© 1997 Wiley-Liss, Inc.

DRUG DEVELOPMENT RESEARCH 40:193–204 (1997)

DDR 644

Research Overview

Allosteric Binding Sites on Muscarinic ReceptorsJohn Ellis*

Departments of Psychiatry and Pharmacology, Penn State University College of Medicine,Hershey, Pennsylvania

ABSTRACT The classical (acetylcholine) binding sites of all five subtypes of muscarinic receptors areknown to be subject to allosteric regulation by a variety of small molecules. The hallmarks of such modula-tion in binding assays are that the allosteric ligands can alter both the affinities and the rates of associationand dissociation of classical ligands. By the use of suitable combinations of allosteric ligands and appropri-ate models, it has been demonstrated that at least some of these ligands act via a single well-defined site.On the basis of protein-modification and mutational studies, it appears that these allosteric ligands bind toa part of the receptor that is extracellular to the classical binding site. The location of the allosteric site islikely the reason that the few ligands that have been found to increase the affinity of classical antagonistsalso cause a dramatic slowing of the kinetics of these classical ligands; the slowing can be so profound as toappear to reverse the increases in affinity. Fortunately, the effects of allosteric ligands on the kinetics ofacetylcholine itself are not so problematic. Recent studies have described allosteric ligands that are ca-pable of enhancing the affinity of acetylcholine in binding and response assays. Ligands of this class mayprove to have quite useful applications, for example in restoring function lost due to depletion of acetyl-choline. Drug Dev. Res. 40:193–204, 1997. © 1997 Wiley-Liss, Inc.

Key words: allosteric; muscarinic receptors; alcuronium; gallamine; cooperativity

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INTRODUCTION

At the beginning of the twentieth century, it wasrecognized that the actions of acetylcholine fell into twoclasses: those that were mimicked by muscarinic andthose that were mimicked by nicotine. Muscarinic ac-tions are typically distinguishable by their slower onsetand longer duration, anatomical localization, and antago-nism by highly selective antagonists. However, it has onlybeen within the past two decades that the complexitiesof muscarinic receptors have come to be appreciated.Until that time it was difficult to argue against the con-clusion that muscarinic receptors were of a single kind[Beld et al., 1975; Inch and Brimblecomb, 1974]. How-ever, with the introduction of newer antagonists such as4DAMP (4-diphenylacetoxy-N-methylpiperidinemethiodide) [Barlow et al., 1976] and then pirenzepine[Hammer et al., 1980], it became apparent that furthersubclassification was warranted. At the same time, it was

observed that some ligands, most notably the neuromus-cular blocker gallamine, interacted with a secondary siteon the receptor [Stockton et al., 1983; Clark andMitchelson, 1976], or with multiple subpopulations [Ellisand Hoss, 1982], or both [Dunlap and Brown, 1983]. Thisadvancing pharmacology was greatly illuminated andaided by the purification of muscarinic receptors fromheart and brain and subsequent cloning of five subtypesof muscarinic receptors [Jones et al., 1992]. The ability todefinitively express the receptor subtypes in isolationhas led to more precise determinations of the selectivi-ties of muscarinic ligands. These determinations, how-ever, have not revealed any highly selective muscarinic

Contract grant sponsor: National Institutes of Health, contractgrant number R01 AG05214.

*Correspondence to: John Ellis, Department of Psychiatry, PennState University College of Medicine, Hershey, PA 17033.

194 ELLIS

ligands; while there are ligands that can reliably discrimi-nate given pairs of subtypes, there are none that exhibitmore than 10-fold selectivity for one subtype over all oth-ers [Jones et al., 1992]. It has been suggested that thislack of selectivity may reflect a high degree of conserva-tion of structure among the subtypes in the vicinity ofthe acetylcholine binding site [Hulme et al., 1990]. If so,it may be expected that agents that act allosterically maybe able to exhibit greater selectivity by virtue of bindingto more distant and presumably less conserved regionsof the receptors. Allosteric ligands can also offer otherimportant advantages, as will be discussed below.

THE NATURE OF THE ALLOSTERIC MODEL

The strength of the evidence for the existence ofallosteric interactions at muscarinic receptors is due, inpart, to the relatively rigid nature of the competitivemodel. Interactions that do not yield the expected re-sults by Schild analysis, dose-ratio additivity, or result-ant analysis can be presumed to be allosteric, providedthat the assumptions of the model are satisfied (e.g., thatequilibrium has been achieved). It is necessary to remem-ber, however, that the possibility of receptor heteroge-neity must also be taken into account. For example,nonlinear Schild plots can be observed in heterogeneoussystems, even when the interactions are competitive[Kenakin, 1992].

The key difference between competitive and allos-teric models is that, in the allosteric model, two ligandscan bind to the receptor simultaneously. The two sitesinvolved need not necessarily be on the same protein,but they appear to be for the cases we will be consider-ing [Musilkova and Tucek, 1995; Poyner et al., 1989]. Thatis, we will not be considering regulation of agonist bind-ing by guanine nucleotides, nor the cooperative modelsthat suggest oligomeric complexes of receptors[Hirschberg and Schimerlik, 1994; Wreggett and Wells,1995]. The evidence suggests that labeled ligands suchas [3H]NMS (N-methylscopolamine) bind to a single siteon muscarinic receptors and that allosteric regulators bindto another site. A model describing this relationship hasbeen presented [Ehlert, 1988a; Stockton et al., 1983] andis shown in Figure 1. For descriptive purposes, in thisreview the site at which NMS, acetylcholine, atropine,etc., bind is called the classical site, and the other sitethe allosteric site.

