Investigation of the Molecular Mechanism of the α7 Nicotinic Acetylcholine Receptor Positive Allosteric Modulator PNU-120596 Provides Evidence for Two Distinct Desensitized States

Abstract

Although ␣7 nicotinic acetylcholine receptors are considered potentially important therapeutic targets, the development of selective agonists has been stymied by the ␣7 receptor's intrinsically low probability of opening (P open ) and the concern that an agonist-based therapeutic approach would disrupt endogenous cholinergic function. Development of ␣7 positive allosteric modulators (PAMs) holds promise of avoiding both issues. N-(5-Chloro-2,4-dimethoxyphenyl)-NЈ-(5-methyl-3-isoxazolyl)-urea (PNU-120596) is one of the most effective ␣7 PAMs, with a mechanism associated, at least in part, with the destabilization of desensitized states. We studied the mechanism of PNU-120596 potentiation of ␣7 receptors expressed in Xenopus laevis oocytes and outside-out patches from BOSC 23 cells. We identify two forms of ␣7 desensitization: one is destabilized by PNU-120596 (D s ), and the other is induced by strong episodes of activation and is stable in the presence of the PAM (D i ). Our characterization of prolonged bursts of single-channel currents that occur with PNU-120596 provide a remarkable contrast to the behavior of the channels in the absence of the PAM. Individual channels that avoid the D i state show a 100,000-fold increase in P open compared with receptors in the nonpotentiated state. In the presence of PNU-120596, balance between D s and D i is dynamically regulated by both agonist and PAM binding, with maximal ion channel activity at intermediate levels of binding to both classes of sites. In the presence of high agonist concentrations, competitive antagonists may have the effect of shifting the balance in favor of D s and increasing ion channel currents.

Critical Molecular Determinants of α7 Nicotinic Acetylcholine Receptor Allosteric Activation

“…1E is a single-channel recording of ␣7 receptor currents in the presence of 10 M GAT107. Bursting activity was observed that was similar to that reported for ACh and PNU-120596 (10).…”

“…The binding of orthosteric agonists alone promote only rare and brief openings, although orthosteric agonists with the PAMs also bound produce occasional protracted bursts of openings occurring from a PAM-sensitive desensitized state (D s ) (10). Shown in Fig.…”

“…GAT904 is an effective allosteric activator and also acts as a direct potentiator (Fig. 6D); however, it was relatively ineffective at producing primed potentiation, either due to relatively reversible PAM-site binding or due to preferential induction of PAM-insensitive desensitization (10). The further distance between the bromine atom and phenyl ring in GAT193 is associated with the loss of activity as an allosteric agonist, but primed potentiation was observed and was larger than that seen with GAT904 (Fig.…”

“…1C). However, this form of activation appears to shift the conformational equilibrium of the receptors toward PAM-insensitive desensitized (D i ) states (10) so that even primed potentiation of subsequent ACh responses is minimal (13).…”

“…PNU-120596, one of the most efficacious of such agents, destabilizes one of the non-conducting states pro-moted by high levels of agonist occupancy and induces a novel conducting state that is long-lived and associated with protracted bursts of openings (10). Data suggest that the binding site for such PAMs is within the transmembrane domains of the receptor (11,12).…”