Positive allosteric modulators of ␣7 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 ␣7 nAChRs (PNU-120596 and LY-2087101). From studies with a series of subunit chimeras, we have identified the transmembrane regions of ␣7 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 ␣7 nAChRs. The amino acids we have identified are located within the ␣-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 EC 20 concentrations of acetylcholine to a tenth of the level seen with wild-type ␣7. Reference to homology models of the ␣7 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 ␣-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 GABA A and glycine receptors. Our findings suggest that this is a conserved modulatory allosteric site within neurotransmitter-gated ion channels.allosteric modulators ͉ neurotransmitter receptor N icotinic 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 ␥-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 ␣7 and ␣8 subunits are important exceptions. Both ␣7 and ␣8 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 ␣7 nAChRs are an important receptor subtype in the mammalian brain (and in other vertebrates), the ␣8 subunit has been identified only in avian species. The 5-HT receptor 3A subunit (5-HT 3A ) has close sequence similarity to nAChR subunits (2) and, like the nAChR ␣7 and ␣8 subunits, is able to generate functional homomeric cationselective ion channels (13). Indeed, the ability of ␣7 and 5-HT 3A subunits to...

ABSTRACT␣4 and ␤2 nicotinic acetylcholine receptor (nAChR) subunits expressed heterologously assemble into receptors with high (HS) and low (LS) sensitivity to acetylcholine (ACh); their relative proportions depend on the ␣4 to ␤2 ratio. In this study, injection of oocytes with 1:10 ␣4/␤2 subunit cDNA ratios favored expression of HS ␣4␤2 nAChRs, as evidenced by monophasic ACh concentration-response curves, whereas injections with 10:1 cDNA ratios favored expression of LS ␣4␤2 receptors. The stoichiometry was inferred from the shifts in the ACh EC 50 values caused by Leu to Thr mutations at position 9Ј of the second transmembrane domain of ␣4 and ␤2. The 1:10 injection ratio produced the (␣4) 2 (␤2) 3 stoichiometry, whereas 10:1 injections produced the (

Acetylcholine (ACh) is an important neurotransmitter in the mammalian brain; it is implicated in arousal, learning, and other cognitive functions. Recent studies indicate that nicotinic receptors contribute to these cholinergic effects, in addition to the established role of muscarinic receptors. In the hippocampus, where cholinergic involvement in learning and memory is particularly well documented, alpha7 nicotinic acetylcholine receptor subunits (alpha7 nAChRs) are highly expressed, but their precise ultrastructural localization has not been determined. Here, we describe the results of immunogold labeling of serial ultrathin sections through stratum radiatum of area CA1 in the rat. Using both anti-alpha7 nAChR immunolabeling and alpha-bungarotoxin binding, we find that alpha7 nAChRs are present at nearly all synapses in CA1 stratum radiatum, with immunolabeling present at both presynaptic and postsynaptic elements. Morphological considerations and double immunolabeling indicate that GABAergic as well as glutamatergic synapses bear alpha7 nAChRs, at densities approaching those observed for glutamate receptors in CA1 stratum radiatum. Postsynaptically, alpha7 nAChRs often are distributed at dendritic spines in a perisynaptic annulus. In the postsynaptic cytoplasm, immunolabeling is associated with spine apparatus and other membranous structures, suggesting that alpha7 nAChRs may undergo dynamic regulation, with insertion into the synapse and subsequent internalization. The widespread and substantial expression of alpha7 nAChRs at synapses in the hippocampus is consistent with an important role in mediating and/or modulating synaptic transmission, plasticity, and neurodegeneration.

Conventional nicotinic acetylcholine receptor (nAChR) agonists, such as acetylcholine, act at an extracellular "orthosteric" binding site located at the interface between two adjacent subunits. Here, we present evidence of potent activation of α7 nAChRs via an allosteric transmembrane site. Previous studies have identified a series of nAChR-positive allosteric modulators (PAMs) that lack agonist activity but are able to potentiate responses to orthosteric agonists, such as acetylcholine. It has been shown, for example, that TQS acts as a conventional α7 nAChR PAM. In contrast, we have found that a compound with close chemical similarity to TQS (4BP-TQS) is a potent allosteric agonist of α7 nAChRs. Whereas the α7 nAChR antagonist metyllycaconitine acts competitively with conventional nicotinic agonists, metyllycaconitine is a noncompetitive antagonist of 4BP-TQS. Mutation of an amino acid (M253L), located in a transmembrane cavity that has been proposed as being the binding site for PAMs, completely blocks agonist activation by 4BP-TQS. In contrast, this mutation had no significant effect on agonist activation by acetylcholine. Conversely, mutation of an amino acid located within the known orthosteric binding site (W148F) has a profound effect on agonist potency of acetylcholine (resulting in a shift of ∼200-fold in the acetylcholine dose-response curve), but had little effect on the agonist dose-response curve for 4BP-TQS. Computer docking studies with an α7 homology model provides evidence that both TQS and 4BP-TQS bind within an intrasubunit transmembrane cavity. Taken together, these findings provide evidence that agonist activation of nAChRs can occur via an allosteric transmembrane site.allosteric potentiation | neurotransmitter receptor

Sazetidine-A has been recently proposed to be a "silent desensitizer" of ␣4␤2 nicotinic acetylcholine receptors (nAChRs), implying that it desensitizes ␣4␤2 nAChRs without first activating them. This unusual pharmacological property of sazetidine-A makes it, potentially, an excellent research tool to distinguish between the role of activation and desensitization of ␣4␤2 nAChRs in mediating the central nervous system effects of nicotine itself, as well as those of new nicotinic drugs. We were surprised to find that sazetidine-A potently and efficaciously stimulated nAChR-mediated dopamine release from rat striatal slices, which is mediated by ␣4␤2* and ␣6␤2* subtypes of nAChR. The agonist effects on native striatal nAChRs prompted us to re-examine the effects of sazetidine-A on recombinant ␣4␤2 nAChRs in more detail. We expressed the two alternative stoichiometries of ␣4␤2 nAChR in Xenopus laevis oocytes and investigated the agonist properties of sazetidine-A on both ␣4(2)␤2(3)and ␣4(3)␤2(2) nAChRs. We found that sazetidine-A potently activated both stoichiometries of ␣4␤2 nAChR: it was a full agonist on ␣4(2)␤2(3) nAChRs, whereas it had an efficacy of only 6% on ␣4(3)␤2(2) nAChRs. In contrast to what has been published before, we therefore conclude that sazetidine-A is an agonist of native and recombinant ␣4␤2 nAChRs but shows differential efficacy on ␣4␤2 nAChRs subtypes.