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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2008 Sep 12;105(38):14686–14691. doi: 10.1073/pnas.0804372105

Potentiation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

Gareth T Young *, Ruud Zwart †, Alison S Walker †, Emanuele Sher †, Neil S Millar *,‡
PMCID: PMC2535569  PMID: 18791069

Abstract

Positive allosteric modulators of α7 nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as potential tools for the treatment of neurological and psychiatric disorders such as Alzheimer's disease and schizophrenia. However, despite the potential therapeutic usefulness of these compounds, little is known about their mechanism of action. Here, we have examined two allosteric potentiators of α7 nAChRs (PNU-120596 and LY-2087101). From studies with a series of subunit chimeras, we have identified the transmembrane regions of α7 as being critical in facilitating potentiation of agonist-evoked responses. Furthermore, we have identified five transmembrane amino acids that, when mutated, significantly reduce potentiation of α7 nAChRs. The amino acids we have identified are located within the α-helical transmembrane domains TM1 (S222 and A225), TM2 (M253), and TM4 (F455 and C459). Mutation of either A225 or M253 individually have particularly profound effects, reducing potentiation of EC20 concentrations of acetylcholine to a tenth of the level seen with wild-type α7. Reference to homology models of the α7 nAChR, based on the 4Å structure of the Torpedo nAChR, indicates that the side chains of all five amino acids point toward an intrasubunit cavity located between the four α-helical transmembrane domains. Computer docking simulations predict that the allosteric compounds such as PNU-120596 and LY-2087101 may bind within this intrasubunit cavity, much as neurosteroids and volatile anesthetics are thought to interact with GABAA and glycine receptors. Our findings suggest that this is a conserved modulatory allosteric site within neurotransmitter-gated ion channels.

Keywords: allosteric modulators, neurotransmitter receptor

Nicotinic acetylcholine receptors (nAChRs) are excitatory neurotransmitter-gated ion channels and members of a superfamily that also includes ionotropic receptors for 5-hydroxytryptamine (5-HT; serotonin), glycine and γ-aminobutyric acid (GABA) (1, 2). Nicotinic receptors are allosteric proteins (3), which have been implicated in a variety of neurological and psychiatric disorders, including Alzheimer's disease, Parkinson's disease, epilepsy, and schizophrenia (4–6). As a consequence, nAChRs are viewed as being important targets for therapeutic drug discovery (7, 8).

Although most nAChR subunits assemble only into heteromeric nAChRs (9, 10), the α7 and α8 subunits are important exceptions. Both α7 and α8 are able to generate functional homomeric nAChRs (11, 12) and have much closer sequence similarity to each other than to other nAChR subunits (2, 9). Whereas there is evidence that α7 nAChRs are an important receptor subtype in the mammalian brain (and in other vertebrates), the α8 subunit has been identified only in avian species. The 5-HT receptor 3A subunit (5-HT3A) has close sequence similarity to nAChR subunits (2) and, like the nAChR α7 and α8 subunits, is able to generate functional homomeric cation-selective ion channels (13). Indeed, the ability of α7 and 5-HT3A subunits to form homomeric receptors has been exploited extensively in studies in which functional α7/5-HT3A subunit chimeras have been constructed and expressed (for example, see refs. 14 and 15).

Nicotinic receptors are targets for a diverse collection of naturally occurring ligands. These include agonists and antagonists isolated from plants, freshwater algae, marine worms and frogs, and the venoms of snakes and predatory marine snails. In addition to this collection of pharmacologically diverse agonists and antagonists, several positive allosteric modulators of nAChRs have been identified (16). Recently, selective positive allosteric modulators of α7-type nAChRs have attracted particular attention, arising in part from the suggestion that they may have potential in the treatment of disorders such as Alzheimer's disease and schizophrenia (16–19). PNU-120596 is a selective potentiator of α7 nAChRs, with little or no activity on most other nAChR subtypes (17, 20). Recent in vivo studies with this and other α7-selective potentiators have provided evidence of effects in cognitive enhancement (18, 19) and for efficacy in models of schizophrenia (17). However, despite the considerable interest in these compounds, their mechanism of action is unclear.

