Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues.

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.

ABSTRACT␣7 nicotinic acetylcholine receptors (nAChRs) have been a puzzle since their discovery in brain and non-neuronal tissues. Maximal transient probability of an ␣7 nAChR being open with rapid agonist applications is only 0.002. The concentration dependence of ␣7 responses measured from transfected cells and Xenopus laevis oocytes shows the same disparity in potency estimations for peak currents and net charge, despite being studied at 1000-fold different time scales. In both cases the EC 50 was approximately 10-fold lower for net charge than for peak currents. The equivalence of the data obtained at such disparate time scales indicates that desensitization of ␣7 is nearly instantaneous. At high levels of agonist occupancy, the receptor is preferentially converted to a ligand-bound nonconducting state, which can be destabilized by the positive allosteric modulator N-(5-chloro-2,4-dimethoxyphenyl)-NЈ-(5-methyl-3-isoxazolyl)-urea (PNU-120596). Such currents can be sufficiently large to be cytotoxic to the ␣7-expressing cells. Both the potentiating effect of PNU-120596 and the associated cytotoxicity have a high temperature dependence that can be compensated for by serum factors. Therefore, despite reduced potentiation at body temperatures, use of type II positive allosteric modulators may put cells that naturally express high levels of ␣7 nAChRs, such as neurons in the hippocampus and hypothalamus, at risk. With a low intrinsic open probability and high propensity toward the induction of nonconducting ligand-bound states, it is likely that the well documented regulation of signal transduction pathways by ␣7 nAChRs in cells such as those that regulate inflammation may be independent of ion channel activation and associated with the nonconducting conformational states.