A number of features of this model are worth not-ing. The first is that the binding of each ligand at equilib-rium is described by two parameters, its affinity for thefree receptor and the cooperativity factor, α. Another isthat the binding of either ligand must affect the affinityof the other ligand by the same amount (α). However,the value of α may be unique for every pair of ligands;that is, α for the NMS–gallamine pair need not be the

same as for the quinuclidinylbenzilate–gallamine pair,nor for the NMS–d-tubocurarine pair. Of course, the af-finity of either ligand for the free receptor is a fixed valueand independent of ligand pairing. Another point is thatvery great negative cooperativity can prevent the forma-tion of LRA under testable concentrations and thereforebe indistinguishable from a competitive interaction. Per-haps most distinctively, if the receptor is prelabeled withL prior to the addition of an excess of a competitive ligand(atropine is typically used), the additional presence of Amay alter the rate of dissociation of L; further, if A equili-brates rapidly with LR, it will affect the rate of dissocia-tion of L with an apparent affinity of αKA. The advantagesand disadvantages of such dissociation assays are dis-cussed further, below.

A number of useful elaborations and modificationsof the basic model of Figure 1 have been presented. Forexample, Lazareno and Birdsall [1995] considered thecase where two ligands compete at the classical bindingsite, while a third ligand interacts only at the allostericsite. This model is appropriate for investigating the allos-teric regulation of an unlabeled classical ligand, by measur-ing effects on the binding of a labeled classical ligand. Theseauthors also provide a detailed accounting of the allostericmodulation of the rate of dissociation of the classicalligand. Tomlinson and Hnatowich [1988] considered thecase where one ligand binds solely to one site, while theother can bind to both sites. This model is appropriatefor cases where it is suspected that the allosteric ligandalso has a competitive component; a necessary aspect ofthis model is that the ligand that can bind to both sites maybe cooperative with itself. Waelbroeck [1994] has extendedthis approach to a model including one purely classical

Fig. 1. Scheme for binding of classical ligand L and allosteric ligand A toseparate sites on receptor R.

ALLOSTERIC BINDING SITES ON MUSCARINIC RECEPTORS 195

ligand, one purely allosteric ligand, and one ligand thatcan interact with both sites.

As the number of parameters in the model grows,the equations describing the binding of the ligands be-come increasingly flexible, so that evaluation of the modelrequires more dimensions in the experimental approach[Tomlinson and Hnatowich, 1988]. The interaction of theneuromuscular blocking agent alcuronium with muscar-inic receptors serves to illustrate the potential pitfalls ofusing very flexible models. In 1990, Tucek and colleaguesreported the first instance of positive cooperativity atmuscarinic receptors, demonstrating that low concentra-tions of alcuronium increased the binding of [3H]NMSto cardiac muscarinic receptors. However, at higher con-centrations, alcuronium inhibited the binding of[3H]NMS. The authors suggested that alcuronium mightbe acting allosterically at low concentrations, but com-petitively at higher concentrations. Studies carried outin our laboratory with cloned m2 receptors replicatedtheir result and found that the model of Tomlinson andHnatowich [1988] could indeed accommodate the datavery nicely (Fig. 2). Subsequently, however, Proska andTucek [1994] showed convincingly that the apparent in-hibition at high concentrations of alcuronium was due toa profound slowing of the kinetics of [3H]NMS binding

and that there was no reason to posit a competitive com-ponent to the actions of alcuronium. Such slowing of thekinetics of primary muscarinic ligands is a nearly univer-sal characteristic of muscarinic allosteric ligands. Theeffect on equilibrium experiments is usually less dramaticwith negatively cooperative ligands because, at the con-centrations that cause profound slowing, the binding ofthe primary ligand has already been greatly inhibited.

THE CAPPED MODEL

The ability of alcuronium to slow the kinetics of NMS sodramatically led Proska and Tucek to formulate anothermodification to the basic model. They suggested that,when bound, allosteric ligands cap the receptor, allow-ing neither association (L+RA→LRA) nor dissociation(LRA→L+RA) of the classical ligand to occur. Use ofthis model can be problematic, however. One reason issimply that the basic model can accommodate arbitrarilyslow kinetics, by setting the appropriate rate constantsas close to zero as necessary; that way, modulators thatdo not induce such slow kinetics (see below) can be ana-lyzed by the same model as those that do. Another casein which the basic model applies more generally is therelationship between the observed rate of dissociation ofthe classical ligand and the concentration of the allos-teric ligand. According to Proska and Tucek [1994], therelationship is

αKAkobs = k0[A] (1)

where kobs is the observed offrate of L in the presence ofa given concentration of A and k0 is the offrate of L in theabsence of A. On the other hand, we have used an equa-tion that differs from equation 1. In the same terminol-ogy given above, it is

m[A]kobs = k0 (1–

[A] + αKA

)(2)

where the new term, m, refers to the maximal effect ofthe allosteric ligand on the rate of dissociation of the clas-sical ligand [Ellis and Seidenberg, 1992]. That is, whenm is 0.9, kobs will approach 0.1k0 at high concentrationsof A, but it will never be slower than that. In the case ofan allosteric ligand that can prevent the dissociation ofthe classical ligand, m would equal 1. In this case, equa-tion 2 can be simplified to

αKAkobs = k0[A] + αKA (3)

which agrees with the form presented by Lazareno andBirdsall [1995].