As has been discussed (16), positive allosteric modulators of α7 nAChRs have been classified as either type I or type II compounds; type I compounds predominantly affect the peak current response, whereas type II compounds affect both peak current responses and the kinetics of agonist-evoked responses (16). PNU-120596 is the prototype member of the type II family, being the first member of this group to be extensively characterized (17, 20). LY-2087101 is a recently identified allosteric potentiator of nAChRs, which is less selective for α7 nAChRs than PNU-120596 (21). Also, in contrast to PNU-120596, LY-2087101 has little effect on the time course of agonist-evoked responses (21), features that are typical of type I positive allosteric modulators.

In the present study, we used subunit chimeras and subunits modified by site-directed mutagenesis with the aim of identifying the binding site for positive allosteric modulators, such as PNU-120596 and LY-2087101. We have identified amino acids located within the α-helical transmembrane domains of the α7 subunit as being critical for potentiation of α7 nAChRs by PNU-120596. These findings have also been extended to demonstrate similar effects of transmembrane mutations on LY-2087101. These findings suggests that nAChR allosteric modulators, such as PNU-120596 and LY-2087101, have a similar mechanism of action and binding site to that of neurosteroids and volatile anesthetics acting on inhibitory GABAA and glycine receptors (22, 23).

Results

PNU-120596 Is a Selective Allosteric Modulator of α7-Like nAChRs.

As was reported in refs. 17 and 20, preapplication of PNU-120596, a positive allosteric modulator of α7 nAChRs, was found to cause a dramatic potentiation of peak agonist-evoked responses and a reduction in the rate of receptor desensitisation with α7 nAChRs expressed in Xenopus oocytes (Fig. 1). In contrast to these effects on α7 nAChRs, we have been unable to detect any significant potentiation by PNU-120596 of agonist-evoked responses with homomeric 5-HT3ARs expressed in Xenopus oocytes (Fig. 1). Our findings, taken together with previous evidence indicating that PNU-120596 displays no activity as an allosteric modulator of α3β4, α4β2, or α9α10 nAChRs (17), support the conclusion that PNU-120596 is a highly selective potentiator of α7 nAChRs.

Fig. 1.

Fig. 1.

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Positive allosteric modulation by PNU-120596 examined by two-electrode voltage-clamp recording in Xenopus oocytes. Representative recordings are shown illustrating agonist-evoked responses obtained in the absence (Left) and presence (Right) of PNU-120596. Data are shown from oocytes expressing the rat α7 subunit (A), mouse 5-HT3A subunit (B), α7V201–5HT3A subunit chimera (C), α74TM-5HT3A chimera (D), and α73TM-5HT3A chimera (E). Data are shown for mouse 5-HT3A subunit but similar data were also obtained with human 5-HT3A. Traces illustrate the influence of PNU-120596 (1 μM) on responses evoked by maximally effective concentrations of agonist; either 1 mM acetylcholine (A and C–E) or 5 μM 1-(3-chlorophenyl)biguanide (B) in the absence (Left) and presence (Right) of PNU-120596. The duration of agonist and PNU-120596 application is illustrated by solid and hatched horizontal lines, respectively. Diagram representations of subunit topology illustrate domains derived from either the rat α7 subunit (black) or mouse 5-HT3A subunit (gray). (Scale bars: vertical, 2 μA; horizontal, 2 s.)

The nAChR α8 subunit, like α7, forms rapidly desensitising homomeric nAChRs when expressed in Xenopus oocytes (12). Although an α8 subunit has not been identified in any mammalian species, the chicken α8 subunit has close sequence similarity to α7 (2, 9). We found that PNU-120596 also acts as a potent positive allosteric modulator of α8 nAChRs, increasing peak responses to a similar extent to that observed with α7. With maximal concentrations of agonist (1 mM acetylcholine), the maximum level of potentiation (fold effects) with PNU-120596 of α7 and α8 nAChRs was not significantly different (9.6 ± 1.7, n = 4 for α7 and 11.0 ± 2.9, n = 5 for α8). We conclude, therefore, that PNU-120596 is a highly selective potentiator of homomeric α7-like nAChRs.

