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J. Biol. Chem., Vol. 275, Issue 25, 18836-18844, June 23, 2000

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Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors^*

Jan Jakubík, Esam E. El-Fakahany^§, and Stanislav Tuek^¶

From the  Institute of Physiology, Academy of Sciences of the Czech Republic, 14220 Prague, Czech Republic and the ^§ University of Minnesota Medical School, Minneapolis, Minnesota 55455

Received for publication, January 16, 2000, and in revised form, March 13, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

After short preincubations with N-[³H]methylscopolamine ([³H]NMS) or R()-[³H]quinuclidinyl benzilate ([³H]QNB), radioligand dissociation from muscarinic M₁ receptors in Chinese hamster ovary cell membranes was fast, monoexponential, and independent of the concentration of unlabeled NMS or QNB added to reveal dissociation. After long preincubations, the dissociation was slow, not monoexponential, and inversely related to the concentration of the unlabeled ligand. Apparently, the unlabeled ligand becomes able to associate with the receptor simultaneously with the already bound radioligand if the preincubation lasts for a long period, and to hinder radioligand dissociation. When the membranes were preincubated with [³H]NMS and then exposed to benzilylcholine mustard (covalently binding specific ligand), [³H]NMS dissociation was blocked in wild-type receptors, but not in mutated (D99N) M₁ receptors. Covalently binding [³H]propylbenzilylcholine mustard detected substantially more binding sites than [³H]NMS. The observations support a model in which the receptor binding domain has two tandemly arranged subsites for classical ligands, a peripheral one and a central one. Ligands bind to the peripheral subsite first (binding with lower affinity) and translocate to the central subsite (binding with higher affinity). The peripheral subsite of M₁ receptors may include Asp-99. Experimental data on [³H]NMS and [³H]QNB association and dissociation perfectly agree with the predictions of the tandem two-site model.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The agonists and competitive antagonists of muscarinic acetylcholine receptors associate with a binding domain that is located within a cavity formed by the seven transmembrane segments of receptor molecules; the cavity opens into the extracellular space (1, 2). Most interactions between muscarinic receptors and their competitive antagonists can be explained in terms of simple bimolecular reactions, whereas the interactions between the receptors and the agonists are complicated by the associated receptor-G protein interactions (3-6). With regard to some antagonists, however, observations have been described that are difficult to explain in terms of simple single-step bimolecular reactions. Based on such observations, it has been proposed that the initial binding of the antagonist quinuclidinyl benzilate (QNB)¹ is followed by a slow change in the conformation of the receptor molecule to a state that binds the ligand more avidly (receptor "isomerization"; Refs. 7-10) and that two "competitive" ligands may bind to the receptor simultaneously (10-12).

The present study of the complexity of the binding of muscarinic antagonists was started within the context of our investigations of allosteric modulations of muscarinic receptors (13-15). We wanted to know how the dissociation of muscarinic antagonists N-[³H]methylscopolamine ([³H]NMS) and [³H]QNB from the receptors is affected by changes in the concentration of the unlabeled ligand added to the system in order to reveal ("induce") the dissociation, by the duration of the preceding radioligand association, and also by the treatment of receptors with a compound known to associate covalently with the classical binding site of muscarinic receptors (benzilylcholine mustard (BCM); see "Experimental Procedures") (16, 17). The data we obtained provide strong support for a model in which there are two tandemly arranged binding sites on each muscarinic receptor molecule, a "peripheral" one and a "central" one. In this model, classical ligands bind first to the former and then move to the latter site. The movement of the ligand from the peripheral to the central site may explain some of the phenomena which had been previously interpreted as consequences of receptor isomerization. The results and interpretations have been published as a symposium abstract (18).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- [³H]NMS and [propyl-³H]propylbenzilylcholine mustard ([³H]PBCM; N-propyl-N-chlorethyl-2-aminoethyl benzilate) were from NEN Life Science Products, and [³H]QNB was from Amersham Pharmacia Biotech. Unlabeled QNB was from RBI (Nattick, MA), and NMS was from Sigma. BCM (N-methyl-N-chlorethyl-2-aminoethyl benzilate) was synthesized in the Institute of Experimental Medicine (St. Petersburg, Russia) and kindly provided by Dr. S. Shelkovnikov (Institute of Experimental Medicine, St. Petersburg, Russia).

