1 Pharmacological studies of a7 nicotinic acetylcholine receptors are confounded by the fact that rapid desensitization to high agonist concentration causes a7 peak responses to occur well in advance of complete solution exchange. For this reason, peak currents are an invalid measure of response to applied agonist concentrations. We show that results comparable to those that have been corrected for instantaneous concentration are obtained if net charge is used as the measure of receptor response. 2 Dose response curves obtained with these methods indicate that a7 receptors are approximately 10 fold more sensitive to agonist than previously reported. The agonists, ACh, choline, cytisine, GTS-21, 4OH-GTS-21 and 4-MeO-CA have the same rank order potency for both human and rat receptors: 4-MeO-CA 4 4OH-GTS-21 4 GTS-21 4 cytisine 4 ACh 4 choline. However, di erences in e cacy exist between rat and human receptors. GTS-21 is more e cacious for rat than human a7 receptors and cytisine more e cacious for human than rat a7 receptors. 3 Choline is the least potent agonist for both human and rat a7, with a potency approximately 10 fold lower than that of ACh. While the EC50 for the activation of a7 receptors by choline (400 ± 500 mM) is outside the normal physiological range (10 ± 100 mM), choline can nonetheless produce detectable levels of channel activation in the physiological concentration range. Since these concentrations are relatively non-desensitizing, the contribution of choline-activated a7 receptor current may play a signi®cant role in the regulation of calcium homeostasis in a7-expressing neurons.

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.

This review covers history underlying the discovery of the molecular mediators of nicotine's effects in the brain and the diversity of the nicotinic acetylcholine receptor (nAChR) subtypes. Models are presented for both their structure and their function as mediators of signal transduction, with special consideration of the differences between the two main subtypes: heteromeric receptors, which are specialized for rapid electrochemical signal transduction, and homomeric α7 receptors, which have come to be implicated in both ionotropic and metabotropic signaling. The review presents perspectives on the pharmacology and therapeutic targeting of nAChRs for the treatment of nicotine dependence or disease.

The α7 nicotinic acetylcholine receptor (nAChR) is a promising drug target for a number of neurological disorders including chronic pain and inflammatory diseases. Since α7 can function as a ligand-gated ion channel, drug development initially focused on ligands that were selective activators of the α7 ion channel. However, the best α7 drugs for chronic pain and inflammation indications may not be ion channel activators but rather “silent agonists”, which bind to the receptor but preferentially induce non-conducting states that modulate signal transduction in non-neuronal cells. One such compound is NS6740. We show that NS6740 selectively induces prolonged desensitization of α7 nAChRs. There are two forms of α7 desensitization that can be distinguished by their sensitivity to the positive allosteric modulators (PAMs). At high concentrations, NS6740 preferentially induces PAM-insensitive desensitization, which over the course of several minutes reverts to the sensitive form. NS6740 was tested in several pain models after in vivo administration in the mouse. Although it had no effects in acute thermal pain, NS6740 induced significant dose- and time-dependent antinociceptive activity in formalin- and acetic acid-induced nociceptive behaviors as well as in the chronic constrictive nerve injury (CCI) model for neuropathic pain. The antinociceptive activity of NS6740 in these models was α7-dependent. In addition, NS6740 administration reversed pain-induced aversion, an important affective component of pain. The time and concentration dependence of the effects were consistent with NS6740 induction of PAM-insensitive non-conducting states, suggesting that signal transduction required for analgesia is accomplished by α7 receptors in that conformation.

3-[(2,4-Dimethoxy)benzylidene]-anabaseine dihydrochloride (DMXBA; GTS-21), an Alzheimer's drug candidate, selectively stimulates ␣7 nicotinic acetylcholine receptors. It rapidly enters the brain after oral administration and enhances cognitive behavior. Less than 1% of orally administered DMXBA is recovered in the urine. We report the identification and characterization of the major phase I metabolites of this drug candidate. Three hydroxy metabolites were generated in vitro by hepatic microsomal O-dealkylation of the two methoxy substituents on the benzylidene ring. They were also found in plasma of rats after oral administration, but at significantly lower concentrations relative to the parent compound. The metabolites displayed similar binding affinities and partial agonist potencies at rat brain ␣7 receptors. However, each displayed a higher efficacy than DMXBA for stimulating rat and human ␣7 receptors.Like DMXBA, the metabolites were weak antagonists at ␣ 4 ␤ 2 receptors. The predicted conformations of the metabolites were nearly identical with that of DMXBA. Ionization of the tetrahydropyridyl nitrogen was essential for high-affinity binding of DMXBA to the ␣7 receptor. The hydroxy metabolites were much more polar than DMXBA, derived from their experimentally estimated octanol/water partition coefficients, and they entered the brain much less readily than DMXBA. Their contributions to the behavioral effects of orally administered DMXBA, if any, would probably be very small during short-term administration. Benzylidene anabaseines pharmacologically similar to the hydroxy metabolites, but which enter the brain more readily, may provide greater stimulation of ␣7 receptors in the whole organism.

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