Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

Abstract

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

Critical Molecular Determinants of α7 Nicotinic Acetylcholine Receptor Allosteric Activation

“…We have also previously reported that DAA is sensitive to MLA, a selective competitive antagonist of ␣7 nAChR, whereas primed potentiation is not (13). The hypothesis that the PAM site for GAT107 is related to that for other efficacious PAMs, such as PNU-120596, is supported by the observation that transmembrane domain mutant M254L that reduces PNU-120596 effects (11) also reduces the PAM activity of 4BP-TQS, the racemic form of GAT107 (14). We hypothesize that although GAT107 has activity at two separate sites, to detect the activity at the DAA site, GAT107 must also work at the PAM site or the PAM site needs to be occupied by another PAM, as described next.…”

“…Studies of ␣7 and related chimeras have instead indicated that the binding sites for efficacious PAMs like PNU-120596 are most likely located within the hydrophobic transmembrane domains (11,12). The PAM activity associated with 4BP-TQS and GAT107 is hypothesized to arise from binding to a site related to that of PNU-120596, due to effects of mutations in the region (14). We have shown however that the DAA of GAT107 can be separated from its PAM activity, and most appear to arise from binding to a novel solvent-accessible site, probably in the extracellular domain.…”

“…Although PNU-120596 in combination with an orthosteric agonist can sporadically induce an extremely large increase in the opening of single channels, it also has the effect of stabilizing alternative desensitized non-conducting conformations and has similar efficacy as the desensitizing agent NS6740 in the same pain models (55)(56)(57). The discovery of the ago-PAM 4BP-TQS (14) and the characterization of its active isomer GAT107 (13) has now brought us to a further threshold for advancing ␣7 drug development.…”

“…Several lines of evidence also support the hypothesis that the DAA site is distinct from the orthosteric agonist site (13,14). To further test this hypothesis, we utilized the "gatekeeper" cysteine mutant L119C (23).…”

“…A new class of ␣7 receptor activators has also recently been discovered, which, in addition to being able to potentiate orthosteric activation, can directly activate the receptor's ion channel through an allosteric mechanism. The prototypical compound with such ago-PAM activity is GAT107, the active isomer of 4BP-TQS (13,14), which is an analog of the PAM TQS. The PAM activity of TQS, as well as the direct allosteric activation (DAA) produced by GAT107, appears to rely on binding to the same transmembrane site as other PAMs of the PNU-120596 type.…”