Cells and Cell Membranes-- Most experiments were performed on membranes of Chinese hamster ovary (CHO) cells stably transfected with the rat gene for muscarinic M₁ receptors (rM₁ membranes; Refs. 19 and 20) and on CHO cells stably transfected with the same gene in which Asp in position 99 had been mutated to Asn (rM₁-D99N membranes; Refs. 19 and 20). Some observations were duplicated on CHO cells stably transfected with the human gene for muscarinic M₂ receptors (hM₂ membranes; Refs. 21 and 22). Cells were grown in plastic dishes in Dulbecco's modified Eagle's medium with 10% fetal calf serum and 0.005% Geneticin (14). They were harvested 7 days after subculturing, washed twice through centrifugation (3 min at 300 × g) and resuspension, and homogenized with an Ultra-Turrax homogenizer in a medium consisting of 136 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 1 mM sodium phosphate buffer (pH 7.4), and 10 mM Na-HEPES buffer (pH 7.4). The homogenate was centrifuged twice for 10 min at 600 × g and combined supernatants were kept frozen at 20 °C. On the day of experiment, membranes were sedimented by 15 min of centrifugation at 60,000 × g, and washed twice by resuspension and recentrifugation (see Ref. 15 for details).

Experiments with Covalently Associating Muscarinic Ligands BCM and [³H]PBCM-- When BCM or PBCM are dissolved in water, their amino moieties give rise to aziridinium ions (the agents become "activated"), and this is followed by the formation of a covalent bond between the ligand and the receptor (17). BCM and [³H]PBCM were kept at 20 °C in a stock solution in ethanol and, before being added to the incubation tubes, they were diluted with water and kept at room temperature for 60-90 min. Their interaction with receptors was stopped by adding 1 mM sodium thiosulfate. In experiments with radioligand association to BCM-pretreated membranes, the products of the reaction between BCM and sodium thiosulfate were removed by washing the membranes with centrifugation, but they remained in the incubation medium in experiments investigating the effect of BCM-pretreatment on radioligand dissociation. Details have been included in the descriptions of individual types of experiments.

Radioligand Binding Experiments-- Measurements of radioligand binding were performed essentially as described (15, 22, 23). Membranes corresponding to 600,000 cells were incubated at 25 °C in a final incubation volume of 0.8 ml. The composition of the incubation medium corresponded to that of the homogenization medium (see above), with added ligands as indicated for individual experiments. Atropine (5 µM) was used to determine the nonspecific binding of [³H]NMS and [³H]QNB. The incubation was terminated by filtration through Whatman GF/C glass fiber filters in a Brandel cell harvester, and the radioactivity retained on the filters was measured by liquid scintillation spectrometry.

Several types of experiments were performed.

1) Experiments were designed to examine how [³H]NMS and [³H]QNB dissociation from their binding sites is affected by differences in the concentration of the unlabeled antagonist applied to induce dissociation. Membranes were preincubated with 100-1400 pM [³H]NMS or with 50 pM [³H]QNB for 60 min, after which unlabeled NMS or QNB were added at varying concentrations (1 µM to 1 mM) and the loss of bound radioactivity with time was followed.

2) Experiments were designed to examine how the dissociation of [³H]NMS and [³H]QNB from their binding sites is affected by shortening the preincubation with the radiolabeled antagonist. Membranes were preincubated with 100 pM [³H]NMS for just 2 min, or with 50 pM [³H]QNB for just 5 min, after which unlabeled NMS or QNB were added at three different concentrations and the time course of the loss of bound radioactivity was followed.

3) Experiments were designed to examine how the dissociation of [³H]NMS is affected by the irreversible ligand BCM (added after the membranes had been prelabeled with [³H]NMS). In these experiments, membranes were preincubated with a suprasaturating (10 nM or 20 nM) concentration of [³H]NMS for 60 min, and then treated with a high concentration (100 nM or 10 µM) of BCM for 5 min. The action of BCM was stopped by adding 1 mM sodium thiosulfate, and the dissociation of [³H]NMS was started by adding 1 µM cold NMS (in experiments with the M₁ receptor subtype) or 5 µM cold atropine (in experiments with the M₂ receptor subtype).

4) Experiments were performed with [³H]NMS, [³H]QNB or [³H]PBCM association to their binding sites. Membranes were incubated with varying concentrations of [³H]NMS (50-400 pM), [³H]QNB (25-200 pM), or [³H]PBCM (20 nM), and the time course of radioligand binding was followed.

5) Experiments were designed to investigate how the association of [³H]PBCM to its binding sites is affected by mild pretreatment of membranes with BCM, and by the presence of unlabeled NMS, carbachol, alcuronium, or gallamine. In these experiments, membranes were preincubated with 5 nM BCM for 15 min, after which the action of BCM was stopped with 1 mM sodium thiosulfate and the membranes were washed by three cycles of centrifugation (15 min at 60,000 × g) and resuspending. The concentration of BCM applied was the lowest one with which it was still possible to achieve (during 15-min treatment) full blockade of the binding of 20 nM [³H]NMS (in experiments with the M₁ receptor subtype) or 5 nM [³H]NMS (in experiments with the M₂ receptor subtype) during a 2-h incubation.