Fig. 2. Deceptive influence of failure to reach equilibrium on the inter-pretation of the effects of alcuronium on the binding of [3H]NMS. Recep-tors (m2 subtype), 0.05 nM [3H]NMS, and the indicated concentrationsof alcuronium were incubated for 90 min at room temperature, thenfiltered. Data were fitted to the model of Tomlinson and Hnatowich [1988],in which NMS binds to one site exclusively with affinity K1, whilealcuronium competes with NMS for that site (with affinity K2) and alsobinds to an allosteric site (with affinity K3). The cooperativity factor for thealcuronium–NMS interaction is β. For this fit, the cooperativity factor forthe alcuronium–alcuronium interaction (α) was arbitrarily fixed at unity.Best-fit values were: K1, 0.2 nM; K2, 5.8 µM; K3, 0.11 µM; β, 0.48. Inspite of the apparent goodness of fit, the model is not appropriate be-cause the system is far from equilibrium at the higher concentrations ofalcuronium (see text).

196 ELLIS

Equation 1 is a valid approximation to equation 3only when A>>αKA, whereas equations 2 and 3 are validover the entire range of A, if A equilibrates rapidly withLR (as noted above). Most important, equations 2 and 3will apply whenever equation 1 applies, but the reverseis not true (see further illustration, in the next section).Also, while slow equilibration of A with LR may lead tocomplex dissociation curves, such complications are notexpected to arise with the relatively low-affinity, nega-tively cooperative allosteric ligands that have been thefocus of most investigations. For example, Lazareno andBirdsall [1995] found that the dissociation of [3H]NMSfit a monoexponential function, whether in the presenceof the negatively cooperative gallamine or positively co-operative strychnine (and even alcuronium).

The most striking failure of the capped model is itsinability to account for allosteric ligands that acceleratethe dissociation of classical ligands, or that only partiallyslow the dissociation of classical ligands. The latter canreverse the effects of other allosteric agents, in much theway that partial agonists can reverse the effects of fullagonists. Both of these effects are illustrated in Figure 3.We have previously shown that obidoxime partially slowsthe dissociation of [3H]NMS from cardiac (m2) muscar-inic receptors and that it can reverse the stronger effectsof gallamine in a concentration-dependent manner thatis exactly consistent with competition at a single site [Ellisand Seidenberg, 1992]. Figure 3 indicates that obidoximeexerts virtually no effect on the dissociation of [3H]QNB(quinuclidinylbenzilate) from m2 receptors, while gal-lamine accelerates and TMB-8 (8-(diethylamino)octyl3,4,5-trimethoxybenzoate) decelerates the dissociation of

[3H]QNB. Furthermore, obidoxime prevents the effectsof both gallamine and TMB-8, most likely by preventingthe binding of these ligands. Other recent studies in ourlaboratory have found that allosteric ligands generallyexert smaller maximal effects on the dissociation of[3H]acetylcholine than they do on the dissociation of[3H]NMS [Gnagey and Ellis, 1996]. This may be relatedto the smaller size and greater flexibility of the acetyl-choline molecule and is very good news for the prospectsof developing allosteric ligands that are positively coop-erative with acetylcholine, but do not also slow its accessto the receptor. In these studies, TMB-8 was particularlyineffective at decelerating the dissociation of [3H]-acetylcholine and could reverse the effect of gallamine,in a similar manner to the actions of obidoxime referredto above.

TESTING THE ALLOSTERIC MODEL

It is apparent from features such as curved Schildplots and modulation of the rate of dissociation of labeledligands that interactions between many of these pairs ofligands are not competitive. Similarly, the capped modelcan be ruled out for at least some allosteric ligands. But,can the basic allosteric model (Fig. 1) be tested rigor-ously, in light of its flexible nature? In fact, a variety ofapproaches have been used that serve this end. A mini-mum test is whether the model can adequately fit ex-perimental data, which has been demonstrated in studieswith many allosteric ligands [Lee and El-Fakahany, 1991b;Stockton et al., 1983]. However, more rigorous ap-proaches are suggested by the model. Although the de-gree of cooperativity between two ligands will be uniquefor each pair of ligands, the affinity of each ligand for thefree receptor should be constant and independent of thenature of any cooperatively interacting ligand. Thus,Ehlert [1988b] found that gallamine showed similar af-finities toward rat cardiac receptors in antagonizing theresponses of oxotremorine-M and BM5 (N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide), and also in in-hibiting the binding of [3H]NMS. Similarly, Waelbroeck etal. [1988] found that gallamine exhibited identical affinitiesin slowing the rates of association (a measure of the affinityof gallamine for the free receptor) of [3H]NMS and[3H]oxotremorine-M with cardiac muscarinic receptors.

