Introduction

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Muscarinic acetylcholine (ACh) receptors belong to the superfamily of receptors that share structural homology with rhodopsin and couple to G proteins. All five subtypes of muscarinic receptors bind the endogenous agonist ACh and have similar affinities for classical competitive antagonists such as atropine and scopolamine. In addition, muscarinic receptors are capable of binding a second ligand at a well defined allosteric site (Ellis and Seidenberg, 1992; Waelbroeck, 1994). Allosteric interactions between ligands are quite common at receptors that possess intrinsic ion channels; a notable example is the -aminobutyric acid_A receptor, with its associated sites for benzodiazepines and other drugs. From another viewpoint, the hallmark of seven-transmembrane-domain (7TM) receptors is the allosteric interaction between agonist and receptor/G protein coupling. However, ligand-ligand allosteric interactions at the external surface of 7TM receptors have been well documented only at muscarinic receptors, ₂-adrenergic receptors, and A₁-adenosine receptors (Birdsall et al., 1995; Leppik et al., 1998). Thus, at present, ACh appears to be the only transmitter for which a complete family of 7TM receptors is sensitive to ligand-ligand allosteric interactions.

The very large number of receptors in the 7TM superfamily and the high degree of conservation of some TM residues throughout the family have allowed for a significant amount of sharing of structural and functional information across receptors (Baldwin et al., 1997; Schwartz et al., 1997). Within subgroups of the superfamily, even greater structure/function homology may exist. For example, within the subset of biogenic amine receptors, which includes the muscarinic receptors, many studies have pointed to the crucial importance of an aspartate residue in TM3 (Strader et al., 1987, 1994). This conclusion has been supported by mutagenic studies of muscarinic receptors (Fraser et al., 1989; Page et al., 1995) and by peptide analysis of receptors labeled by irreversible muscarinic agonists and antagonists (Curtis et al., 1989; Kurtenbach et al., 1990; Spalding et al., 1994). On the other hand, the rarity of ligand-ligand interactions at 7TM receptors and the lack of irreversible allosteric ligands have hampered progress in delineating the structural features of the binding site(s) for muscarinic allosteric agents. Nonetheless, a number of observations have suggested that muscarinic allosteric ligands bind near to the extracellular entrance to the ACh-binding pocket of the receptor. This location is in agreement with the nearly universal property of these ligands to dramatically slow the kinetics of binding of classical muscarinic antagonists (Stockton et al., 1983; Proska and Tucek, 1994); the binding of allosteric ligands also appears to protect the classical binding site from protein-modifying reagents (Jakubik and Tucek, 1994).

Gallamine was the first muscarinic allosteric ligand to be identified (Clark and Mitchelson, 1976; Stockton et al., 1983) and has been the ligand studied most intensively. All of the mutagenic studies of the muscarinic allosteric site that have been published to date have used gallamine as the allosteric ligand (Lee et al., 1992; Ellis et al., 1993; Leppik et al., 1994; Matsui et al., 1995). Of these studies, two have taken advantage of the subtype selectivity of gallamine. We examined chimeric receptors composed of pieces of high-affinity subtype sequence (M₂) embedded in a background of low-affinity sequence (M₅). From these studies, we identified a small portion of the receptor sequence, which included the third outer loop (o3; between TM6 and TM7), that seemed to be uniquely important in conferring M₂/M₅ selectivity toward gallamine (Ellis et al., 1993). Leppik et al. (1994) found that they could markedly reduce affinity toward gallamine by replacing a four-residue segment of o2 in the M₂ subtype with the corresponding sequence of the M₁ receptor. From one point of view, this finding seemed to conflict with our chimeric studies, because different epitopes were implicated in the binding of gallamine to the M₂ receptor. However, based on comparative sequences, we have noted that the combined results might be explained by the presence of a common gallamine-binding epitope within o2 if it were present in both M₂ and M₅, but absent in M₁ (Ellis, 1997).

This study extends our chimeric study by identifying a specific residue in o3 that influences gallamine binding. It also confirms and extends the importance of the sequence identified by Leppik et al. (1994) in o2. Finally, it appears likely that each of the muscarinic receptor subtypes derives affinity toward gallamine from one or the other of these epitopes in the outer loops, except for M₂, in which both epitopes appear to be important. Some of these results have been reported recently in preliminary form (Ellis et al., 1999).

