Abstract
Xanomeline is a potent agonist that is functionally selective for muscarinic M1 receptors. We have shown previously that a significant fraction of xanomeline binding to membranes of Chinese hamster ovary (CHO) cells expressing the M1 receptors occurs in a wash-resistant manner and speculated that this persistent binding likely does not take place at the primary binding site on the receptor. In the present work we investigated in depth the pharmacological characteristics of this unique mode of xanomeline binding and the effects of this binding on the interaction of classical competitive ligands with the receptor in CHO cells that express the M1 muscarinic receptor. Onset of persistent binding of xanomeline to the M1 muscarinic receptor was fast and was only slightly hindered by atropine. Its dissociation was extremely slow, with a half-life of over 30 h. Although persistently bound xanomeline strongly inhibited binding of the classical antagonistN-methylscopolamine (NMS) to the receptor, there are multiple indications that this is not the result of competition at the same binding domain. Namely, wash-resistant binding of xanomeline only slightly slowed the rate of NMS association, but enhanced the rate of NMS dissociation. Moreover, preincubation with xanomeline followed by extensive washing brought about an apparent decrease in the number of NMS binding sites. Our findings are best interpreted in terms of allosteric interactions between xanomeline-persistent binding to the M1 muscarinic receptor and competitive ligands bound to the classical receptor binding site.
Muscarinic receptors are widely distributed throughout the body, mediate a variety of functions, and consequently represent potential pharmacological targets in many disorders and diseases (Hulme et al., 1990; Caulfield, 1993; Wess, 1996). Five subtypes of muscarinic receptors are known to exist in mammals (Caulfield and Birdsall, 1998). Currently, there are markedly more subtype-selective muscarinic antagonists available than agonists. There has been active search for potent muscarinic agonists with selectivity for the M1 receptor subtype with the aim of pharmacological application of such agonists to compensate for acetylcholine deficiency in Alzheimer's disease because the M1 receptor is important for learning and memory (McKinney and Coyle, 1991). One of the promising compounds is 3-[3-hexyloxy-1,2,5-thiadiazo-4-yl]-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline) (Bodick et al., 1997; Veroff et al., 1998; Shannon et al., 2000). Xanomeline behaves as an agonist with a high potency, and is functionally selective for the M1 subtype of muscarinic receptors (Ward et al., 1995; Bymaster et al., 1998; Ward et al., 1998; Wood et al., 1999). It has been shown recently in our laboratory that a significant part of xanomeline binding to the M1 receptors is wash-resistant, resulting in persistent activation of receptors even after extensive washing (Christopoulos et al., 1998).
A hypothetical interpretation of xanomeline binding has been proposed (Christopoulos et al., 1998, 1999, 2000) whereby xanomeline possibly interacts reversibly with the classical binding site on the receptor, but also binds to a secondary site, “exosite”, in a wash-resistant manner. The objective of the present study was to carefully test the hypothesis that wash-resistant binding of xanomeline does not take place at the primary binding site on the M1muscarinic acetylcholine receptor. Experiments were designed so that one of the two components of xanomeline binding (either the reversible or the wash-resistant one) was measured predominantly at a time. Additional experiments were performed to exclude experimental artifacts that might contribute to xanomeline wash-resistant binding.
The data we obtained confirm the concept that xanomeline displays two substantially different modes of binding to the M1 muscarinic receptors: 1) reversible binding conforming to an interaction at the receptor primary binding site; and 2) wash-resistant, very stable binding that takes place somewhere else. The latter mode of binding is accompanied by marked allosteric modulation of the interaction of competitive ligands with the receptor.
We had previously speculated that wash-resistant binding of xanomeline to the M1 muscarinic receptor might take place at an exosite on the receptor, similar to the mode of binding of salmeterol to β2-adrenergic receptors (Coleman et al., 1996). The present data, however, do not conclusively support this mode of xanomeline binding.
Materials and Methods
Reagents
[3H]NMS was from PerkinElmer Life Sciences (Boston, MA). Atropine was from Sigma-Aldrich (St. Louis, MO). Xanomeline tartrate was a kind gift from Eli Lilly & Co. (Indianapolis, IN).