Allosteric Potentiation by PNU-120596 Is Influenced by nAChR Transmembrane Regions.

To examine the influence of subunit domains on potentiation by PNU-120596, we have examined its ability to act as an allosteric modulator of a series of subunit chimeras containing regions derived from the nAChR α7 subunit (which is potentiated by PNU-120596) and the 5-HT3A subunit (which is not). Three previously described subunit chimeras (α73TM-5HT3A, α74TM-5HT3A and α7V201–5HT3A) (15) gave agonist-evoked responses when expressed in Xenopus oocytes but displayed no allosteric potentiation by PNU-120596 at concentrations up to 30 μM (Fig. 1), suggesting that amino acids located within the transmembrane domains (and, in particular, within TM1-TM3) of the nAChR α7 subunit are required for potentiation by PNU-120596.

Identification of Amino Acids Required for Allosteric Modulation by PNU-120596.

An amino acid sequence alignment of the transmembrane regions of nAChR α7 and α8 subunits (both of which are potentiated by PNU-120596) with that of the mouse and human 5-HT3A subunit (which are not) identified a large number of amino acid differences that might be responsible for the selective allosteric modulation by PNU-120596 [supporting information (SI) Fig. S1]. Of these, several amino acids were selected from each of the four transmembrane domains for examination by site-directed mutagenesis. In each case, amino acids within the rat α7 subunit were altered to the corresponding amino acid in the 5-HT3A subunit (either mouse or human).

For all mutated α7 nAChRs, the influence of a range of concentrations of PNU-120596 was examined on a submaximal (EC20) concentration of acetylcholine (Fig. 2 and Fig. S2). In some cases (e.g., D265I, V267T, C448L, and M450H), mutations resulted in nonfunctional receptors, but in all cases where functional responses were detected, there was little or no difference in agonist (acetylcholine) potency, as determined by its EC20 concentration in the absence of allosteric modulator (see legend to Fig. S2 for details).

Fig. 2.

Fig. 2.

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The influence of point mutations in α7 transmembrane domains on potentiation by PNU-120596. Data represent amino acid mutations located within TM1 (A), TM2 (B), TM3 (C), and TM4 (D). In all cases, dose-response data are presented for a range of concentrations of PNU-120596 (0.1–30 μM) on responses evoked by a submaximal (EC20) concentration of acetylcholine. For comparison, dose-response curves for wild-type α7 nAChRs are illustrated as a dotted line. Data shown are G211V (inverted filled triangles) (A), S222M (filled squares), L230Y (filled circles) and A225D (filled triangles) (A); M253L (filled circles) and M260L (filled squares) (B); Q272V (filled circles) and S276V (filled squares) (C); and F455A (filled circles), T456V (filled triangles), and C459Y (filled squares) (D). Data are means ± SEM of five to seven independent experiments, each from different oocytes.

Several of the amino acids mutated in this study had no significant effect on the ability of PNU-120596 to potentiate α7 responses (G211V, L230Y, M260L, Q272V, S276V, and T456V; Fig. 2 and Table 1). Other mutations (S222M, A225D, M253L, F455A, and C459Y) caused significant decreases in the level of potentiation observed with PNU-120596 (Fig. 2 and Table 1). Of these, two mutations (A225D and M253L) caused the most substantial decrease in the ability of PNU-120596 to potentiate α7 responses (Fig. 2 and Table 1). Whereas responses to EC20 concentrations of acetylcholine were potentiated by 36.7 ± 4.1 (fold effects) in wild-type α7 receptors, maximal levels of potentiation observed in α7 receptors containing the A225D and M253L mutations were 3.2 ± 0.3 and 4.1 ± 0.8 (fold effects), respectively (≈10% of the level of potentiation of wild-type).