	Experimental Procedures

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Materials. Atropine sulfate and gallamine triethiodide were obtained from Sigma Chemical Co. (St. Louis, MO). Labeled N-methylscopolamine chloride ([³H]NMS; 82-85 Ci/mmol) was obtained from NEN-DuPont (Boston, MA).

Mutagenesis and Expression. Site-directed mutagenesis was performed with muscarinic receptor DNA in pcD plasmids, either with the Altered Sites II kit (Promega, Madison, WI) or the QuikChange kit (Stratagene, Inc., La Jolla, CA). Briefly, oligonucleotides containing the desired base changes were synthesized and allowed to anneal with a vector containing the appropriate muscarinic receptor DNA sequence. A high-fidelity polymerase then extended these synthetic oligonucleotides. With the Altered Sites II kit, an appropriate region of the receptor gene was first cloned into the pAlter-Ex1 vector, and single-stranded DNA was produced with a helper phage. After mutagenesis, mutated double-stranded DNA was cloned back into the pcD vector. With the QuikChange kit, mutagenesis was conducted directly in the pcD vector, and parental DNA was digested by a methylation-specific endonuclease. In either case, mutations were confirmed by sequencing.

Dissociation Binding Assays. Binding assays were conducted in 5 mM PB at 25°C. Membranes (~30 µg of protein in 1 ml) were prelabeled with 1 nM [³H]NMS for 30 min. Dissociation of the labeled ligand was initiated by the addition of 3 µM atropine, with or without the indicated concentration of gallamine, and the incubation was allowed to continue for the appropriate time. The incubation was terminated by filtration through S & S no. 32 glass fiber filters (Schleicher & Schuell, Keene, NH), followed by two rinses with 40 mM PB (0°C). Nonspecific binding was determined by the inclusion of 3 µM atropine during the prelabeling period.