Cells and Cell Membranes
Experiments were performed on whole cells or membranes of Chinese hamster ovary (CHO) cells stably transfected with the human gene for muscarinic M1 receptors (kindly provided by Dr. M. Brann, University of Vermont Medical School, Burlington, VT). Cells were grown in plastic dishes in Dulbecco's modified Eagle's medium with 10% calf serum and 0.005% Geneticin. They were harvested by mild trypsinization 7 days after subculturing, washed twice through centrifugation (3 min at 300g), and resuspended in HEPES medium (110 mM NaCl, 5.3 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 25 mM glucose, 20 mM HEPES, and 58 mM sucrose, with pH adjusted to 7.4 and osmolarity to 340 mOsM). Membranes were prepared by homogenization with a Polytron PT3000 homogenizer (Kinematica, Littau-Luzern, Switzerland) and centrifugation for 20 min at 30,000g. Pellets were kept frozen at −20°C.
Experimental Design of Binding Experiments
Two main types of experiments were performed to study xanomeline-reversible and wash-resistant binding. In one type of experiment, xanomeline was present during incubation with [3H]NMS. In the first type of binding experiments, xanomeline was present during incubation simultaneously with [3H]NMS. In these experiments, changes in [3H]NMS binding induced by xanomeline were due both to xanomeline, which associated with receptors in a reversible manner, and to xanomeline, which associated in the wash-resistant manner. In the second type of experiments, membranes had been first pretreated with xanomeline, after which they were washed and incubated with [3H]NMS without xanomeline. In these experiments, changes in [3H]NMS binding induced by xanomeline were only due to xanomeline, which was attached to the receptors in the wash-resistant manner.
Pretreatment of Cells or Membranes with Xanomeline
In experiments designed to measure the wash-resistant binding of xanomeline, cells or membranes were diluted in the HEPES medium to a final concentration of 3 to 10 million cells or corresponding membranes per milliliter and incubated with xanomeline at 37°C for 1 h (except measurements of xanomeline association where incubation time varied). Pretreatment was terminated with 10-fold dilution of sample with HEPES medium, and brief centrifugation (cells, 1 min at 1000g; membranes, 10 min at 37,000g). Excess xanomeline was removed by centrifugation and resuspension in 10 times the incubation volume of HEPES medium. After incubation at room temperature for 30 min. Samples were centrifuged and supernatant was discarded. Dilution, incubation, and centrifugation were repeated three times.
Radioligand Binding
Measurements of radioligand binding were performed essentially as described (Jakubı́k et al., 1995). Cells or membranes corresponding to 300,000 to 1,000,000 cells were incubated at 37°C in a final incubation volume of 0.8 ml. The incubation medium corresponded to the HEPES medium described above, with added ligands as indicated for specific experiments. Atropine (5 μM) was used to determine nonspecific binding of [3H]NMS. The incubation was terminated by filtration through GF/C glass fiber filters (Whatman, Maidstone, UK) in a cell harvester (Brandel, Inc., Gaithersburg, MA), and the radioactivity retained on the filters was measured by liquid scintillation spectrometry.
Saturation Binding Experiments
Membranes were preincubated for 1 h in the absence or presence of xanomeline at concentrations of up to 1 mM. Then membranes were centrifuged and washed three times with fresh HEPES medium. After that, membranes were incubated for 1 h with [3H]NMS (36 pM-4 nM). In another type of experiment, untreated membranes were simultaneously incubated with both [3H]NMS and xanomeline. Equations 1 and 2 under “Treatment of Data” were fitted to the resulting data sets.
Competition-Type Experiments
Membranes were preincubated for 30 min in the absence or presence of xanomeline in concentrations ranging from 1 pM to 3.16 mM. Then the membranes were centrifuged and washed with HEPES medium three times and incubated for 1 h with 125, 250, 500, or 1000 pM [3H]NMS. Alternatively, untreated membranes were incubated with [3H]NMS in the simultaneous presence of increasing concentrations of xanomeline (1 pM-3.16 mM). Equation 3 under Treatment of Data was fitted to the resulting data sets.
Kinetic Experiments
Three kinds of kinetic experiments were performed: 1) kinetics of xanomeline wash-resistant binding; 2) kinetics of xanomeline association to the receptor in the presence of atropine; and 3) kinetics of [3H]NMS association to and dissociation from the receptor in the simultaneous presence of xanomeline, and [3H]NMS association to and dissociation from membranes pretreated with xanomeline.