Radioligand Binding Properties of rM₁ and rM₁-D99N Receptors-- Mutant rM₁-D99N receptors differed from the wild-type rM₁ receptors in their lower affinity for classical muscarinic antagonists [³H]NMS and [³H]QNB, in accordance with earlier findings (19, 20). The K_d values for the binding of [³H]NMS were 118 and 464 pM for the rM₁ and rM₁-D99N cells, respectively, and the corresponding K_d values for the binding of [³H]QNB were 43.7 and 88.9 pM, respectively. B_max values expressed in fmol/10⁶ cells were 7.8 and 8.1 for the binding of [³H]NMS and 9.4 and 10.7 for the binding of [³H]QNB to membranes from the rM₁ and rM₁-D99N cells, respectively.

Treatment of Data-- Our observations led us to conclude that there are two binding sites for "classical" antagonists and agonists on each muscarinic receptor: a peripheral one to which the ligands bind first, and a central one to which they move subsequently. Corresponding "two-site" model is proposed under "Discussion," together with a set of differential equations defining its kinetics. With the use of the KINSIM (24) and FITSIM (25) program for chemical kinetics, these equations have been collectively fitted to data from experiments shown in Figs. 1-7, and the best fit kinetic constants obtained have been summarized in Table II. Individual data points in Figs. 1-7 are means of three experiments performed with incubations in triplicate. The curves in these figures have been drawn by computer using the proposed model and the best fit kinetic constants from Table II.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Dissociation of [³H]NMS and [³H]QNB from M₁ Muscarinic Receptors in the Presence of Different Concentrations of Unlabeled NMS or QNB-- rM₁ membranes were preincubated with 100 pM [³H]NMS (Fig. 1A) or 400 pM [³H]NMS (Fig. 1B) for 60 min, or with 50 pM [³H]QNB for 90 min (Fig. 3A), and dissociation of the radioligand was then induced by adding different concentrations of unlabeled NMS (Fig. 1) or QNB (Fig. 3). It can be seen from Fig. 1 (A and B) that the rate of the dissociation of [³H]NMS from rM₁ membranes was fastest after the addition of 1 µM NMS, and slowest after the addition of 1 mM NMS. As shown in Fig. 1A, 62% of originally bound [³H]NMS dissociated during 10 min after the addition of 1 µM NMS, but only 35% dissociated after the addition of 1 mM NMS (see Table I for numerical values at selected data points). A similar relationship between the rate of dissociation of [³H]QNB from rM₁ membranes and the concentration of unlabeled QNB was observed (Fig. 3A). Sixty min after the start of dissociation, 38% and 16% of originally bound [³H]QNB dissociated in the presence of 1 µM and 1 mM QNB, respectively. It is evident that the rate of dissociation of labeled ligands is inversely related to the concentration of the unlabeled ligands applied to reveal dissociation.

View larger version (37K): [in this window] [in a new window]   Fig. 1.   [3H]NMS dissociation from rM1 receptors. Membranes of cells expressing rM1 receptors were preincubated with 100 pM [3H]NMS (A) or 400 pM [3H]NMS (B) for 60 min, after which unlabeled NMS was added at three different concentrations: 0.001 mM (), 0.1 mM (), or 1 mM (), and the incubation was arrested at times indicated on the abscissa. The time course of dissociation is shown in the left part of the figures. Ordinate, percentage of binding at time 0 (before the addition of unlabeled NMS). Dotted lines have been drawn by computer as best fits for radioligand dissociation from a single-site model, whereas solid lines describe dissociation from the tandem two-site model. Graphs in the middle column are plots of residuals for the single-site model, and those in the right-hand column are plots of residuals in the tandem two-site model. Data are means of three experiments with incubations performed in triplicate.

Stippled curves in the left parts of Figs. 1 and 3 have been computed as best fits for an assumed monoexponential time course of dissociation. Solid lines correspond to the two-site model described under "Discussion" and to the kinetic constants summarized in Table II. It is apparent that the fit between individual data points and the computed curves is much better for the two-site model. This is confirmed by the plots of residuals shown in the right halves of Figs. 1 and 3.

View this table: [in this window] [in a new window]   Table II Values of rate constants for radioligand association and dissociation and of equilibrium dissociation constants obtained by fitting the set of differential equations described under "Tandem Two-site Model" of "Discussion" to experimental data See Fig. 11 for explanation of the meaning of individual constants. KI = k1/k+1; KII = k2/k+2; KIII = k3/k+3;  = KIII/KI; Kd = KI × KII.