	Results

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The sequences of the M₂ and M₅ subtypes of muscarinic receptors show a large degree of identity in the TM6 region, but less in the o3 region. Of the 31 amino acids in the segment of the receptors that was swapped in chimeric receptor (CR) 4 in a previous study (Ellis et al., 1993), all but 9 are identical (Fig. 1). A series of mutant M₅ receptors was created, such that one of these nine residues was mutated in isolation in each receptor, except that the Val  Ile mutation at the position corresponding to M₂⁴¹⁷ was not tested. Additionally, in the two cases in which there are adjacent differences between M₂ and M₅ (i.e., the positions corresponding to M₂^409-410 and M₂^414-415), double mutations were also created. Thus, a total of 10 mutated M₅ receptors were investigated. These discrete mutations were evaluated for their effects on the ability of gallamine to allosterically regulate the receptors, relative to gallamine's effects on the wild-type M₂ and M₅ receptors and the chimeric M₂/M₅ receptor CR4 (Fig. 2). Most of these mutations resulted in receptors with characteristics that did not differ significantly from the wild-type M₅ receptor. Three mutations resulted in significantly different affinities. The replacement of the M₅ Ser with Asn (at position M₂⁴¹⁰) gave a very low affinity for gallamine (Fig. 3). This anomalous result may be related to the importance of this position in constitutive activation of the receptor (Spalding et al., 1997), but clearly cannot be the explanation for gallamine's preference for the M₂ subtype over the M₅ subtype (see Discussion). The replacement of the M₅ Lys with Pro (at position M₂⁴¹⁵) resulted in a receptor with an affinity equal to that of CR4. This raised the possibility that the unique structural effects of proline might be an organizing factor in the preference of gallamine for the M₂ subtype (see Discussion). However, in this case, the double (adjacent) mutation provides additional insight. Where M₂^414-415 is Ala-Pro, the homologous M₅ sequence is Asp-Lys. Mutating the M₅ aspartate to alanine had no effect on gallamine's affinity by itself, but that mutation prevented the effect of the Lys  Pro mutation (Fig. 3). Thus, it appears that the insertion of the proline into M₅ allows an anomalous interaction with the aspartate that does not occur in the wild-type M_5. Furthermore, this interaction cannot be responsible for the greater affinity toward the M₂ subtype, because M₂ lacks that aspartate (see Discussion). This leaves us with the asparagine at M₂⁴¹⁹. When the valine residue at this position in the M₅ receptor is replaced by asparagine, the resulting receptor exhibits an affinity approximately equal to that of CR4. The sequences of the o3 regions of the five muscarinic receptor subtypes are shown in Fig. 4. The M₁ and M₄ receptors contain acidic amino acids at the positions corresponding to the M₂⁴¹⁹ Asn, whereas the corresponding M₃ residue is lysine. Previous studies with chimeric M₂/M₃ receptors have demonstrated that swapping regions containing the o3 loops between these two subtypes yields results that are in agreement with the M₂/M₅ chimeric receptors (Ellis et al., 1993). Substitutions at the M₂⁴¹⁹ position produced results that are consistent with these previous studies. Replacement of the asparagine in M₂ by lysine markedly reduced the affinity toward gallamine, whereas replacement of the lysine in M₃ by asparagine markedly increased the affinity toward gallamine (compare Fig. 5, A and B). On the other hand, the acidic amino acid aspartate is associated with somewhat higher affinity toward gallamine than is asparagine, in both M₂ and M₄ (compare Fig. 5, A and C); the affinity of M₂ toward gallamine also is slightly enhanced when the asparagine is replaced by the glutamate found in M₁ (Fig. 5A).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Amino acid residues that differ between the M2 and M5 subtypes, in TM6 and o3. The circled letters represent the M5 sequence. The numbered letters denote the sequence (and residue number) of the M2 receptor where it differs from the M5 receptor.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Gallamine's allosteric effects on mutated M5 receptors. The abilities of gallamine to slow the rate of dissociation of [3H]NMS from the various receptors were determined as described in Experimental Procedures. A, gallamine's potencies at the wild-type M2 and M5 receptors and at CR4, described previously (Ellis et al., 1993). B-D, representative experiments performed with mutated M5 receptors in which one or two residues have been replaced with the homologous residue from the M2 receptor. Summary data are presented in Fig. 3. The numbers refer to the M2 receptor to be compatible with the diagram in Fig. 1. For example, 401 indicates a receptor that has M5 sequence everywhere except for the threonine in the middle of TM6, which has been replaced by alanine.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Apparent affinities for gallamine at mutated M5 receptors. Experiments were performed as in Fig. 2 and analyzed as described in Experimental Procedures. The apparent affinities obtained from those analyses were expressed as the negative logarithm (pKapp) and are shown as the mean ± S.E. of three to five experiments. The x-axis represents the position of the mutation according to the M2 numbering scheme, and the substitutions made at each symbol are indicated in boxes (see also Fig. 1) In two cases, double mutants were studied. They were assigned average positions and are connected to the related single mutants by solid lines. For example, position 414.5 indicates a receptor that is M5 sequence everywhere, except that the Asp-Lys residues in o3 have been replaced by Ala-Pro.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Muscarinic receptor sequences of o3 and adjacent TMs. The positions of the shaded residues are homologous to the asparagine located at position 419 in the M2 receptor.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Mutations at homologous positions in the o3 regions of muscarinic receptor subtypes yield consistent effects on gallamine's affinities. Mutations were created at the position shaded in Fig. 4. Data are representative of at least three similar experiments. A, gallamine's affinity for the M2 receptor is markedly reduced when the asparagine at position 419 is replaced by its M3 homolog (lysine), but somewhat enhanced when an aspartate or glutamate is present, as in M4 and M1, respectively. B, gallamine's affinity for the M3 receptor is markedly enhanced by the opposite mutation of that shown above, i.e., replacement of the M3 lysine by asparagine. C, gallamine's affinity for the M4 receptor is somewhat reduced by the replacement of aspartate by asparagine.

Although our chimeric studies did not indicate an involvement of o2 in the subtype specificity of gallamine for M₂ over M₅, other studies have suggested that a cluster of acidic amino acids in this region of the M₂ receptor is related to gallamine's affinity. Leppik et al. (1994) found that the replacement of the M₂ sequence Glu-Asp-Gly-Glu (Fig. 6) by the corresponding M₁ sequence (Leu-Ala-Gly-Gln) resulted in a significant reduction in affinity toward gallamine. We have confirmed their result (Fig. 7B) and extended it to the reverse substitution in M₁. That is, insertion of the acidic sequence into the M₁ receptor results in a significant increase in affinity toward gallamine (Fig. 7A). These regions of the M₁ and M₄ receptors appear to be equivalent with regard to gallamine's allosteric actions, because replacement of the M₄ sequence by the M₁ sequence produced no effect (Fig. 7C). We have suggested that the reason that this region was not implicated in our M₂/M₅ chimeras may be that the M₅ subtype contains a similar gallamine-binding epitope in this region (Ellis, 1997). In agreement with this suggestion, the affinity toward gallamine was reduced markedly when the acidic amino acids in this region of the M₅ subtype (Asp-Glu) were replaced by the corresponding M₁ sequence (Gly-Gln) (Fig. 7D).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Muscarinic receptor sequences of o2 and adjacent TM regions. As in Fig. 4, the shaded regions lie in homologous positions.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Mutations in o2 regions of muscarinic receptor subtypes yield consistent effects on gallamine's affinities, with a different pattern from that seen in o3. Mutations were created within the positions shaded in Fig. 6. Data shown are representative of at least three similar experiments. A, insertion of the acidic sequence (Glu-Asp-Gly-Glu) from M2 into the M1 subtype leads to an increase in affinity toward gallamine. B, conversely to A, removal of the acidic sequence leads to a reduction in affinity toward gallamine. C, unlike the case in B, replacement of the M4 sequence by M1 sequence has no effect on affinity toward gallamine. D, replacement of the two acidic residues in this region of the M5 receptor (Asp-Glu) by the corresponding M1 sequence (Gly-Gln) markedly reduces the affinity toward gallamine.