To measure the rate of the wash-resistant association of xanomeline to the receptors, intact cells were incubated with xanomeline at concentrations up to 0.1 mM for up to 30 min. Then cells were briefly centrifuged and extensively washed three times with HEPES medium. After that, cells were incubated for 1 h with [3H]NMS and the loss of [3H]NMS caused by xanomeline binding was taken as a measure of the wash-resistant association of xanomeline. To measure the dissociation rate of the wash-resistant bound xanomeline, membranes were incubated for 30 min with 0.1 mM xanomeline, centrifuged, and washed three times with HEPES medium. After that, the wash-resistant bound xanomeline was allowed to dissociate in a 50-fold excess of HEPES medium for up to 24 h. After dissociation, membranes were again extensively washed three times with HEPES medium and incubated with [3H]NMS. The binding of [3H]NMS increased in parallel with the dissociation of the wash-resistant bound xanomeline and served as the indicator of its rate.
To measure the effects of the wash-resistant bound xanomeline on [3H]NMS association, membranes were preincubated for 1 h in the absence or presence of xanomeline at concentrations of up to 10 μM. Then they were centrifuged and washed three times with HEPES medium. After that, 125 pM [3H]NMS was added and its association was followed for up to 30 min. Alternatively, [3H]NMS association to untreated membranes was studied in the simultaneous presence of xanomeline. Equations 4 and 5under “Treatment of Data” were fitted to the resulting data sets. To measure the effects of xanomeline on [3H]NMS dissociation, membranes were preincubated in the absence or presence of xanomeline at concentrations of up to 10 μM. Then they were centrifuged and washed three times with HEPES medium. After that, membranes were labeled with 125 pM [3H]NMS for 1 h. The dissociation of [3H]NMS was started by addition of 1 μM atropine. Alternatively, membranes were prelabeled with [3H]NMS for 1 h, and [3H]NMS dissociation was initiated by atropine added either alone or simultaneously with xanomeline. Equations 6 and 7under “Treatment of Data” were fitted to the resulting data sets.
To measure the effects of atropine on the association rate of xanomeline wash-resistant binding, membranes were preincubated with atropine for 30 min and then xanomeline was added and allowed to associate for 5 to 30 min. After that, membranes were centrifuged and extensively washed with HEPES medium three times, and incubated for 1 h with [3H]NMS. The rate of the wash-resistant association of xanomeline was determined according to the rate at which the binding of [3H]NMS diminished dependent on the duration of the preincubation with xanomeline.
Treatment of Data
Equations for saturation, association, and dissociation built in GraphPad Prism, version 3.00 (GraphPad Software, San Diego, CA) were fitted to the data. The equations were as follows.
Saturation Binding Experiments.
After subtraction of nonspecific binding eqs. 1 and 2 were fitted to the data:
Competition-Type Experiments.
After subtraction of nonspecific binding and normalization (to express the binding of [3H]NMS in the presence of xanomeline as percentage of the binding in the absence of xanomeline), eq. 3 was fitted to the data:
Kinetic Experiments.
Association experiments. After subtraction of nonspecific binding, eqs. 4 and 5 were fitted to the data:
Dissociation experiments.
After subtraction of nonspecific binding eqs. 6 and 7 were fitted to the data:
The error distributions for individual constants were verified according to Christopoulos (1998). Only IC50 (eq.3) has log-normal error distribution. Error distributions for other constants conform to normal Gaussian distribution. GraphPad Prism was also used for statistical analysis of the results.
Results
The characteristics of xanomeline binding to M1 muscarinic receptors were determined indirectly according to changes in [3H]NMS binding. Previous reports from this laboratory (Christopoulos et al., 1998, 1999) have shown that a fraction of xanomeline binding is wash-resistant. Therefore, two kinds of experiments were performed to distinguish between the mode of xanomeline reversible and wash-resistant binding. In one type of experiment xanomeline was present during incubation with [3H]NMS and in the other membranes were first pretreated with xanomeline then washed and incubated with only [3H]NMS. Although in the former type of experiment the changes in [3H]NMS binding are due to both xanomeline wash-resistant and reversible binding, the changes in [3H]NMS binding in the latter type of experiment are only due to xanomeline wash-resistant binding, allowing us to distinguish between these two modes of xanomeline binding to M1 muscarinic receptors. In preliminary experiments, we tested whether the observed xanomeline-persistent binding is an experimental artifact due to the inefficiency of the washing protocol, or is the result of locking residual xanomeline into a high-affinity agonist-binding receptor conformation. Accordingly, membranes were treated with xanomeline, washed, and the supernatant from the last wash was tested for its ability to change [3H]NMS binding. The results of these experiments were negative (data not shown). Furthermore, wash-resistant binding of xanomeline to membranes was found to be unaffected by the addition of a stable GTP analog, GppNHp (data not shown).