Another approach is to demonstrate that the allos-teric effects are due to interaction with a specific site, bydemonstrating competition between two allostericligands. As mentioned above, we have shown that com-binations of obidoxime and gallamine affect the dissocia-tion of [3H]NMS from cardiac muscarinic receptors in amanner consistent with their interaction with a singleallosteric site [Ellis and Seidenberg, 1992]. Proska andTucek [1995] have disputed the conclusions of this study,based on two arguments derived from their model of re-

Fig. 3. Reversal of allosteric effects of gallamine and TMB-8 byobidoxime. Dissociation of [3H]QNB from m2 muscarinic receptors wasmeasured as described previously [Ellis et al., 1991]. Gallamine (1 µM)accelerated the dissociation, whereas TMB-8 (10 µM) decelerated it (openbars). Obidoxime (1 mM) exerted no effect by itself (not shown), butmarkedly attenuated the effects of the other two agents (filled bars).

ALLOSTERIC BINDING SITES ON MUSCARINIC RECEPTORS 197

ceptor capping (above). Their first argument was that wehad implicitly assumed that the receptor is not cappedand that it is possible for [3H]NMS to escape from theLRA complex via the path LRA→L + RA. This argu-ment is erroneous for two reasons. Our analysis is appli-cable to both capped and uncapped models (see [Lazarenoand Birdsall, 1995]); the only requirement is that the al-losteric ligand equilibrates sufficiently rapidly (as dis-cussed above). Furthermore, the very data we wereanalyzing refutes the capping model, because the cap-ping model is incapable of explaining the relief of theeffect of gallamine by increasing concentrations ofobidoxime. Their second argument was that there is nodata that the rate of dissociation of [3H]NMS is propor-tional to occupancy of the allosteric site; they also citetheir own data (Table 2 of [Proska and Tucek, 1994]) insupport of their argument. In fact, both our data and theirdata support the occupancy assumption. We were ableto simultaneously fit a complex data set of the effect ofgallamine, the effect of obidoxime, and the reversal byobidoxime of the effect of gallamine with an occupancy-based model (Fig. 2 of [Ellis and Seidenberg, 1992]). Theyshowed that their data was described well by equation 1(above; see their equation 10 and Table 4 in the appendixof [Proska and Tucek, 1994]). As noted above, at the (high)concentrations of A that they used, equation 1 is an ap-proximation to equation 3, which is the occupancy equa-tion; this relationship between equations 1 and 3 isgraphically illustrated in Figure 4. In summary, neitherof their arguments holds up under scrutiny. Subsequentstudies have also demonstrated competition betweenmuscarinic allosteric modulators in equilibrium bindingstudies. Waelbroeck [1994] found that d-tubocurarinecould partially reverse inhibition by gallamine of[3H]NMS binding, due to the much lower degree of nega-tive cooperativity between d-tubocurarine and [3H]NMS.Once again, this interaction is particularly striking be-cause d-tubocurarine, which by itself moderately inhibitsthe binding of [3H]NMS, can increase the binding of[3H]NMS under the right conditions (i.e., in the presencegallamine). Furthermore, the interaction was well-describedby a model in which gallamine and d-tubocurarine inter-acted only with the allosteric site; other ligands testedwith d-tubocurarine required more complex models.Proska and Tucek [1995] have also found that positively(alcuronium, strychnine) and negatively (gallamine) co-operative allosteric modulators appear to interact com-petitively at the same allosteric site.

Yet another approach is to compare equilibrium anddissociation assays. As described above, analysis of equi-librium assays can provide both the affinity of the allos-teric ligand for the free receptor (KA) and the degree ofcooperativity between the allosteric ligand and the clas-sical ligand (α). On the other hand, analysis of the disso-

ciation assays yields the apparent affinity (Kapp) with whichthe allosteric ligand modulates the rate of dissociation ofthe classical ligand. If the model is appropriate, and ifthe allosteric ligand equilibrates rapidly with the com-plex between receptor and classical ligand, Kapp will beequal to αKA. This seems to be a particularly good testfor detecting concomitant competitive interactions of theallosteric ligand, because the equilibrium assay wouldbe sensitive to competition, while the dissociation assaywould not be. It can be seen in Figure 5 that the interac-tion between [3H]NMS and gallamine at the m2 recep-tor does satisfy this criterion. That is, the average valuesfrom the dissociation data and the equilibrium data donot differ significantly. Other studies have yielded simi-lar results, although without reporting statistical analy-ses [Lazareno and Birdsall, 1995; Leppik et al., 1994; Leeand El-Fakahany, 1991b].

Thus, there seems to be overwhelming evidence thatgallamine is a strictly allosteric ligand at the m2 muscarinicreceptor under a variety of conditions. The evidence is alsostrong for some other ligands, especially d-tubocurarine,alcuronium, and strychnine. Extensive testing has so farproved to be more difficult at the other subtypes.

Fig. 4. Best fits of equations 1 (capped model, solid curve) and 3 (dashedcurve) concerning the effect of alcuronium on the rate of dissociation of[3H]NMS from m2 muscarinic receptors. Data were taken from Table 2of Proska and Tucek [1994]. The value for k0 was set at 0.500 min-1 andeach equation was independently fitted to the data points shown. Thetwo equations gave very similar estimates of αKA: 124 nM for equation 1and 129 nM for equation 3. However, at lower concentrations ofalcuronium, the resulting curves are quite different. Equation 3 exhibits asmooth sigmoidal shape, in agreement with the fact that kobs is equiva-lent to k0 at very low concentrations of alcuronium. Equation I, on theother hand, rises without bound at low concentrations of alcuronium;the expanded scale of the inset confirms that equation 1 is a good ap-proximation to equation 3 at very high concentrations of alcuronium.