The effects of mutation on the dissociation rate of [³H]NMS (k₀) and on the parameters m and K_app are summarized in Table 1. The affinity of [³H]NMS did not appear to be affected very much by these mutations but was not determined at every construct; this affinity does not enter into the analysis of the dissociation data generated in this study in any way. A few mutations did produce small but noticeable changes in k₀ and m, but we do not think that they are relevant to this study (see Discussion).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Two different approaches have been adopted in previous mutagenic studies of the binding of gallamine to muscarinic allosteric sites. One approach has assumed that there is a core binding component common to all five subtypes, analogous to the aspartate residue in TM3 of biogenic amine receptors; investigators using this approach have targeted likely conserved residues (Lee et al., 1992; Matsui et al., 1995). Another approach, which we have used, assumes that the ligand adopts a common orientation at each subtype, regardless of whether there is a common binding component. This strategy uses chimeric receptors to look for epitopes that may represent structural bases for subtype-specific differences in affinity. Although this chimeric approach cannot locate a common core binding component directly, it does offer the advantage of providing a natural structure to mutagenic studies. In our case, we expected to be able to increase the affinity of low-affinity M₅ and M₃ receptors by providing a structural feature from the high-affinity M₂ receptor, and we found that to be the case. We also demonstrated the reciprocal effect of making the converse chimera (Ellis et al., 1993). On the other hand, mutations of conserved residues are very much more likely to reduce affinity than to increase affinity, and there is no "converse" mutation to be considered. Because of this, it is more difficult to discriminate specific disruption of a binding epitope from a nonspecific deleterious effect on the receptor.

Leppik et al. (1994) adopted an intermediate approach, mutating both conserved and subtype-specific residues. The most striking effect of their study was found by an essentially chimeric approach, wherein they converted a four-residue sequence of the M₂ receptor to its M₁ counterpart and significantly reduced affinity toward gallamine. For the reasons outlined above, we were anxious to examine the converse chimera. In this study, we have observed the same affinity-reducing effect of eliminating the acidic (EDGE) sequence from the M₂ receptor that had been reported. Moreover, we found a similarly significant increase in affinity toward gallamine upon inserting that acidic sequence into the homologous position of the M₁ receptor. These results are entirely consistent with the suggestion that gallamine adopts essentially the same orientation at the M₁ and M₂ subtypes.

Another advantage of the chimeric approach is that after an important region of the receptor structure has been identified, the essential component may be found by making smaller chimeric inserts or point mutations; the nature of the mutations is dictated by the comparative sequences of the receptors or subtypes being studied. Thus, in this work, we built on our M₂/M₅ chimeric study by converting individual residues in the M₅ receptor to the corresponding M₂ residue (Figs. 4 and 5). Only three of these point mutations affected the affinity toward gallamine to a significant extent. The first of these, the substitution of asparagine for serine near the top of TM6, reduced affinity toward gallamine. It is immediately apparent that this result cannot be taken as support for the involvement of this residue in the higher affinity of M₂. However, it is worth noting that many mutations at this residue in M₅ have been reported to induce constitutive activity, although asparagine was one of the few amino acids that was not tested in that study (Spalding et al., 1997). Nonetheless, induction of receptor activity undoubtedly entails conformational changes quite remote from the top of TM6, and mutations at this position might be especially capable of interfering indirectly with gallamine's binding.