Kinetics of Xanomeline Wash-Resistant Binding.
Whole cells rather than membranes were used in these experiments to shorten centrifugation times and expedite the washing procedure as much as possible. To measure the association rate of xanomeline-persistent binding to M1 receptors, intact CHO cells expressing the receptor were incubated with xanomeline at concentrations ranging from 10−5.5 to 10−4 M for up to 30 min. Then cells were briefly centrifuged and extensively washed three times. After that cells were incubated for 1 h with [3H]NMS and residual [3H]NMS binding was used to measure xanomeline wash-resistant binding. The association of xanomeline was fast (Fig. 1). Thus, xanomeline obliterated from 17 (3.2 μM) to 62% (100 μM) of the binding sites in a wash-resistant manner, even when cells were spun down and washed immediately after xanomeline addition. Fitting eq. 4 to the data in Fig. 1 resulted in an observed association rate (Kobs) of 0.22, 0.27, 0.32, and 0.32 min−1 at 3.2, 10, 32, and 100 μM xanomeline, respectively. Note that the slope of the relationship between the observed rate of association (Kobs) of xanomeline wash-resistant binding and xanomeline concentration is markedly shallower than what would be expected for a simple bimolecular interaction (Fig. 1B). Moreover, there was no increase in value of the observed rate of association when xanomeline concentration was increased from 32 to 100 μM (Fig. 1B).
To measure the dissociation rate of xanomeline wash-resistant binding, membranes from cells expressing M1 receptors were incubated for 30 min with 10 μM xanomeline, spun down, and washed three times with buffer. After that, wash-resistant xanomeline was allowed to dissociate in excess buffer for up to 24 h. After dissociation, membranes were again extensively washed three times and subsequently incubated with [3H]NMS. Residual [3H]NMS binding was used to calculate remaining wash-resistant xanomeline binding. As shown in Fig.2, preincubation of membranes with xanomeline resulted in reduction of [3H]NMS binding to less than 20% of control. When xanomeline was allowed to dissociate for 24 h, [3H]NMS binding returned only to 45% of control.
Saturation Binding Experiments.
In saturation experiments (Fig. 3), increasing concentrations of [3H]NMS were added to membranes in the continued presence of various concentrations of xanomeline (Fig. 3A) or to membranes that had been pretreated with xanomeline at various concentrations for 30 min, followed by removal of xanomeline (Fig. 3B). In either case, incubation of membranes with [3H]NMS was for 1 h. The results of nonlinear regression analysis of the data in Fig. 3 are summarized in Table 1. Equations describing binding to either one (eq. 1) or two (eq. 2) binding sites were fitted to the data. [3H]NMS binding was adequately described by a single homogeneous population of sites (eq. 1) in all cases except in the presence of 0.1 μM xanomeline, where eq. 2 gave a significantly better fit than eq. 1 (F = 17.47,P < 0.01). Under both experimental conditions, xanomeline decreased maximum binding capacity (BMAX) and increased equilibrium dissociation constant (Kd; diminished affinity) of receptors for [3H]NMS. Higher concentrations of xanomeline were required in the preincubation/washing paradigm to produce effects similar to those in continued presence of xanomeline (Fig. 3, A and B). Moreover, effect of preincubation/washing with xanomeline on Kd approached a maximal limit (Fig. 3B; Table 1). This phenomenon was not observed in the simultaneous presence of xanomeline and [3H]NMS (Fig. 3A).
Competition-Type Experiments.
In competition-type experiments (Fig. 4), binding of 125, 250, 500, and 1000 pM [3H]NMS was measured either in the presence of increasing concentrations of xanomeline, or after the membranes were pretreated with increasing concentrations of xanomeline for 30 min and washed. In both cases membranes were incubated for 1 h with [3H]NMS. Potency of xanomeline to inhibit [3H]NMS binding when continuously present was approximately 2 orders of magnitude higher than the inhibitory potency of the persistent component of xanomeline binding. Equation 3 under “Treatment of Data” was fitted to the resulting data sets. Results of nonlinear regression analysis are summarized in Table 2. Although increasing [3H]NMS concentration caused a significant increase in xanomeline IC50, this shift was slightly smaller than what would be expected in competitive interaction. Thus, increasing [3H]NMS concentration from 125 to 1000 pM resulted in a change in xanomeline IC50 of 2.24-fold (preincubation/washing) and 2.34-fold (simultaneous presence), which is slightly but significantly lower than a 2.77-fold shift predicted by the Cheng-Prusoff equation (Cheng and Prusoff, 1973) for competitive interaction. Interestingly, although the simultaneous presence of xanomeline caused complete inhibition of [3H]NMS binding, the inhibition of [3H]NMS binding produced by pretreatment with xanomeline was incomplete (92% for 500 and 1000 pM [3H]NMS) (Fig. 4).