Table 1.

Influence of α7 point mutations upon potentiation by PNU-120596

Subunit/mutation Location Fold potentiation by PNU120596 EC50, μM
Wild type 36.7 ± 4.1 1.5 ± 0.2
G211V TM1 35.0 ± 2.2 1.3 ± 0.5
S222M TM1 25.7 ± 1.4* 2.8 ± 0.4
A225D TM1 3.2 ± 0.3 *** 5.0 ± 4.0 ***
L230Y TM1 40.1 ± 5.3 2.4 ± 0.3
M253L TM2 4.1 ± 0.8 *** 1.9 ± 0.2
M260L TM2 28.5 ± 2.0 1.6 ± 0.2
D265I TM2 Non-functional
V267T TM2 Non-functional
Q272V TM3 38.4 ± 0.2 1.6 ± 0.2
S276V TM3 40.3 ± 2.7 1.0 ± 0.3
C448L TM4 Non-functional
M450H TM4 Non-functional
F455A TM4 24.7 ± 3.8* 2.8 ± 0.5
T456V TM4 38.3 ± 1.7 1.4 ± 0.3
C459Y TM4 15.4 ± 1.3 *** 2.4 ± 0.8
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Data are means of three to seven independent experiments, ± SEM Statistical significance, determined by ANOVA and Tukey's test: *, P < 0.05; ***, P < 0.001.

Mutations were also introduced into the 5-HT3A subunit in an attempt to confer sensitivity to PNU-120596. Two mutations (D241A and L269M) were constructed in the mouse 5-HT3A subunit (see Fig. S1 legend for an explanation of 5-HT3A amino acid numbering). These mutations are analogous to mutations of A225 and M253 in the α7 subunit (the two mutations that had the greatest effect on the sensitivity of α7 nAChRs to PNU-120596) but mutated 5-HT3A subunits containing these mutations (either singly or as a double mutation) did not show significant levels of potentiation by PNU-120596 (data not shown).

Type I and Type II Allosteric Modulators.

PNU-120596 has been classified as a type II positive allosteric modulator (16, 20) because it causes both a slowing of the rate of desensitisation and potentiates peak agonist-evoked responses (17). In addition, as shown in ref. 20, PNU-120596 is able to induce the opening of desensitised α7 receptors (see also Fig. S3B). In contrast, previous studies with LY-2087101 (21) suggest that it is typical of type I positive allosteric modulators, in that it is able to potentiate peak agonist-evoked responses of α7 nAChRs (21) (albeit with somewhat lower maximal fold effect than PNU-120596), but with little effect on the rate of receptor desensitisation (Fig. S3C). We have also found that, unlike PNU-120596, LY-2087101 displays little ability to facilitate opening of α7 nAChRs after desensitisation with high concentrations of acetylcholine (Fig. S3B), a property that has not, to our knowledge, been examined in other studies (21). Because mutations A225D and M253L had a particularly pronounced effect on potentiation of α7 responses by PNU-120596 (maximum potentiation was 8.1 ± 0.8% and 10.3 ± 2.3%, respectively, of the level observed with wild-type α7 nAChRs), we examined the influence of these mutations on potentiation by LY-2087101 (Fig. S3D). As had been observed with PNU-120596, both mutations significantly reduced levels of potentiation by LY-2087101 (maximum potentiation was 36.7 ± 5.8% and 12.4 ± 4.8%, respectively, of the level observed with wild-type α7 nAChRs; Fig. S3E).

Molecular Docking Simulations.

Inspection of homology models of the α7 subunit (24, 25), and of the 4Å resolution structure of the Torpedo nAChR from which they were derived (26, 27), reveals an intrasubunit cavity between the four transmembrane α-helices (Fig. S4). The cavity is open at its extracellular end but closes approximately half way into the lipid bilayer, and is lined by inwardly facing amino acid side chains, many of which are charged or polar residues. The five amino acids identified in this study (S222, A225 M253, F455, and C458) line the base and sides of the cavity and have side chains pointing toward its interior (Fig. 3A). In contrast, those amino acids that had no significant effect on potentiation by allosteric modulators have side chains that point away from the interior of the cavity or are located toward the ends of the α-helices, either below the cavity or above the central part of it (Fig. 3A).