Dissociation of [³H]NMS and [³H]QNB from Membranes Containing Muscarinic M₁ Receptors with D99N Mutation-- In experiments shown in Fig. 2A, 66% of originally bound [³H]NMS dissociated from the rM₁-D99N membranes 10 min after the start of dissociation in the presence of 1 µM NMS and 49% in the presence of 1 mM NMS. This compares to 62% and 35%, respectively, in the case of membranes with wild-type rM₁ receptors (Fig. 1A). In experiments with [³H]QNB, 83% of the originally bound radioligand dissociated from rM₁-D99N membranes 60 min after the addition of 1 µM QNB and 75% after the addition of 1 mM QNB (Fig. 3B); these values contrast with those observed in the case of membranes with wild-type rM₁ receptors (38% and 16%, respectively; Fig. 3A). Apparently, the deceleration of dissociation produced by high concentrations of unlabeled ligands (NMS or QNB) is smaller at mutated D99N receptors than at receptors of the wild type. In addition, the D99N mutation brings about a general increase in the rate of [³H]QNB (but not [³H]NMS) dissociation.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   [3H]NMS dissociation from rM1-D99N receptors. Membranes of cells expressing rM1-D99N receptors were preincubated with 350 pM [3H]NMS (A) or 1400 pM [3H]NMS (B) for 60 min, after which unlabeled NMS was added at three different concentrations: 0.001 mM (), 0.1 mM (), or 1 mM (), and the incubation was arrested at times indicated on the abscissa. The time course of dissociation is shown in the left part of the figures. Ordinate, percentage of binding at time 0 (before the addition of unlabeled NMS). Dotted lines have been drawn by computer as best fits for radioligand dissociation from a single-site model, whereas solid lines describe dissociation from the tandem two-site model. Graphs in the middle column are plots of residuals for the single-site model, and those in the right-hand column are plots of residuals in the tandem two-site model. Data are means of three experiments with incubations performed in triplicate.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   [3H]QNB dissociation from rM1 (A) and rM1-D99N (B) receptors. Membranes were preincubated with 50 pM [3H]QNB for 90 min, after which unlabeled QNB was added at three different concentrations: 0.001 mM (), 0.1 mM (), or 1 mM (), and the incubation was arrested at times indicated on the abscissa. The time course of dissociation is shown in the left part of the figures. Ordinate, percentage of binding at time 0 (before the addition of unlabeled QNB). Dotted lines have been drawn by computer for radioligand dissociation from a single-site model, whereas solid lines describe dissociation from the tandem two-site model. Graphs in the middle column are plots of residuals for the single-site model, and those in the right-hand column are plots of residuals in the tandem two-site model. Data are means of three experiments with incubations performed in triplicate.

Dissociation of [³H]NMS and [³H]QNB from Membranes after Short Preincubations-- In experiments with radioligand dissociation shown in Fig. 4, [³H]NMS was permitted to associate with receptors for only 2 min, compared with 60 min of preincubation in Fig. 1. Similarly, in experiments shown in Fig. 5, [³H]QNB was permitted to associate with the receptors for only 5 min, compared with 90 min of preincubation in Fig. 3. It can be seen that the dissociation of both [³H]NMS and [³H]QNB was about 10 times faster after the short than after the long preincubations. Interestingly, the rate of dissociation of either [³H]NMS or [³H]QNB was not changed in experiments with short preincubations by increases in the concentration of the same unlabeled ligand (NMS or QNB) from 1 µM to 1 mM (overlapping data; not shown). This is in contrast to our findings following prolonged prelabeling (Figs. 1 and 3). Furthermore, the time course of dissociation was monoexponential after short preincubations (Figs. 4 and 5) but not after long preincubations (Figs. 1 and 3). The D99N mutation had no effect on the rate of dissociation of [³H]NMS (Fig. 4) but accelerated the dissociation of [³H]QNB (Fig. 5), although not as much as in experiments with long preincubations.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   [3H]NMS dissociation from rM1 (A) and rM1-D99N (B) receptors after preincubation lasting only 2 min. The concentration of [3H]NMS during preincubation was 100 pM for rM1 and 350 pM for rM1-D99N. [3H]NMS dissociation was induced by the addition of 1 µM unlabeled NMS at time 0. Data obtained after the addition of 0.1 or 1 mM NMS have not been included because they completely overlap with those shown here. Ordinate, percentage of binding before the start of dissociation. Data are means of three experiments with incubations performed in triplicate.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   [3H]QNB dissociation from rM1 (A) and rM1-D99N (B) receptors after preincubation lasting only 5 min. The concentration of [3H]QNB during preincubation was 50 pM. [3H]QNB dissociation was induced by the addition of 1 µM unlabeled QNB at time 0. Data obtained after the addition of 0.1 or 1 mM QNB have not been included because they completely overlap with those shown here. Ordinate, percentage of binding before the start of dissociation. Data are means of three experiments with incubations performed in triplicate.