Effects of Xanomeline on Kinetics of [3H]NMS Binding.
To measure the effects of xanomeline on [3H]NMS association, membranes were preincubated in the absence or presence of xanomeline at concentrations up to 10 μM. Then membranes were centrifuged and washed three times with buffer. After that, 125 pM [3H]NMS was added and its association was followed for up to 30 min (Fig.5B). For membranes not pretreated with xanomeline, xanomeline was added simultaneously with [3H]NMS (Fig. 5A). Note that concentrations of xanomeline used in Fig. 5A are lower than those used in Fig. 5B, so that the ratio of the applied concentrations to IC50 values of xanomeline under the two experimental conditions is the same. Also, note that the concentrations of xanomeline (0.1 and 1 μM) used simultaneously with [3H]NMS are expected to exert minimal wash-resistant interactions. Equations 4 and 5 under Treatment of Data were fitted to the resulting data sets. Association of [3H]NMS to receptors in the absence of xanomeline was well described by eq. 4 as an association with a single homogeneous population of binding sites. According to two-way analysis of variance, both membrane preincubation with xanomeline and xanomeline presence during incubation of membranes with [3H]NMS significantly slowed the rate of [3H]NMS association. When present simultaneously with [3H]NMS, xanomeline at concentration of 0.1 μM drug (i.e., at a concentration corresponding to its IC50 under these conditions) slowed the rate of [3H]NMS association by 2.8-fold. This is consistent with competition between xanomeline and [3H]NMS for the receptor. In contrast, the wash-resistant bound xanomeline (at 10 μM, i.e., a concentration corresponding to the IC50 of the wash-resistant component) slowed the rate of [3H]NMS association by only 16%. This is in spite of decreasing [3H]NMS equilibrium binding by 45%. Taken together, this is clearly inconsistent with a competitive mode of inhibition of [3H]NMS binding by prebound xanomeline.
To measure the effects of persistently bound xanomeline on [3H]NMS dissociation, membranes were preincubated for 30 min in the absence or presence of xanomeline at concentrations of up to 10 μM, centrifuged, and washed three times with buffer. After that membranes were labeled with 125 pM [3H]NMS for 1 h. Dissociation of [3H]NMS was started by the addition of 1 μM atropine. In another set of experiments, membranes were prelabeled with [3H]NMS for 60 min and dissociation was initiated by adding atropine either alone or together with xanomeline. The concentrations of xanomeline used in experiments involving the preincubation with the drug were higher than those in experiments in which xanomeline was applied simultaneously with atropine, in view of the difference between the potencies of xanomeline determined in the two paradigms (Fig. 4; Table 2). Equations 6 and 7 under “Treatment of Data” were fitted to the resulting data sets, and the results are summarized in Fig. 6 and Table3. The dissociation of [3H]NMS from membranes pretreated with xanomeline was significantly faster than from nontreated membranes. In contrast, no significant difference was observed between the rate of dissociation started with atropine alone or with a mixture of atropine and xanomeline. These data indicate that the interaction between [3H]NMS and persistently bound xanomeline is substantially different from the interaction between [3H]NMS and reversibly bound xanomeline.
Effects of Atropine on Xanomeline Wash-Resistant Binding.
To measure the effects of occupancy of the classical primary receptor binding site on the association rate of xanomeline wash-resistant binding, membranes were preincubated with receptor-saturating and -supersaturating concentrations of atropine for 30 min then xanomeline was added and allowed to associate for up to 30 min. After that, membranes were centrifuged, extensively washed three times with buffer, and incubated for 1 h with [3H]NMS. The results are summarized in Fig. 7. Atropine (1 μM) slowed the association of both 10 and 100 μM xanomeline with its wash-resistant binding site, but did not prevent it. Increasing the concentration of atropine over 1 μM did not cause further slowing of xanomeline association. Thus, xanomeline is able to gain access to its wash-resistant binding site, even if the primary receptor-binding site is completely blocked.