Fig. 3.

Fig. 3.

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Computer docking simulations. (A) Homology model of the α7 transmembrane domain based on the 4Å structure of the Torpedo nAChR α subunit (26), illustrating the location of mutated amino acids. The backbone of the four transmembrane α-helices (TM1-TM4) are colored gray. Side chains of amino acids that, when mutated, had a significant effect on potentiation by allosteric modulators are shaded red. Amino acids that had little or no effect when mutated are shaded blue. The model is shown from a side-on view (Right) and as viewed from above, looking down from the extracellular face of the lipid membrane (Left). In Right, part of the TM3 domain has been omitted, to avoid obscuring amino acid side chains. (B) The lowest energy (highest predicted binding affinity) docked position of PNU-120596 within the Cheng α7 homology model (25) is illustrated. The position of the five amino acids identified by site-directed mutagenesis as being important in potentiation of PNU-120596 are also shown.

Computational docking simulations (28) were performed with three previously described α7 subunit homology models (24, 25, 29). Our aim was to examine whether this intrasubunit cavity is a plausible binding site for nAChR potentiators and where within the α7 homology model PNU-120596 and LY-2087101 would be predicted to bind. Initial docking simulations were performed with the transmembrane region of two α7 homology models (24, 25) based on the 4Å closed structure of the Torpedo nAChR (27), here referred to as the “Cheng” and “Taly” models. In both homology models, the most favourable docked position of PNU-120596 and LY-2087101 (that of lowest predicted binding free energy and, hence, highest predicted affinity; see Table S1) were in very close proximity (within 6Å) of the five amino acids that, when mutated, exert a significant effect on potentiation (for example, see Fig. 3B). In addition, we examined an open channel model of the α7 transmembrane region that was generated by normal mode analysis from the closed model described by Taly et al. (24, 29). Interestingly, comparison of computer docking simulations with these equivalent closed and open channel models predicted higher affinity binding to the open channel model than the equivalent closed channel model (Table S1).

In all three homology models we examined (Cheng, Taly closed, and Taly open), PNU-120596 and LY-2087101 were predicted to bind with higher affinity when flexibility was permitted in side chains of the five amino acids identified by site-directed mutagenesis (Table S1). Of the homology models we examined, PNU-120596 and LY-2087101 were predicted to bind with highest affinity to the Cheng model (25). Even without allowing flexibility of receptor side chains in this model, the predicted binding free energy (ΔG) of PNU-120596 and LY-2087101 within the Cheng homology model was −8.0 kcal/mol and −8.6 kcal/mol, respectively (equivalent to predicted binding affinities of 1 μM and 0.5 μM). By allowing receptor side-chain flexibility during the docking simulations of the five amino acids identified by site-directed mutagenesis, both PNU-120596 and LY-2087101 were predicted to bind with substantially lower predicted free energy (and higher affinity): −11.7 kcal/mol (3 nM) and −13.4 kcal/mol (0.1 nM), respectively (Table S1).

Discussion

We have identified amino acids located within the transmembrane domains of the nAChR α7 subunit that exert a dramatic influence on the ability of PNU-120596 and LY-2087101 to potentiate agonist-evoked responses. Studies with α7/5-HT3A subunit chimeras indicated that transmembrane domains TM1-TM3 are essential in influencing potentiation by positive allosteric modulators (Fig. 1). Electrophysiological studies with mutated α7 subunits support the conclusion that amino acids within TM1-TM3 are the principal determinants of potentiation by allosteric modulators. Indeed, the greatest influence on potentiation by PNU-120596 was observed in α7 subunits containing mutations at either A225 (in TM1) or M253 (in TM2). However, a role for TM4 as an additional determinant of potentiation by allosteric modulators is provided by mutagenesis studies with amino acids within TM4 (F455 and C459), a conclusion that is also supported by homology modeling and computer docking simulations.