Association of [³H]NMS and [³H]QNB with rM₁ and rM₁-D99N Receptors-- As shown in Fig. 6, there was little difference between the time courses of [³H]NMS association with rM₁ and rM₁-D99N receptors as predicted by the one-site model and the two-site model, and as actually observed. On the other hand, the time course of [³H]QNB association to the rM₁ receptors (Fig. 7A) was clearly different for the one-site and the two-site models, and the actual observations corresponded to what had been predicted by the two-site model. This is best seen from the plots of residuals in the right half of Fig. 7A.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   [3H]NMS association with rM1 (A) and rM1-D99N (B) receptors. [3H]NMS was added at time 0, and its binding was followed for up to 30 min (graphs on the left). The concentration of [3H]NMS was 50 pM (lowest curves), 100 pM (middle curves), or 400 pM (upper curves). Ordinate, bound [3H]NMS (fmol/106 cells). Dotted lines have been drawn by computer as best fits for association according to a single-site model, whereas full lines correspond to best fits in the tandem two-site model. Graphs in the middle and on the right are plots of residuals for the single-site and tandem two-site model, respectively. Data are means of three experiments with incubations perfomed in triplicate.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   [3H]QNB association with rM1 (A) and rM1-D99N (B) receptors. [3H]QNB was added at time 0, and its binding was followed for up to 60 min (graphs on the left). The concentration of [3H]QNB was 25 pM (lowest curves), 50 pM (middle curves), or 200 pM (upper curves). Ordinate, bound [3H]QNB (fmol/106 cells). Dotted lines have been drawn by computer as best fits for association in the single-site model, whereas solid lines correspond to best fits in the tandem two-site model. Graphs in the middle and on the right are plots of residuals for the single-site and tandem two-site model, respectively. Data are means of three experiments with incubations performed in triplicate.

Association of [³H]PBCM with rM₁ and rM₁-D99N Receptors-- [³H]PBCM is known to covalently associate with the classical binding sites of muscarinic receptors with high specificity and in an atropine-sensitive manner (19, 26, 27). Fig. 8A reveals a conspicuous difference between the time courses of [³H]PBCM association to the membranes of cells expressing the wild-type and the mutant receptors. While the association of [³H]PBCM to mutant rM₁-D99N receptors reached a maximum after about 45 min, the association to wild-type rM₁ receptors continued during subsequent 2 h of observation and reached a nearly 2-fold value in comparison with the mutant, although the density of the [³H]NMS binding sites (as determined in experiments with [³H]NMS saturation binding) was virtually the same in the two types of membranes (see legend to Fig. 8).

View larger version (18K): [in this window] [in a new window]   Fig. 8.   [3H]PBCM association to membranes containing M1 receptors. A, comparison of [3H]PBCM association to rM1 () and rM1-D99N () receptors. B, association of [3H]PBCM to rM1 receptors under control conditions (), or after mild pretreatment of receptors with BCM (abolishing the binding of NMS, ). The association of [3H]PBCM after mild pretreatment with BCM was also followed in the presence of 1 mM carbachol (), 1 mM NMS (), or 1 mM alcuronium (). Ordinate, bound [3H]PBCM (fmol/106 cells). Data are means of three or four experiments with incubations performed in triplicate. Bmax for the binding of [3H]NMS to rM1 receptors was 7.8 fmol/106 cells, and that for the binding of [3H]NMS to rM1-D99N was 8.1 fmol/106 cells.

Pretreatment of cells with BCM under conditions that were just sufficient to completely prevent the binding of [³H]NMS diminished but did not prevent subsequent binding of [³H]PBCM (Fig. 8B). The rate of [³H]PBCM association to receptors mildly pretreated with BCM was substantially diminished (but not blocked) by 1 mM NMS or 1 mM carbachol, and virtually stopped by 1 mM alcuronium (an allosteric modulator of muscarinic receptors).

Association of [³H]PBCM with hM₂ Receptors-- Membranes of cells expressing hM₂ receptors were incubated for 2 h with suprasaturating concentrations of [³H]NMS (5 nM) or [³H]PBCM (20 nM), and the binding of radiolabel was measured (Fig. 9, left two columns). It was more than twice as high after incubations with [³H]PBCM than with [³H]NMS, suggesting that one molecule of the receptor associates with more than one molecule of [³H]PBCM. In subsequent experiments (Fig. 9, right part), membranes were preincubated with 5 nM BCM for 15 min, after which BCM was reacted with sodium thiosulfate and the membranes were washed and incubated with [³H]NMS, or with [³H]PBCM and several orthosteric or allosteric ligands. While the binding of [³H]NMS was completely blocked by BCM pretreatment, [³H]PBCM still bound to membranes, and its binding was lower by approximately half than that in control membranes. This "extra" binding of [³H]PBCM was diminished by 100 µM NMS, 3 mM carbachol, 100 µM alcuronium, or 100 µM gallamine.