198 ELLIS

LOCATION OF THE ALLOSTERIC SITE

The ability of highly charged ligands such as gal-lamine to inhibit the responses of intact cells has beendocumented for some time [Ehlert, 1988b; Clark andMitchelson, 1976] and suggests that the allosteric site lieson the extracellular face of the receptor or within a pocketformed by the receptor. Many studies have indicated thatthe binding site for acetylcholine and competitive antago-nists lies within a pocket formed by the transmembraneregions of the receptor [Hulme et al., 1990]. Peptide la-beling studies and mutational studies both point to anaspartate residue in TM3 (the third transmembrane re-gion from the N-terminal of the receptor) as a crucialcomponent for the binding of classical muscarinic ligands[Spalding et al., 1994; Fraser et al., 1989; Curtis et al.,1989]. A growing body of less direct evidence suggeststhat allosteric ligands bind closer to the extracellular sur-face of the transmembrane pocket. As discussed above,most allosteric ligands do dramatically slow the kineticsof classical ligand binding, an effect consistent with a

Fig. 5. Comparison of the effects of gallamine on NMS binding to m2receptors under equilibrium and dissociating conditions. A: The specificbinding of [3H]NMS was determined under equilibrium conditions in theabsence or presence of gallamine. The data points shown are from arepresentative experiment and the entire set of points was fitted simulta-neously to the model shown in Figure 1, according to the equation

RTLB(L,A) =

KA + AL + KL

AKA +

α

where B is the concentration of bound [3H]NMS; L is the concentrationof free [3H]NMS; A is the concentration of gallamine; RT is the total con-centration of receptors; KL is the affinity of NMS for the free receptor; KA

is the affinity of gallamine for the free receptor; and α represents thedegree of cooperativity between L and A. The concentrations of gallamineused were (from left to right) 0, 0.01, 0.03, 0.1, 0.3, 1, and 3 µM. Best-fit

values were α = 107, KA = 0.0067 µM, KL = 0.078 nM, RT = 0.019 nM.The inset shows a Schild-type representation of the same data, in whichthe apparent affinity of [3H]NMS was determined separately for eachcurve. The y-axis is log(affinity ratio -1) and the x-axis is log[gallamine], inµM. B: The rate of dissociation of [3H]NMS was determined in the pres-ence of the indicated concentrations of gallamine. The data points fromthis representative experiment were fitted to the equation

kobs mA

k0

= 1 –A + Kapp

where A is the concentration of gallamine, m is the maximal reduction ofthe rate constant that can be exerted by L, and Kapp is the affinity ofgallamine for the NMS-bound form of the receptor. The best-fit valueswere m = 0.93, Kapp = 0.45 µM. Analysis of means ±SEM for three ofeach of the above experiments found α = 120 ± 7, KA = 4.9 ± 0.9 nM,and p( KA) = 6.25 ± 0.05, while pKapp = 6.42 ± 0.21; analysis by t-testfound that p( KA) did not differ significantly from pKapp (t = 0.79; 4 df, notsignificant).

partial or complete blocking of the entrance. Alcuroniumand gallamine have also been found to protect the classi-cal binding site against the effects of protein modifyingreagents, possibly by limiting their access to the bindingpocket [Jakubik and Tucek, 1994].

Several studies have employed mutagenic tech-niques to attempt to locate residues essential to the al-losteric action of gallamine. Two different approacheshave been taken in these studies, both based on thepremise that allosteric ligands must bind essentially simi-larly to the different muscarinic receptor subtypes. Onone hand, one would expect to find a core binding com-ponent that is common to all of the subtypes that couldbe located by investigating conserved residues. On theother hand, one would expect that subtype-specific com-ponents would contain auxiliary binding sites that wouldbe responsible for selectivities of ligands; if all subtypesbind the ligand in roughly the same orientation, swap-ping these components may swap the selectivities also.

Lee et al. [1992] examined the influences of three

ALLOSTERIC BINDING SITES ON MUSCARINIC RECEPTORS 199

conserved aspartate residues on the interaction of gal-lamine with the m1 receptor. Substitution of asparagineat asp71, asp99, or asp122 produced relatively small changesin the affinity of gallamine toward the free receptor, andno change toward the NMS-bound receptor. Matsui etal. [1995] mutated 21 conserved residues in the externalloops and external portions of the transmembrane heli-ces of the m1 receptor. They found that changing Trp101

(predicted to lie barely within TM3) or Trp400 (just out-side TM7) to alanine produced marked reductions in theaffinity of gallamine for the free receptor, with littlechange in the degree of cooperativity between gallamineand NMS. The authors also noted the analogy to the im-portance of aromatic residues in the acetylcholine bind-ing site of acetylcholinesterase.

We have taken the subtype-selectivity approach.Because gallamine has much higher apparent affinity forthe m2 receptor than for the m3 or m5 subtypes, we madeuse of chimeric receptors in which portions of the m3 orm5 receptors were replaced by the homologous m2 se-quence [Ellis et al., 1993]. We found that the affinity ofgallamine was significantly raised when (and only when)the chimeric receptors included a short (31-residue)stretch of the m2 sequence; this sequence is predicted toinclude nearly all of TM6 and nearly all of the third outerloop. Interestingly, this sequence is immediately adja-cent to the tryptophan (position 400 in the m1 receptor)implicated by Matsui et al. [1995]. Furthermore, it alsoincludes a site where mutation-induced constitutive ac-tivation has been demonstrated in the m5 receptor[Spalding et al., 1995], emphasizing the importance ofthis region to receptor function.