Examination of α7 homology models (25, 29) and the 4Å resolution structure of the Torpedo nAChR (27) indicates that all of the amino acids that were found to exert a significant effect on potentiation by PNU-120596 (S222, A225, M253, F455, and C459) are located centrally within the transmembrane helices and point toward an intrasubunit cavity located between the four α-helical domains (Fig. 3A and Fig. S4). In contrast, amino acids that have no effect on potentiation point away from the central cavity or are located away from the central region of the α-helical domains (Fig. 3A). To examine the ability of allosteric modulators to bind within this intrasubunit cavity, computer docking simulations were performed with homology models containing the four α-helical domains from three previously described α7 homology models. A “blind docking” approach (30) was used in which no assumptions were made about where within the transmembrane domain ligands might be expected to bind. Not only did computer docking simulations suggest that it is possible for PNU-120596 and LY-2087101 to fit within the cavity located between the four α-helices of the α7 homology model, but with all of the models examined, the most favourable docked positions of PNU-120596 and LY-2087101 (those of the lowest predicted binding free energy) were in very close proximity (within 6Å) to all five of the amino acids that we have shown by site-directed mutagenesis to influence allosteric potentiation of α7 nAChRs (for example, see Fig. 3B).

Of the two α7 closed conformation homology models we examined, PNU-120596 and LY-2087101 were predicted to bind with the highest affinity to an α7 homology model described by Cheng et al. (25). The main difference between the two models is that the model described by Taly et al. (24) was generated from the Torpedo α subunit by applying fivefold averaging and energy minimization. In contrast, the Cheng model was based on the entire Torpedo nAChR and did not apply fivefold symmetry. Our docking simulations with the Cheng model were, however, performed with the subunit that was modeled on the Torpedo α subunit [“subunit A” in the Torpedo structure (27)]. Interestingly, comparisons of open and closed receptor models derived from the same homology model (29) predicted that binding of PNU-120596 and LY-2087101 was of higher affinity to a homology model of the receptor open channel structure (Table S1). Also, in all of the homology models we examined, PNU-120596 and LY-2087101 were predicted to bind with higher affinity when flexibility was permitted in side chains of the five amino acids identified by site-directed mutagenesis (Table S1).

There is evidence, from studies of nAChRs purified from the Torpedo electric organ, that transmembrane α-helices, particularly TM2, adopt a different conformation in the open and closed states (31). Without wishing to over-interpret the results of the docking simulations described in the present study, these results provide support for the conclusion that the amino acids we have identified are involved directly in the binding of positive allosteric modulators such as PNU-120596 and LY-2087101. They also suggest that these allosteric effectors may bind with higher affinity to the open channel. It is possible that, by binding at a site located between the four transmembrane α-helices, allosteric modulators reduce the energy barrier for transitions between the closed and open receptor conformations or decrease the probability of the receptor entering a desensitized state.

Docking of these ligands into our homology model enables us to hypothesize about which features of the site are important for potentiation by allosteric ligands. All of the amino acids we have identified line the walls and base of an intrasubunit cavity. Mutation of these amino acids would both have the effect of altering the dimensions of the cavity, whereas A225D (the mutation that had the most dramatic effect in this study) would also introduce a negative charge, hence altering its hydrophobicity. In all of the homology models we have examined by computer docking simulations, the position at which PNU-120596 and LY-2087101 are docked with the lowest predicted binding energy lies in very close proximity to the five amino acids we have identified by site-directed mutagenesis. When docked in its most energetically favourable orientation, PNU-120596 was positioned such that its isoxazole heterocyclic group was in close proximity to F455 (in TM4) and its larger halogenated aromatic group positioned higher in the intrasubunit cavity (Fig. 3 and Fig. S4). In its most favourable docked orientation, LY-2087101 was in a similar position to that adopted by PNU-120596, with its thiophene heterocyclic group in close proximity to F455 and its halogenated aromatic group positioned higher in the cavity (Fig. S4).