View larger version (23K): [in this window] [in a new window]   Fig. 9.   [3H]PBCM and [3H]NMS binding to membranes containing hM2 receptors, without (left) and with (right) mild pretreatment with BCM. Ordinate, bound radioligand (fmol/mg protein). Left part, membranes were incubated for 2 h with 5 nM [3H]NMS (column 1) or 20 nM [3H]PBCM (column 2). Right part, membranes were mildly pretreated with BCM (5 nM for 15 min), which was then inactivated with 1 mM sodium thiosulfate. The membranes were washed by three centrifugations, and afterward preincubated (60 min) and incubated (120 min) with the following additions: column 3, no addition for preincubation, 5 nM [3H]NMS for incubation; column 4, no addition for preincubation, 20 nM [3H]PBCM for incubation; column 5, 0.1 mM NMS for preincubation, 20 nM [3H]PBCM for incubation; column 6, 3 mM carbachol for preincubation, 20 nM [3H]PBCM for incubation; column 7, 0.1 mM alcuronium for preincubation, 20 nM [3H]PBCM for incubation; column 8, 0.1 mM gallamine for preincubation, 20 nM [3H]PBCM for incubation. Data are means ± S.E. of three or four experiments with incubations performed in triplicate. Statistical significance: *, p < 0.05, ***, p < 0.01, for the difference between the binding of [3H]PBCM and [3H]NMS in the left part, and between the binding of [3H]PBCM alone and in combination with other ligands in the right part.

Deceleration of [³H]NMS Dissociation from rM₁ and hM₂ Receptors by BCM-- In experiments shown in Fig. 10, rM₁ membranes (Fig. 10A), rM₁-D99N membranes (Fig. 10B), and hM₂ membranes (Fig. 10C) were preincubated with [³H]NMS for 60 min, and the dissociation of [³H]NMS was started by the addition of unlabeled NMS or atropine at time 0. Portions of the membranes were exposed to BCM for 5 min in the end of the preincubation with [³H]NMS, before the addition of the unlabeled ligand. The exposure to BCM brought about a decrease (by close to 40%) of the initial [³H]NMS labeling and at the same time prevented further dissociation of [³H]NMS in the wild-type (rM₁ and hM₂), although not in the mutant (rM₁-D99N) receptors.

View larger version (17K): [in this window] [in a new window]   Fig. 10.   Effect of BCM on [3H]NMS dissociation from membranes of cells expressing rM1 receptors (A), rM1-D99N receptors (B), and hM2 receptors (C). Membranes had been prelabeled for 60 min with 20 nM [3H]NMS (A and B) or 10 nM [3H]NMS (C) and then (in the continuing presence of [3H]NMS) treated with 100 nM BCM (A and B) or 10 µM BCM (C) for 5 min. The reaction was stopped by adding 1 mM sodium thiosulfate, and the dissociation of [3H]NMS was started by adding 1 µM cold NMS (A and B) or 5 µM atropine (C) (). Samples not exposed to BCM () were investigated in parallel. Data are means of three experiments with incubations performed in triplicate.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Main Findings and Their Interpretation-- Five observations appear to be most important conceptually.

1) After 60 or 90 min of preincubation, the rate of [³H]NMS or [³H]QNB dissociation from receptors was slowed down by increases in the concentration of unlabeled NMS or QNB, applied to reveal dissociation. A similar phenomenon has already been noted in work with atropine and other competitive antagonists (28-30), without a definite explanation. It seems apparent that unlabeled NMS or QNB bind to receptors simultaneously with [³H]NMS or [³H]QNB (thus creating ternary NMS-receptor-[³H]NMS or QNB-receptor-[³H]QNB complexes and slowing down dissociation), and yet that they prevent [³H]NMS and [³H]QNB reassociation.

2) If the prelabeling of the receptors had been shortened from 60 or 90 min to just 2 or 5 min, the dissociation of [³H]NMS or [³H]QNB was much faster and was not slowed down by higher concentrations of unlabeled NMS or QNB. It seems apparent that, during the short preincubation, the labeled ligand binds to a low affinity site (hence the fast dissociation rate after a short preincubation), while it translocates to a high affinity site during the long preincubation (hence the slow dissociation rate during a long preincubation). The unlabeled NMS or QNB have no effect on the rates of [³H]NMS or [³H]QNB dissociation after short preincubations because they cannot bind to the site that enables them to hinder the dissociation. Most probably, they do so when they sit on the low affinity site and prevent the departure of the labeled ligands from the high affinity site. However, the high affinity sites only become occupied by [³H]NMS or [³H]QNB after a long preincubation.

3) The time course of [³H]NMS and [³H]QNB dissociation was monoexponential after short, but not after long, preincubation.

4) Covalently associating [³H]PBCM detected a higher number of binding sites than the losely associating [³H]NMS.

5) BCM blocked [³H]NMS dissociation from prelabeled receptors.