Leppik et al. [1994] adopted an intermediate ap-proach, relative to conserved versus subtype-specificresidues. They mutated every acidic amino acid in thetransmembrane and extracellular regions of the m2 re-ceptor (except asp103 in TM3), plus two in the intracellu-lar region. If the residue was conserved among muscarinicsubtypes, they converted it to the corresponding amine.If it was not conserved, they altered it to be the homolo-gous residue of the m1 receptor, especially residues 172–175 of m2, which were mutated from EDGE to LAGQ.This three residue substitution led to an 8-fold reductionin the affinity toward gallamine. Whether the reversesubstitution would improve the affinity of gallamine to-ward the m1 receptor is unknown. It is worth noting thatthe involvement of the EDGE motif in the binding ofgallamine to m2 receptors would not be inconsistent withour chimeric studies. It can be seen in Figure 6 that ev-ery subtype except m1 has at least one acidic amino acidin that region; m5 has two. Also, the greater flexibility ofthe second outer loop, compared to the transmembranehelices, might provide less constraint on the exact posi-tions of the residues. It would be interesting to investi-

gate the effect of introducing or abolishing acidic resi-dues at these sites in each of the muscarinic receptorsubtypes.

Thus, none of these studies suggests that any trans-membrane acidic amino acid provides a crucial ionic in-teraction with any of the cationic groups of gallamine thatwould be analogous to the aspartate of TM3 (e.g., resi-due 103 in m2). They do strengthen the suggestion thatgallamine in particular may interact predominantly withresidues that are extracellular to the binding site for clas-sical ligands. The involvement of the tryptophans at ornear the tops of TM3 and TM7 and the analogy to acetyl-cholinesterase are especially interesting. The active siteof acetylcholinesterase lies at the bottom of a deep pocket,and the quaternary nitrogen of acetylcholine orients soas to interact with a tryptophan residue. There is an al-losteric, or peripheral, site nearer the entrance to thepocket at which a number of quaternary modifiers bind;this site also appears to involve a tryptophan residue thataccommodates quaternary ligands [Harel et al., 1993]. Infact, gallamine and d-tubocurarine are allosteric modu-lators of acetylcholinesterase and display similar sensi-tivities to ionic strength at that site as they do at themuscarinic allosteric site [Changeux, 1966].

It remains to be seen whether gallamine proves tobe a representative muscarinic allosteric ligand, in termsof the molecular location of its binding site. Our experi-ence so far suggests that it may not be, at least as far asthe residues involved in subtype selectivity are con-cerned. That is, we have so far found no other modulatorthat is uniquely affected by that 31-residue chimeric sub-stitution. For example, TMB-8 is especially sensitive to asubstitution that includes part of TM4, all of the secondouter loop, all of TM5, and part of the third inner loop(data not shown). Nonetheless, the ability of obidoximeto reverse the effects of both gallamine and TMB-8(Fig. 3) suggests that they are competitive with eachother (at the allosteric site). There is another muscar-inic ligand that has been shown to be sensitive to thesame part of the receptor as gallamine, namely thepirenzepine analog UH-AH 37 (6-chloro-5,10-dihydro-

Fig. 6. Alignment of sequences of muscarinic receptor subtypes aroundthe EDGE motif mutated by Leppik et al. [1994]. See text for discussion.

200 ELLIS

5-[(1-methyl-4-piperidyl)acetyl]-11H-dibenzo-[b,e]-[1,4]diazepine-11-one) [Wess et al., 1992]. Although pre-vious studies of UH-AH 37 have been entirely consistentwith a competitive mode of interaction, the combinationof its preferential affinity for the m5 subtype over m2and its sensitivity to that 31-residue segment of the re-ceptor led us to reinvestigate the possibility of allostericinteractions. We found that UH-AH 37 does indeed in-teract allosterically with NMS at muscarinic receptors,but with a reverse selectivity to that found in assays atequilibrium. That is, UH-AH 37 markedly slowed therate of dissociation of [3H]NMS from m2 receptors withan apparent affinity of about 3 µM, whereas its effects onm5 receptors were weaker and of lower affinity; further-more, different epitopes were implicated in the subtype-selectivities found in equilibrium and dissociation assays.These discrepancies suggest that UH-AH 37 interactswith muscarinic receptors both competitively and allos-terically (manuscript in preparation). The main featuresof the mutagenic studies of the muscarinic allosteric sitethat have been published to date are summarized sche-matically in Figure 7.

THE DIVERSITY OF MUSCARINICALLOSTERIC LIGANDS

It should be apparent from the preceding sectionsthat gallamine is currently the best characterized musca-rinic allosteric ligand. Other ligands that have been ex-tensively investigated and will undoubtably continue tobe useful in defining the allosteric site(s) are d-tub-ocurarine, alcuronium, and obidoxime. These compoundsillustrate the striking promiscuity among many agentsthat act at the extracellular sites to which acetylcholinebinds. Thus, gallamine, d-tubocurarine, alcuronium, andmany other neuromuscular blockers are allosteric mus-carinic ligands, as are many acetylcholinesterase inhibi-tors and reactivators (e.g., obidoxime). The structures ofthese ligands are shown in Figure 8. Strychnine, whichalso exhibits positive cooperativity with NMS, is alsoshown to illustrate its relationship to alcuronium.

