarine. The three black traces are for 20, 107, and 1050 ms after removal of (+)-tubocurarine before application of 100 µM ACh.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   The time course of onset and recovery of inhibition for 20 nM pancuronium (A) and 150 nM (+)-tubocurarine (B). Data are from the experiments shown in Figs. 5 and 6.

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View larger version (16K): [in this window] [in a new window]   Fig. 8.   The concentration-dependence of onset () and recovery () rates for pancuronium (A) and (+)-tubocurarine (B). The solid lines are fits of the data to eqs. 4 and 5. The fit parameters are given in Table 1.

	Discussion

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(+)-Tubocurarine and pancuronium are potent neuromuscular blockers used during general anesthesia to provide muscle relaxation. Both drugs have a long history of in vitro and in vivo investigations.

We examined the effects of these antagonists on outside-out patches containing embryonic mouse nAChRs using a two-tube protocol to measure equilibrium inhibition by antagonists. We found IC₅₀ values of 5.5 and 41 nM for pancuronium and (+)-tubocurarine, respectively. Our data did not allow us to determine the low affinity constants with any precision. These results are in good agreement with published data on BC₃H1 cells and the QF18 cell line expressing embryonic mouse nAChRs. Bungarotoxin displacement experiments on BC₃H1 cells reveal pancuronium affinities of 9.1 and 69 nM (Sine and Taylor, 1981). Similar experiments on QF18 cells reveal pancuronium affinities of 8 and 95 nM and (+)-tubocurarine affinities of 71 and 1100 nM (Steinbach and Chen, 1995; Fletcher and Steinbach, 1996). Functional measurements reveal similar affinities. In BC₃H1 cells, Na⁺ permeability was inhibited by 7.4 nM pancuronium with a Hill coefficient of 1.16 (Sine and Taylor, 1981). In QF18 cells, ion currents were inhibited by 5 nM pancuronium with a Hill coefficient of 1.23 (Fletcher and Steinbach, 1996) and 56 nM (+)-tubocurarine with a Hill coefficient of 1.06 (Steinbach and Chen, 1995).

In agreement with other researchers (Steinbach and Chen, 1995; Fletcher and Steinbach, 1996), we found that (+)-tubocurarine, but not pancuronium, acts as a partial agonist on embryonic mouse nAChR (single channel data not shown). (+)-Tubocurarine activates receptors in the absence of ACh and also increases channel activity at low concentrations of ACh. The latter effect is caused by activation of hetero-liganded receptors containing ACh at one binding site and (+)-tubocurarine at the other. The efficacy of both of these opening pathways is very low (Steinbach and Chen, 1995). In rapid perfusion experiments with 100 µM ACh, the baseline currents in the presence and absence of (+)-tubocurarine are indistinguishable. Thus, it is unlikely that these opening pathways interfere with out kinetic measurements.

We used the three-tube perfusion protocol to measure the kinetics of antagonist inhibition. In an onset experiment, a patch was exposed to a drug for varying intervals before application of ACh. The shortest interval that could reliably be attained was 10 ms. This allowed for rates <50/s to be measured; the fastest rate we observed in these experiments was 30/s. Pancuronium and (+)-tubocurarine have significantly different association rates: 2.7 and 1.2 × 10⁸ M¹ s¹, respectively. The drugs also differ in their dissociation rates: 2.1/s and 5.9/s, respectively. The kinetic results are consistent with the equilibrium results in that the ratio of dissociation rate to association rate is in good agreement with the IC₅₀ values (Table 1).

These antagonist association rates are comparable with or greater than the association rate for ACh determined by single channel recording [embryonic, 4 × 10⁷ M¹ s¹ (Zhang et al., 1995)¹; adult, 1.7 × 10⁸ M¹ s¹ (Akk and Auerbach, 1996)]. The agonist tetramethylammonium has an even slower association rate. It has been suggested that agonist binding is not diffusion limited (GulFreund, 1972) and may involve multiple interactions between agonist and protein before the binding site is reached (Zhang et al., 1995). Structural studies of the nAChR suggest the presence of narrow tunnels leading to agonist binding pockets within the  subunits (Miyazawa et al., 1999). Large antagonist molecules may not be able to diffuse freely through such tunnels. We hypothesize that antagonists prevent the access of agonists to the binding pocket by binding near the entrance to the tunnel. In this way, antagonists would reach their binding site without encountering the diffusion barriers seen by agonists. If bound agonist had a reciprocal effect on antagonist association, this model would mimic classical competitive antagonism.

Comparison of Our Kinetic Results with Results from Other Researchers. Measurements of ligand binding kinetics are complicated by fast desensitization. In addition, studies on tissue preparations may lead to apparent slow rates because of limited access of the ligand to its binding site. These problems may be overcome by using rapid perfusion techniques on small preparations. Ultimately, the resolution of the perfusion system determines the fastest rates that can be measured. The study by O'Leary et al. (1994) of (+)-tubocurarine binding to nAChR was limited by the speed with which a whole oocyte can be perfused. They noted that the observed recovery of currents from inhibition by (+)-tubocurarine had the same time course (3-5 s) as the bath exchange in their experimental setup. Aoshima et al. (1992) also used oocytes, but extracted kinetic information from the acceleration of desensitization after coapplication of agonist and antagonist. They estimated the dissociation of (+)-tubocurarine and pancuronium to be 70 and 39/s, respectively.

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