All of these observations are easily explained on the generalizing assumption that there are two tandemly arranged binding sites for classical antagonists on each receptor molecule, a peripheral and a central one, and that the antagonists first bind to the peripheral site, from which they subsequently translocate to the central site. While the receptor-antagonist association is weak as long as the antagonist is attached to the peripheral site (unless a covalent bond is formed, as in the case of BCM and PBCM), it becomes strong after the antagonist moves to the central site. During the procedure of filtration and washing, the radioligand is more easily lost from the peripheral sites (i.e. a proportion of the non-covalent binding to them remains undetected), while the loss from the central sites is smaller. Moreover, if the peripheral sites become occupied by high concentrations of the unlabeled antagonist (added to the medium to induce dissociation of the radioligand), the rate at which the radioligand is able to leave the central sites is further slowed down.

Tandem Two-site Model-- A system with the features just outlined has been schematically depicted in Fig. 11. In this system, antagonist (A) binding may be described by the following set of differential equations:

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)

(Eq. 5)

The total amount of bound antagonist (Y) corresponds to the sum of [AR] + [RA] + 2 × [ARA]. Consequently,

(Eq. 6)

We used the KINSIM and FITSIM program and fitted these differential equations to data obtained in experiments with [³H]NMS and [³H]QNB association to and dissociation from rM₁ and rM₁-D99N binding sites. In view of the complexity of the system, it was not possible to obtain separate values for k₊₃ and k₃ (describing the association and dissociation of [³H]NMS or [³H]QNB to and from the peripheral site of a receptor whose central site is already occupied by [³H]NMS or [³H]QNB, respectively). Instead, we could only obtain best fit values of the k₃/k₊₃ ratio, which we call K_III in Table I and which describes the affinity for [³H]NMS or [³H]QNB of the peripheral site of a receptor whose central site had already been occupied by the same ligand. If the presence of the ligand at the central site had no effect on the affinity of the peripheral site for the same ligand, K_III should be equal to K_I. The K_III values were consistently higher than the K_I values, however, which indicates that the binding of [³H]NMS or [³H]QNB to the central site diminishes the binding of the same ligand to the peripheral site (a negative cooperative effect). The extent of negative cooperativity has been expressed as factor  (31), taking that  = K_III/K_I, but its nature has not been clarified. We speculate that it depends on direct (steric and coulombic) interaction between the two ligand molecules.

View larger version (20K): [in this window] [in a new window]   Fig. 11.   Antagonist (A) binding to muscarinic receptor (R) according to the tandem two-site model.

The best-fit values of individual parameters have been listed in Table II. Values designed as K_d correspond to the product of K_I × K_II and would represent the affinity of the receptor for [³H]NMS if no formation of ternary complexes (receptor plus two molecules of NMS bound simultaneously) occurred.

Figs. 1-3 demonstrate excellent agreement between experimental data and curves for [³H]NMS and [³H]QNB dissociation computed according to these values. After short preincubations (Figs. 4 and 5), the dissociation of [³H]NMS and [³H]QNB (presumably occurring from the peripheral sites) was fast and obeyed the k_off values computed for the respective sites of the tandem two-site model. While the difference between curves for [³H]NMS association according to the single-site and the tandem two-site model was small (Fig. 6), it is apparent from Fig. 7A that the correspondence between actual data concerning the association of [³H]QNB and theoretical prediction was much better for the tandem two-site model than for the single-site one. The tandem two-site model offers good explanation of why BCM post-treatment stops [³H]NMS dissociation (Fig. 10). It also enables to explain the difference between the extent of receptor labeling with [³H]NMS and [³H]PBCM (Figs. 8 and 9).

It is generally believed that the aspartate residue in position 105 (Asp-105; within the third transmembrane segment) of muscarinic M₁ receptors (and in homologous positions of the other muscarinic receptor subtypes) plays a key role in the binding of positively charged orthosteric muscarinic ligands to receptors (32-36). In all muscarinic receptor subtypes, there is another Asp present at the extracellular end of the third transmembrane domain (position 99 in the M₁ receptor subtype; see Ref. 37), with a less well defined functional role. The observation that BCM virtually lost its ability to prevent [³H]NMS dissociation from rM₁ receptors with D99N mutation suggests that Asp-99 may be a part of the peripheral domain of the supposed tandem binding site. The finding that the rates of [³H]NMS and [³H]QNB dissociation from the D99N mutant receptors are less dependent on the concentration of the unlabeled ("excess") ligand than those from the wild-type receptors supports such view. It has been noted that Asp-99 plays a role in the interactions between the allosteric and orthosteric binding sites (20), but the question of topographical relations between the binding site for allosteric modulators and the peripheral part of the tandem orthosteric site requires special investigation.