In the next set of experiments, we investigated in more detail the concentration dependence of the wash-resistant binding of xanomeline in the absence and presence of atropine. Membranes were preincubated with atropine for 30 min then xanomeline was added at increasing concentrations and incubation continued for additional 10 min. Membranes were washed three times with HEPES medium then incubated for 1 h with [3H]NMS. The results are shown in Fig. 8. Again, the presence of receptor-saturating concentrations of atropine resulted in only a small (approximately half an order of magnitude) shift in IC50 of xanomeline wash-resistant binding, and there was no significant difference in the effects of different concentrations of atropine used. The observation that the effects of atropine on the formation of xanomeline wash-resistant binding reach a maximal limit implicates that there is an allosteric interaction between atropine binding to the classical binding site and the wash-resistant xanomeline binding component (Jakubı́k et al., 1995)
Discussion
Previous work from this laboratory unfolded unique features of the interaction of xanomeline with the M1 subtype of acetylcholine muscarinic receptors. Although the majority of xanomeline binding to this receptor is readily reversible, a distinct binding component is wash-resistant (Christopoulos et al., 1998). The wash-resistant binding of xanomeline results in persistent activation of the receptor in an atropine-sensitive manner. Previous data were tentatively interpreted in terms of an interaction of xanomeline with a hypothetical secondary binding site on the receptor (an exosite), from which xanomeline interacts with the classical binding site to cause long-lasting receptor activation. In the present work we investigated the properties of the persistent binding of xanomeline in more depth. The main results of our experiments may be briefly summarized as follows: Wash-resistant binding of xanomeline is fast and occurs with an apparent affinity in the micromolar range. Its dissociation is extremely slow, with a half-life of over 30 h. Inhibition of binding of the classical competitive antagonist NMS by persistently bound xanomeline was accompanied by a slight slowing down of the rate of NMS association and marked enhancement of its rate of dissociation. There was also an apparent decrease in the number of NMS binding sites in saturation experiments. Atropine bound to the orthosteric binding site only slightly hindered the wash-resistant association of xanomeline with the receptor, and this effect reached a limit regardless of further increasing atropine concentrations.
Taken together, our data provide strong support to the notion that xanomeline-persistent binding does not take place at the classical primary binding site on the M1 muscarinic receptor. More importantly, this mode of binding results in allosteric modulation of interaction of competitive ligands at the receptor primary binding domain.
Control experiments were designed to eliminate the possibility that residual, unwashed xanomeline contributes to the observed persistent binding. In these experiments the supernatant from the last wash failed to affect [3H]NMS binding, indicating that xanomeline wash-resistant binding is not due to insufficient removal of free xanomeline. Furthermore, wash-resistant binding of xanomeline to membranes was not affected by the addition of the stable GTP analog GppNHp, indicating that it is not due to a locking of xanomeline by receptors in a high-affinity agonist binding state. However, we cannot at present exclude the possibility that xanomeline partitioned in the cell membrane contributes to its observed binding profile.
The rate of formation of the wash-resistant xanomeline binding to M1 muscarinic receptors was quite fast, reaching a steady state in 10 min. The half-life of this type of xanomeline binding exceeded 24 h. Although persistent binding of xanomeline has features of near-irreversible binding, data in Fig. 1A indicate that it is not truly irreversible because it reaches a concentration-dependent plateau instead of progressing indefinitely with time. Additional insight is needed but is difficult to obtain in the absence of commercially available radiolabeled xanomeline.
Several pieces of evidence support our previously proposed hypothetical interpretation whereby xanomeline-persistent binding involves its interaction with a domain(s) different from the classical receptor binding site. This is clearly revealed by the fact that xanomeline was capable of exerting wash-resistant binding even when the classical binding site was completely obliterated by atropine, albeit at a slightly slower rate and lower potency. Interestingly, both the rate and the magnitude of xanomeline-persistent binding to the M1 receptor remained constant when the concentration of atropine was increased from 1 μM (receptor saturating concentration) to 100 μM (suprasaturating concentration).