The replacement of lysine by proline in the o3 region of M₅ resulted in a marked increase in affinity toward gallamine. Because proline can sometimes have unique effects on protein structure (MacArthur and Thornton, 1991), it seemed possible that this proline might play an important organizing role in o3. However, a more direct explanation is available. In M₂, the sequence at 414 to 415 in o3 is Ala-Pro, whereas the corresponding residues in M₅ are Asp-Lys. Mutation of the M₅ aspartate to alanine has no effect on gallamine binding by itself, but prevents the effect of the lysine to proline mutation (Fig. 3). Thus, it appears that the insertion of the proline into M₅ serves to make the adjacent aspartate available to gallamine, and that the mutations at these positions cannot be relevant to the binding of gallamine to either wild-type receptor. It is worth noting that mutations of the pair of residues corresponding to M₂^409-410 do not interact in this way. The substitution of asparagine for serine (in M₅) has been described (above) to reduce affinity toward gallamine. Substitution of isoleucine for valine at the adjacent position had no effect by itself and did not interfere with the effect of the serine to asparagine mutation (Fig. 3).

Only one other mutation in M₅ produced a marked increase in affinity toward gallamine, namely, the substitution of asparagine for valine near the end of o3, just above TM7. We have shown previously that swapping this region of the receptor between M₂ and M₃ produces predictable and converse effects on affinity toward gallamine (Ellis et al., 1993). Point mutations at this position mimicked the effects seen in the chimeric receptors; i.e., replacing lysine with asparagine at that position in the M₃ receptor markedly increased affinity toward gallamine, whereas replacing asparagine with lysine in M₂ reduced affinity toward gallamine (see Figs. 4 and 5). Interestingly, an asparagine residue in TM7 of -adrenergic and some 5-hydroxytryptamine receptors, as well as mutant ₂-adrenergic receptors and 5-hydroxytryptamine receptors, has been implicated in interactions with -adrenergic antagonists (Suryanarayana et al., 1991; Guan et al., 1992; Adham et al., 1994). These studies have suggested the importance of an interaction between asparagine and the oxygen atom of an alkoxy group that links to an aromatic ring (Suryanarayana et al., 1991). Gallamine and many other muscarinic allosteric ligands possess just such alkoxy groups. Glennon et al. (1996) have used mutagenesis and a series of propranolol analogs to conclude that propranolol forms two hydrogen bonds with that asparagine. The spacing between the ether oxygen and the hydroxyl group of propranolol is similar to that between the ether oxygen and the quaternary nitrogen of gallamine, but it remains to be determined whether two interactions can occur between gallamine and the asparagine at M₂⁴¹⁹.

The M₁ and M₄ subtypes have acidic residues at this position (Fig. 4). Replacing the aspartate of M₄ with asparagine leads to a small reduction in affinity toward gallamine; replacing the asparagine of M₂ with aspartate or glutamate leads to similar small increases in affinity toward gallamine (Fig. 5). This set of mutations suggests that the interaction between aspartate or glutamate and gallamine's quaternary nitrogen is somewhat stronger than the interaction(s) with asparagine.

We have previously suggested that the epitope in o2 was not apparent with the M₂/M₅ chimeric receptors because M₅ also possesses that binding component (Ellis, 1997). Supporting that idea, the replacement of the acidic amino acids in that region of M₅ (Asp-Glu) with the corresponding M₁ residues (Gly-Gln) dramatically reduced the already low affinity toward gallamine. However, replacement of the M₄ sequence in this region by M₁ sequence has no effect on affinity toward gallamine, in spite of the presence of an acidic amino acid (at a different position) in M₄ (see Figs. 6 and 7).

Our findings in o2 are in agreement with the conclusion of Leppik et al. (1994) that the M₂^172-175 sequence presents an important epitope for gallamine that is lacking in M₁. Furthermore, our data suggest that the M₅ subtype also possesses this epitope, whereas M₄ does not. The locations of acidic residues in M₂, M₄, and M₅ in this region point most strongly to the glutamate at M₂¹⁷⁵, although that remains to be confirmed. If so, M₃ would also be expected to derive affinity toward gallamine from this residue (Fig. 6). In the o3 region, the residue involved in gallamine's affinity corresponds to the asparagine at M₂⁴¹⁹ (Fig. 4). The presence of valine (M₅) or lysine (M₃) appears to be associated with lower affinity for gallamine, whereas aspartate (M₄) or glutamate (M₁) is somewhat more favorable toward gallamine binding. Thus, in both regions the findings are in agreement with the grouping of affinities toward gallamine among the muscarinic subtypes, because M₁ and M₄ have similar affinities, whereas M₃ is similar to M₅ (Ellis et al., 1991, 1993).