It would be interesting to identify amino acids that, when mutated, confer on the 5-HT3A subunit sensitivity to positive allosteric modulators. Initial studies with D241A and L269M mutations in the 5-HT3A subunit failed to confer sensitivity to PNU-120596. It is possible, therefore, that multiple additional mutations might be required. Likely candidates might be other amino acids in 5-HT3A, which are equivalent to those identified by mutagenesis of α7 (Fig. S1) or additional amino acids that computer modeling suggest lie close to the predicted binding site of PNU-120596 and LY-2087101. Examples of such amino acids are S234 and A449 (equivalent to C218 and I458 in α7) (see Fig. S1 legend for an explanation of 5-HT3A amino acid numbering).

Although LY-2087101 and PNU-120596 both act as allosteric potentiators of α7 nAChRs, there are differences in the extent to which they modulate the kinetics of agonist-evoked responses. In this respect they are typical of what has been described as type I and type II allosteric potentiators (16). In addition, as we have shown, LY-2087101 differs from PNU-120596 in the extent to which it can cause opening of α7 nAChRs after desensitisation (Fig. S3B). The evidence we have obtained suggests that both PNU-120596 and LY-2087101 bind to a broadly similar site within the intrasubunit transmembrane cavity. Although both compounds act to potentiate agonist-evoked responses, the difference in their ability to modulate receptor desensitisation may reflect subtle but important differences in their interaction with this binding site. Indeed, although mutation M253L had an equally profound effect on both allosteric modulators, the A225D mutation exerted effects of significantly different magnitude on PNU-120596 and LY-2087101 (Fig. S3E). The Monod–Wyman–Changeux model (32) has been used extensively to explain the function of allosteric proteins and allosteric effectors (for example, see refs. 3, 4, and 33). With reference to this model, it seems plausible that type II allosteric modulators such as PNU-120596 (16, 20), which exert a profound effect on the time course of agonist-evoked responses may decrease the allosteric constants for transitions between the open and desensitized states (3, 33, 34). In addition, both allosteric modulators examined in this study potentiate peak agonist-evoked responses and, as such, may be influencing allosteric transitions between resting and open states.

These findings strongly indicate that allosteric modulators, such as PNU-120596 and LY-2087101, cause potentiation of agonist-evoked α7 responses by binding within the nAChR transmembrane region. On the basis of similar studies with subunit chimeras and/or site-directed mutagenesis, similar conclusions have been made concerning the location of allosteric ligand binding for neurosteroids and volatile anesthetics acting on GABAA and glycine receptors (22, 23, 35). Indeed, two of the amino acids we have identified in this study, M253 (in TM2) and C459 (in TM4), lie in positions exactly analogous to amino acids that have been identified as influencing the binding of allosteric potentiators to inhibitory GABAA or glycine receptors (22, 23, 35). It has been proposed that the binding site for allosteric modulators such as volatile anesthetics, located between the four transmembrane domains, corresponds to a water-filled cavity (26, 36). Recent photoaffinity labeling studies with purified Torpedo nAChR has identified interactions of photoaffinity ligands, including anesthetics, with several transmembrane sites, thought not at the amino acids identified in the present study (37, 38).

In contrast to the findings reported here (and from studies of neurosteroids and volatile anesthetics acting on GABAA and glycine receptors), there is evidence that some allosteric effectors bind to sites outside the transmembrane region. Allosteric potentiators, such as galantamine and physostigmine, are thought to interact with a site located in the nAChR extracellular domain (39–41), perhaps analogous to the site for potentiation of GABAA receptors by benzodiazepines (41). It appears, therefore, that nAChRs and other Cys-loop receptors can be modulated by allosteric effectors acting at multiple independent sites.