The observation that the wash-resistant binding of xanomeline was minimally affected by a complete blockade of the classical binding site demonstrates that an initial interaction of xanomeline with the classical binding site is not a prerequisite for its persistent binding somewhere else. It should be noted, however, that it was previously shown that atropine blocks activation of the receptor induced by the wash-resistant prebound xanomeline (Christopoulos, 2001). Two explanations seem plausible to reconcile these observations. First, persistently bound xanomeline might activate the receptor by an allosteric action (Jakubı́k et al., 1996). Atropine, being an inverse agonist (negative antagonist) stabilizes the receptor in an inactive conformation to suppress its activation by xanomeline. Second, as previously proposed, persistently bound xanomeline might also interact with the receptor classical site in a direct but not as-yet understood way (Christopoulos et al., 1999), which would make receptor activation sensitive to atropine.
There is previous evidence of avid interaction of certain β2-adrenoreceptor agonists with an exosite on the receptor (Clark et al., 1996; Coleman et al., 1996). Although our present findings strongly suggest that persistent binding of xanomeline to the M1 muscarinic receptor does not take place at the conventional primary binding site on the receptor, certain anomalies in the characteristics of xanomeline binding described herein are at odds with the involvement of a saturable secondary binding site that exhibits simple bimolecular mass-action kinetics. Most importantly, there is marked discrepancy between the apparent affinity of xanomeline wash-resistant binding derived from kinetic (Figs. 1 and 2) and equilibrium (Figs. 3 and 4) experiments. The fast association and slow dissociation in Figs. 1 and 2 implicate nanomolar affinity (ranging between 0.7 and 15 nM) for xanomeline wash-resistant binding. On the other hand, apparent affinity of xanomeline-persistent binding obtained in equilibrium experiments in Figs. 3 to 5 is in the micromolar range. It is difficult at the present time to offer a highly satisfactory explanation for such discrepancy, particularly because a spontaneous time-dependent conversion of xanomeline-persistent binding from an initial high-affinity to a late lower affinity state is not thermodynamically feasible. As a matter of speculation, however, one plausible interpretation would implicate negative allosteric modulation of xanomeline-persistent binding by xanomeline binding to the orthosteric binding site. Due to different kinetics of the two modes of xanomeline binding, one would anticipate that the magnitude of such allosteric modulation changes over the time. If xanomeline wash-resistant binding is faster than its reversible interaction at the orthosteric binding site then this mode of binding should exhibit initial high affinity in absence of the dampening effects of xanomeline bound at the primary site. Upon approaching equilibrium, however, a higher proportion of xanomeline binds to the orthosteric site and allosterically decreases affinity of wash-resistant binding of the drug. Another related anomalous observation is the nonlinear relationship between xanomeline concentration and the observed rate of its persistent binding (Fig. 1B). A simple bimolecular mass-action mechanism of binding to a defined saturable site should result in a linear relationship. Moreover, persistently bound xanomeline brings about a loss of the apparent number of NMS binding sites, instead of the anticipated progressive decrease in ligand affinity without influencing its maximal binding.
Taken together, these observations suggest quite complex mechanisms, and perhaps stoichiometry, of formation of xanomeline-persistent binding to the M1 muscarinic receptor. Future experiments using radiolabeled xanomeline will be of paramount importance toward understanding these mechanisms.
Binding of lower concentrations of xanomeline is completely reversible in nature. In contrast to the wash-resistant component of xanomeline binding, several pieces of evidence suggest that its reversible binding is competitive with ligands that interact with the receptor classical binding site. One salient feature of our present data and the proposed interpretation is that reversible and persistent interactions of xanomeline (taking place at low and high concentrations, respectively) influence binding of competitive receptor ligands differently. It is to be noted, that high concentrations of xanomeline will exert both modes of binding to the M1 muscarinic receptor, unless the reversible component of xanomeline binding is eliminated or blocked. In vivo, this could be attained by administering a high dose of xanomeline, followed by a waiting period that allows for metabolism of free xanomeline. Alternatively, xanomeline would bind primarily in a wash-resistant manner in presence of drugs that interact competitively with the classical binding site on the M1muscarinic receptor [e.g., anticholinergics, antidepressants, or antipsychotics (Richelson, 1999)].
Footnotes
-
This work was supported by National Institutes of Health Grant NS25743 (to E.E.E.-F.) and Grant Agency of the Czech Republic Grant GP305/01/D119 (to J.J.).
- Abbreviations:
- NMS
- N-methylscopolamine
- CHO
- Chinese hamster ovary
- Received December 11, 2001.
- Accepted February 18, 2002.
- The American Society for Pharmacology and Experimental Therapeutics