Biphasic time courses of [³H]PBCM binding in Fig. 8 (A and B) raise the question of what determines the rate at which the irreversible ligand associates with the supposed peripheral and central parts of the orthosteric tandem site. We speculate that the association is rapid if it occurs to an empty receptor, or if it occurs at the peripheral site of a receptor the central site of which had already ("beforehand") been occupied. In Fig. 8A, [³H]PBCM binds rapidly but non-covalently to the peripheral site and is quickly translocated to the central site where it stays long enough to make covalent bond. The association of the second molecule of [³H]PBCM with the peripheral site is slowed down. In the D99N mutant receptor, there is little binding at the peripheral site and the fast association concerns the central site. In Fig. 8B, BCM entered empty receptors during the preincubation and associated covalently with their central sites. Subsequent exposure to [³H]PBCM brought about fast covalent association of the radioligand with the peripheral sites. It may be noted that l mM NMS was not very efficient in preventing the binding of [³H]PBCM in the experiments shown in Fig. 8B; this may reflect the fact that the affinity of the peripheral sites for NMS is low in receptors in which the central sites had already been occupied by another ligand.

The tandem two-site model assumes that the ligand moves with regard to the receptor after the first association occurred. In a different context, a similar view has already been proposed by Saunders and Freedman (38), who suggested that muscarinic agonists first contact Asp-105 of the M₁ receptors and then translocate to Asp-71 (in the middle of the second transmembrane segment). Acetylcholine is believed to undergo post-binding translocation between the peripheral and the central binding sites of acetylcholinesterase (39). The tandem two-site model provides explanation for most of the phenomena that had been interpreted in terms of receptor "isomerization" (see Introduction); it is indeed likely that ligand translocation is accompanied by some change in receptor conformation.

In an earlier study, Kurtenbach et al. (32) tried to distinguish whether [³H]PBCM associates with Asp-99 or Asp-105 of the M₁ receptors and concluded that the association is mainly with Asp-105, but they (see also Ref. 40 by Curtis et al.) could not exclude that some association with Asp-99 does occur. On the other hand, Fraser et al. (19) observed that the binding of [³H]PBCM to M₁ receptors was strongly diminished after Asp-99 had been mutated to Asn, perhaps reflecting the loss of the peripheral binding site, which we assume to be responsible for the decrease of [³H]PBCM binding observed in the present work (Fig. 8).

The tandem two-site model agrees with earlier kinetic observations indicating that the initial complex formed during [³H]NMS association with the receptor has a low affinity, and that the affinity with which the ligand is bound increases with time (41). An interesting aspect of the tandem two-site arrangement is that it may explain some of the differences in the results of measurements of the numbers of muscarinic binding sites encountered in literature. In particular, higher numbers of muscarinic binding sites have been discovered frequently when [³H]QNB rather than [³H]NMS was applied for receptor labeling (42-44). Differences in results seem to depend on differences in the speed with which the ligand-receptor association becomes stabilized by ligand translocation from the peripheral to the central binding domain, and in the ease with which double occupancy of receptors by different ligands occurs.

Some of our observations could perhaps be interpreted on an alternative assumption, namely that the receptors work as dimers or oligomers (44, 45) with asymmetric properties of their binding sites, and that dimerization is impaired in D99N receptors. It would be difficult to explain, however, why the dissociation of [³H]NMS and [³H]QNB is fast after short preincubations and slow after long preincubations, and why the dissociation of [³H]NMS is blocked by an after-treatment with BCM.

In conclusion, the assumption that the binding of muscarinic ligands to the classical binding site of the M₁ and M₂ muscarinic receptor subtypes is a dynamic process involving ligand translocation between two tandemly arranged binding domains provides excellent explanation of several phenomena that are difficult to interpret otherwise, and deserves further investigation.

	ACKNOWLEDGEMENTS

We thank Dr. Lucie Baáková for help with cell cultures and Dana Ungerová for unfailing technical assistance.

	FOOTNOTES

^* This work was supported by grants from the Grant Agency of the Czech Republic (309/96/1287 and 309/99/014) and by NIH Fogarty International Collaboration Award (2-R03-TW00171).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Inst. of Physiology AV CR, Vídeská 1083, 14220 Prague, Czech Republic. Tel.: 420-2-4752620; Fax: 420-2-4752488; E-mail: tucek@biomed.cas.cz.

Published, JBC Papers in Press, April 3, 2000, DOI 10.1074/jbc.M000112200

	ABBREVIATIONS

The abbreviations used are: QNB, R()-quinuclidinyl benzilate; BCM, benzilylcholine mustard (= N-methyl-N-chlorethyl-2-aminoethyl benzilate); CHO, Chinese hamster ovary; hM₂ receptors, muscarinic receptors of the M₂ subtype present in membranes of CHO cells stably expressing corresponding human gene; NMS, N-methylscopolamine; PBCM, propylbenzilylcholine mustard (= N-propyl-N-chloroethyl-2-aminoethyl benzilate); rM₁ receptors, muscarinic receptors of the M₁ subtype present in membranes of CHO cells stably expressing corresponding rat gene.

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