Botero Cid et al. [1994] have carried out a system-atic study of the obidoxime analog, TMB 4 (1,1´-trimethylenebis[4-(hydroxyiminomethyl)pyridiniumbromide). They found that the addition of a dichlorobenzylsubstituent to one end of the molecule dramatically in-creased potency in dissociation assays (using [3H]NMS).Surprisingly, however, truncation of the opposite side ofthe molecule, even to the point of eliminating thepyridinium moiety on that side, did not reduce affinitydramatically in that assay. Additionally, the compounds

Fig. 7. Schematic representation of features of muscarinic receptors thathave been implicated in gallamine’s allosteric interactions. The tryptophanresidues (predicted to be located just inside TM3 and just outside TM7)are conserved among muscarinic receptors. The “EDGE” motif in thesecond outer loop (between TM4 and TM5) is found only in m2 (see Fig.6). The ruled area indicates the location of the 31-residue segment thatappears to be responsible for gallamine’s selectivity for m2 over m5. Theaspartate of TM3 that is conserved in biogenic amine receptors is alsoshown. Fig. 8. Structures of muscarinic allosteric ligands.

ALLOSTERIC BINDING SITES ON MUSCARINIC RECEPTORS 201

that lacked the pyridinium group altogether on the distalside, or that had a terminal phenyl group in its place,appeared to possess the greatest degree of negativecooperativity against [3H]NMS.

Small molecules from other pharmacological classeshave also been found to modulate muscarinic receptorbinding or function allosterically. These drugs have beenreviewed [Henis et al., 1989; Birdsall et al., 1987; Leeand El-Fakahany, 1991a], and include ion channel block-ers, antiarrhythmic drugs, and ganglionic blockers. Batra-chotoxin probably acts via intracellular components, andits effects are sensitive to GppNHp [Cohen-Armon et al.,1985]; on the other hand, the allosteric effects of gallamine[Lee and El-Fakahany, 1991b; Ellis et al., 1991] andalcuronium [Musilkova and Tucek, 1995] on receptorbinding seem to be independent of G protein function.Most cardioselective muscarinic antagonists have beenshown to exert allosteric effects. Although commonlyconsidered competitive, even the M1 selective antago-nist pirenzepine has been suggested in some studies tobind to an allosteric site [Kenakin and Boselli, 1989; Chooet al., 1985]; it is possible that it may share the propertiesof its analog, UH-AH 37 (described above).

A variety of larger molecules are known to interactallosterically with muscarinic receptors. Heparin, dext-ran, and trypan blue probably interact with intracellularportions of the receptor to interfere with G-protein cou-pling [Gerstin et al., 1992]. These are not specificallymuscarinic effects, as other G-protein-coupled receptorsare similarly affected [Huang et al., 1990; Willuweit andAktories, 1988]. Polycations have also been found to ex-ert allosteric effects at muscarinic receptors. Thus,polyarginine, polylysine, polyornithine, and protaminewere all found to slow the dissociation of [3H]NMS fromcardiac muscarinic receptors [Hu et al., 1992]. We havefound that polyethylenimine, commonly used to treatglass fiber filters to reduce nonspecific binding in recep-tor binding assays, slows the rate of dissociation of clas-sical muscarinic ligands; this feature may be an addedbenefit with ligands that dissociate fairly rapidly. How-ever, unlike gallamine, TMB-8, THA, alcuronium, andother small ligands, the effects of polyethylenimine, prota-mine, and polyarginine on ligand dissociation are notreversed by obidoxime, which suggests that thepolycations do not bind in the same way that the smallerligands do (unpublished observations).

There is a family of snake venom toxins that havebeen found to bind to muscarinic receptors with highaffinity. Five toxins have been isolated from the venom ofthe Eastern green mamba (MTx1, MTx2, MTx3, MTx4,and m1-toxin), and others have been purified from theWestern green mamba and the black mamba [Jerusalinskyand Harvey, 1994]. These toxins are 64–66-residue pep-tides related to the acetylcholinesterase-inhibiting toxin,

fasciculin; there is a high degree of sequence conserva-tion among these muscarinic toxins. MTx1 and MTx2exhibit highest affinity for the m1 subtype, where theyact as selective agonists. The m1-toxin binds irreversiblywith very high affinity to m1 receptors, and reversiblywith lower affinity at m4 receptors [Max et al., 1993a,b].The sizes of these toxins may offer multiple opportuni-ties for binding to the receptor. It appears that the 125I-labeled MTx1 toxin binds to the classical muscarinic site[Waelbroeck et al., 1996], while the m1-toxin recognizesthe allosteric site [Max et al., 1993b]. A number of otherproteins and peptides affect muscarinic receptor bind-ing and function (see [Tucek and Proska, 1995]); theseinclude protamine (above), dynorphin [Hu and El-Fakahany, 1993], and the eosinophil major basic proteinthat has been implicated in the enhancement ofbronchoconstriction in asthma, due to allosteric antago-nism of m2 receptors [Jacoby et al., 1993].