Synthetic peptides based on the o2 region of the M₂ muscarinic receptor have been found to bind to autoantibodies from patients with dilated cardiomyopathy. These autoantibodies inhibit radioligand binding to M₂ receptors in an atropine-sensitive, but apparently noncompetitive, manner. The autoantibodies also appear to activate the receptor, again in an atropine-sensitive manner (Matsui and Fu, 1998). Interactions between these antibodies and allosteric muscarinic ligands do not appear to have been investigated. We have found that many allosteric ligands are sensitive to our M₂/M₅ chimeric receptors in which o2 has been swapped (J. Ellis and M. Seidenberg, in preparation). However, this almost certainly implicates a different portion of the o2 loop, because gallamine was insensitive to this same chimeric receptor. Additionally, no allosteric ligand other than gallamine has demonstrated a dramatic and selective sensitivity to the o3 chimera (i.e., CR4). This is probably related to the structural diversity of presently known muscarinic allosteric ligands. Future studies should reveal whether structurally related allosteric ligands (Gharagozloo et al., 1999; Nassif-Makki et al., 1999) share common epitopes.

Values for k₀, m, and K_app were compiled and presented in Table 1. The effects of mutations on pK_app have already been discussed extensively above. The effects of the mutations on k₀ and m were found to be less dramatic and less consistent, and are more difficult to interpret for several reasons. One of the largest effects was the N  K mutation in M₂, which slowed k₀ by a factor of ~2. The reverse substitution, K  N in M₃, accelerated k₀ by approximately the same amount, but the V  N mutation in M₅ had no effect at all. Furthermore, whereas the asparagine speeds dissociation in M₃, the D  N mutation in M₄ slows k₀ by almost as much, and the converse N  D mutation in M₂ has no effect on k₀. The kinetics of [³H]NMS binding must be sensitive to the overall structure of the binding pocket; it does not seem surprising that even slight kinks in that pocket might have small effects that are difficult to predict or interpret. Because m combines with k₀ to define the dissociation of [³H]NMS from the ternary complex, it suffers from much the same difficulties. The most obvious effect on m arises from the S  N mutation in M₅ near the top of TM6 (Table 1; Fig. 1). As noted above, this mutation was the only one in the M₅ TM6-o3 series to reduce affinity toward gallamine, and many mutations at this serine have been reported to activate the receptor (Spalding et al., 1997).

All of the present studies have been performed in a hypotonic buffer, which has been shown by a number of laboratories to enhance the affinities of many allosteric ligands, especially gallamine (Ellis et al., 1991; Waelbroeck, 1994; Trankle et al., 1996). Thus, it is not inconceivable that gallamine's allosteric affinity might be affected by different residues in a more physiological buffer. However, we do not think this is likely, based on the available evidence. For example, in a more nearly isotonic buffer (50 mM PB), Leppik et al. (1994) observed a very similar change in affinity attributable to the M₂ EDGE  LAGQ mutation, as well as a similar degree of slowing of the dissociation of [³H]NMS. Also, we have found the affinity of the allosteric ligand alcuronium to be sensitive to a unique portion of the receptor, whether assayed in hypotonic or physiological buffers (JE and MS, unpublished observations).

It should be noted that this study has used dissociation assays exclusively. The great advantage of this assay is that it ensures that only allosteric effects will be observed. The disadvantage is that the affinity of gallamine for the unliganded receptor cannot be extracted from these data. However, to extract that affinity requires confidence that the simple allosteric model applies, and thus far this model has only been tested rigorously at the wild-type M₂ receptor (Waelbroeck, 1994; Ellis, 1997; Ellis and Seidenberg, 1999).

In summary, we have investigated two epitopes involved in gallamine's allosteric subtype specificity at muscarinic receptors. Our results suggest that gallamine adopts a similar orientation at the different subtypes and continue to support the concept that muscarinic allosteric ligands bind to the outermost portions of these receptors. The M₁ and M₄ subtypes appear to derive affinity toward gallamine from acidic residues in the o3 region, whereas the M₃ and M₅ receptors seem to derive affinity from acidic residues in o2. Gallamine binds to the M₂ subtype with the highest affinity, and both o2 and o3 appear to be involved in this interaction.