We consider it reasonable to conclude that the amino acids we have identified by site-directed mutagenesis exert a direct effect on the binding of allosteric modulators, such as PNU-120596 and LY-2087101. An issue concerning the identification of ligand binding sites by approaches such as site-directed mutagenesis is that it is possible that mutations are having an effect (e.g., by an induced conformational change) at a distant binding site. However, given the highly clustered location of the amino acids we have identified, together with the support provided by homology modeling and computer docking simulations, the most parsimonious explanation of our data would seem to be that the amino acids we have identified correspond to the binding site for α7 allosteric modulators, such as PNU-120596 and LY-2087101. Together with studies that have examined the potentiation of GABA and glycine receptors (22, 23, 35, 36), we conclude that the intrasubunit cavity, located between four transmembrane α-helices, corresponds to a highly conserved modulatory site of Cys-loop neurotransmitter-gated ion channels.

Methods

Materials.

PNU-120596 (17) was obtained from Tocris Bioscience. LY-2087101 (21) was synthesised by Eli Lilly. All other chemicals were obtained from Sigma.

Subunit cDNAs, Subunit Chimeras, and Site-Directed Mutagenesis.

Rat nAChR α7 subunit cDNA was obtained from J. Patrick (Baylor College of Medicine, Houston, TX). Chick nAChR α8 subunit cDNA was obtained from J. Lindstrom (University of Pennsylvania, Philadelphia). Mouse 5-HT3A subunit cDNA was obtained from D. Julius (University of California, San Francisco). Human 5-HT3A subunit cDNA was obtained from E. Kirkness (Institute for Genomic Research, Rockville, MD). Subunit chimeras α73TM-5HT3A, α74TM-5HT3A, and α7V201–5HT3A, which are described in ref. 15, contain domains derived from the rat nAChR α7 subunit and mouse 5-HT3A subunit. Site-directed mutagenesis was performed using the QuikChange mutagenesis kit (Stratagene). Mutated cDNA constructs, in plasmid expression vector pcDNA1neo (Invitrogen), were verified by nucleotide sequencing, using the Big Dye Terminator Cycle Sequencing kit and ABI Prism 3100-Avant automated sequencer according to the manufacturer's instructions (Applied Biosystems).

Xenopus Oocyte Electrophysiology.

Xenopus laevis oocytes were isolated and defolliculated as described in ref. 42. For α7 and 5-HT3A, oocyte nuclei were injected with cDNAs cloned downstream from a CMV promoter in plasmid expression vectors (42). For α8, cytoplasmic injection of cRNA was used to enhance levels of functional expression and cRNA was synthesised using mMESSAGE mMACHINE T7 transcription kit (Ambion). Oocytes were injected with 2–20 ng of DNA or 2 ng of cRNA per oocyte in a volume of 18.4 nl, using a Drummond variable volume microinjector. Two electrode voltage-clamp recordings were performed as described in ref. 42.

Computer Docking Simulations.

Computational molecular docking of PNU-120596 and LY-2087101 were performed with the transmembrane region of previously described human α7 homology models (25, 29), using AutoDock 4 (28). To avoid bias, a blind docking approach was used (30) in which no assumptions were made concerning where within the transmembrane region ligands might be expected to bind. Ligands were given flexibility during the docking simulation, whereas the receptor model was treated either as being rigid or with flexibility allowed in selected side chains. Predicted Gibbs free energy of binding (ΔG) was calculated as described (28, 43), as was the predicted equilibrium constant for binding (Keq), using the equation Keq = e − ΔG/RT (where R = gas constant, and T = absolute temperature).

Supplementary Material

Supporting Information
supp_105_38_14686__index.html (635B, html)

Acknowledgments.

We thank David Julius, Ewan Kirkness, Jon Lindstrom, and Jim Patrick for providing cDNAs used in this study; Xiaolin Cheng and Antoine Taly for providing α7 homology models; René Frank and Andrias O'Reilly for assistance with molecular docking simulations; and Izumi Yamamoto for assistance with site-directed mutagenesis. This work was supported by an Industrial CASE PhD studentship that was cofunded by the Biotechnology and Biological Sciences Research Council (to G.T.Y.) and Eli Lilly & Co., Ltd.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0804372105/DCSupplemental.

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