A few ligands that are structurally related toalcuronium have been investigated. As noted above,strychnine has been found to share the positively coop-erative nature of alcuronium (vs [3H]NMS). Lazareno andBirdsall [1995] aptly suggested that alcuronium can beconsidered a “functionalized dimer” of strychnine. Twoother compounds that bear close similarity to strychninehave been evaluated at cardiac receptors, eburnamonineand vincamine [Proska and Tucek, 1996]. Both exertedallosteric effects and eburnamonine (but not vincamine)exhibited positive cooperativity. All these agents are nega-tively cooperative with acetylcholine. Recently, however,brucine (10,11-dimethoxystrychnine) and several relatedcompounds have been shown to be positively coopera-tive toward acetylcholine [Lazareno et al., 1997;Gharagozloo et al., 1997]. These allosteric ligands pro-duce subtype-selective increases in the affinity of acetyl-choline in binding and responses (see below).

CONCLUSIONS

There is no doubt that there are allosteric sites onmuscarinic receptors. Progress in the understanding ofthese sites is accelerating, driven by the factors reviewedabove: better appreciation and application of relevantmodels in the design and interpretation of experiments;use of pharmacological and structural approaches to thedetermination of the location of binding sites for differ-ent ligands; and the beginnings of correlation betweenstructure and activity.

The growing evidence for the existence of thesesites has suggested for some time the possibility that a setof ligands could be found that would raise or lower theaffinity of acetylcholine at specific muscarinic receptorsubtypes. This scenario is analogous to the interactionsof agonists and inverse agonists at the benzodiazepinesites that regulate the interaction of γ-aminobutyric acid

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(GABA) with GABAA receptors [Ehlert et al., 1983]. Thepotential advantages of allosteric modulators over com-petitive agonists and antagonists stem from two consid-erations. The first is that the effects of allosteric drugscan have inherent ceilings, based on the specific level ofcooperativity (α) between the given drug and the endog-enous agonist. Thus, the effect of the appropriate drug(defined as the one with the appropriate α) will be lessdose-dependent than are the effects of competitiveagents. The second consideration is that an allostericagent that enhances the affinity of the endogenous ago-nist but does not itself activate the receptor can preservethe spatial and temporal patterning of presynaptic sig-nalling. In our case, the activity of a specific muscarinicreceptor subtype would be enhanced only when andwhere acetylcholine is released. This is exactly the resultthat has been reported with brucine at the m1 muscar-inic receptor. The application of brucine alone did notactivate the receptor but potentiated the concomitantapplication of a low concentration of acetylcholine[Birdsall et al., 1997]. The possible application of such adrug to a disease of transmitter depletion is obvious. Ad-ditionally, the nature of allosteric interactions may allowthe development of drugs with a unique type of selectiv-ity dubbed “absolute selectivity” [Lazareno et al., 1997].That is, an allosteric agent might exhibit neutral cooperativity(α = 1) toward acetylcholine at all receptor subtypes ex-cept for the target subtype; this selectivity would be inde-pendent of the concentration of the allosteric ligand.

The two-state model of receptor activation has re-ceived renewed attention with regard to G-proteincoupled receptors, due in part to the discovery of muta-tions that lead to constitutively active receptors [Samamaet al., 1993; Ren et al., 1993; Kjelsberg et al., 1992]. Inthis model, the receptor spontaneously interconvertsbetween an inactive state and an active state [Leff, 1995].Ligands shift the equilibrium between these states ac-cording to their preferential affinities; an agonist derivesefficacy from binding to the active state with higher af-finity than to the inactive state. In many receptor sys-tems, the inactive state predominates in the absence ofligand to the extent that it is difficult to distinguish an-tagonists that prefer the inactive state (sometimes calledinverse agonists or negative antagonists) from those thatbind without preference for either state; such neutralantagonists are capable of antagonizing the effects of bothagonists and inverse agonists. Constitutive activity hasbeen demonstrated in muscarinic receptors, by mutation[Burstein et al., 1996; Spalding et al., 1995] and by over-expression of G-proteins [Burstein et al., 1995]. This en-hanced muscarinic activity has been shown to be reducedby all antagonists tested so far, but it can also be furtherenhanced by agonists. Inverse agonism of muscarinicantagonists has also been observed with wild-type re-

ceptors in cell culture [Jakubik et al., 1995] and withphysiological systems [Soejima and Noma, 1984].

Allosteric ligands can fit into this scheme by stabi-lizing one state or the other [Ehlert, 1986]. However, itbecomes difficult to reconcile certain empirical and theo-retical considerations with only two states. For example,allosteric ligands that enhance the binding of one antago-nist, presumably by further stabilizing the ground state,should not inhibit the binding of other antagonists, butthey do [Tucek et al., 1990]; allosteric ligands that inhibitthe binding of antagonists should enhance the binding ofagonists, but they don’t [Gnagey and Ellis, 1996]. Also,agonist-enhancing ligands would be expected to displayagonist activity themselves; as discussed above, it is ex-pected to be most useful for enhancing agents to exert noeffect on response when applied, as has been found forbrucine (above). Finally, some allosteric agents that arenegatively cooperative toward acetylcholine have beenfound to activate muscarinic receptors by themselves[Jakubik et al., 1996]. These observations and consider-ations suggest that there may be two classes of states (ac-tive and essentially inactive), but that each class may berepresented by multiple conformations of the receptor,as noted by Kenakin [1996]. In this case, the actions ofallosteric ligands have the potential to be quite diverse, bothin terms of their interactions with specific classical ligandsand in terms of their interactions with the free receptor.

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