The a3b4 nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the peripheral and central nervous systems, including in airway sensory nerves. The nAChR subtype transduces the irritant effects of nicotine in tobacco smoke and, in certain brain areas, may be involved in nicotine addiction and/or withdrawal. Menthol, a widely used additive in cigarettes, is a potential analgesic and/or counterirritant at sensory nerves and may also influence nicotine's actions in the brain. We examined menthol's effects on recombinant human a3b4 nAChRs and native nAChRs in mouse sensory neurons. Menthol markedly decreased nAChR activity as assessed by Ca 21 imaging, 86 Rb 1 efflux, and voltage-clamp measurements. Coapplication of menthol with acetylcholine or nicotine increased desensitization, demonstrated by an increase in the rate and magnitude of the current decay and a reduction of the current integral. These effects increased with agonist concentration. Pretreatment with menthol followed by its washout did not affect agonist-induced desensitization, suggesting that menthol must be present during the application of agonist to augment desensitization. Notably, menthol acted in a voltageindependent manner and reduced the mean open time of single channels without affecting their conductance, arguing against a simple channel-blocking effect. Further, menthol slowed or prevented the recovery of nAChRs from desensitization, indicating that it probably stabilizes a desensitized state. Moreover, menthol at concentrations up to 1 mM did not compete for the orthosteric nAChR binding site labeled by [ 3 H]epibatidine. Taken together, these data indicate that menthol promotes desensitization of a3b4 nAChRs by an allosteric action.

These findings suggest that non-nicotine constituents in these EC liquids do not contribute to their reinforcement-enhancing effects. However, they may attenuate nicotine's acute aversive/anhedonic and/or toxic effects, which may moderate the abuse liability and/or toxicity of ECs.

The 42 nicotinic acetylcholine receptor (nAChR) is important in central nervous system physiology and in mediating several of the pharmacological effects of nicotine on cognition, attention, and affective states. It is also the likely receptor that mediates nicotine addiction. This receptor assembles in two distinct stoichiometries: (4)(2) and (4)(2), which are referred to as high-sensitivity (HS) and low-sensitivity (LS) nAChRs, respectively, based on a difference in the potency of acetylcholine to activate them. The physiologic and pharmacological differences between these two receptor subtypes have been described in heterologous expression systems. However, the presence of each stoichiometry in native tissue currently remains unknown. In this study, different ratios of rat 4 and2 subunit cDNA were transfected into human embryonic kidney 293 cells to create a novel model system of HS and LS 42 nAChRs expressed in a mammalian cell line. The HS and LS nAChRs were characterized through pharmacological and biochemical methods. Isolation of surface proteins revealed higher amounts of 4 or2 subunits in the LS or HS nAChR populations, respectively. In addition, sazetidine-A displayed different efficacies in activating these two receptor stoichiometries. Using this model system, a neurophysiological "two-concentration" acetylcholine or carbachol paradigm was developed and validated to determine 4/2 subunit stoichiometry. This paradigm was then used in layers I-IV of slices of the rat motor cortex to determine the percent contribution of HS and LS 42 receptors in this brain region. We report that the majority of 42 nAChRs in this brain region possess a stoichiometry of the (4)(2) LS subtype.

AT-1001 [N-(2-bromophenyl)-9-methyl-9-azabicyclo[3.3.1] nonan-3-amine] is a high-affinity and highly selective ligand at a3b4 nicotinic cholinergic receptors (nAChRs) that was reported to decrease nicotine self-administration in rats. It was initially reported to be an antagonist at rat a3b4 nAChRs heterologously expressed in HEK293 cells. Here we compared AT-1001 actions at rat and human a3b4 and a4b2 nAChRs similarly expressed in HEK 293 cells. We found that, as originally reported, AT-1001 is highly selective for a3b4 receptors over a4b2 receptors, but its binding selectivity is much greater at human than at rat receptors, because of a higher affinity at human than at rat a3b4 nAChRs. Binding studies in human and rat brain and pineal gland confirmed the selectivity of AT-1001 for a3b4 nAChRs and its higher affinity for human compared with rat receptors. In patch-clamp electrophysiology studies, AT-1001 was a potent partial agonist with 65-70% efficacy at both human and rat a3b4 nAChRs. It was also a less potent and weaker (18%) partial agonist at a4b2 nAChRs. Both a3b4 and a4b2 nAChRs are upregulated by exposure of cells to AT-1001 for 3 days. Similarly, AT-1001 desensitized both receptor subtypes in a concentration-dependent manner, but it was 10 and 30 times more potent to desensitize human a3b4 receptors than rat a3b4 and human a4b2 receptors, respectively. After exposure to AT-1001, the time to recovery from desensitization was longest for the human a3b4 nAChR and shortest for the human a4b2 receptor, suggesting that recovery from desensitization is primarily related to the dissociation of the ligand from the receptor.

Phantasmidine, a rigid congener of the well-known nicotinic acetylcholine receptor agonist epibatidine, is found in the same species of poison frog ( Epipedobates anthonyi). Natural phantasmidine was found to be a 4:1 scalemic mixture, enriched in the (2a R,4a S,9a S) enantiomer by chiral-phase LC-MS comparison to the synthetic enantiomers whose absolute configurations were previously established by Mosher's amide analysis. The major enantiomer has the opposite S configuration at the benzylic carbon to natural epibatidine, whose benzylic carbon is R. Pharmacological characterization of the synthetic racemate and separated enantiomers established that phantasmidine is ∼10-fold less potent than epibatidine, but ∼100-fold more potent than nicotine in most receptors tested. Unlike epibatidine, phantasmidine is sharply enantioselective in its activity and the major natural enantiomer whose benzylic carbon has the 4a S configuration is more active. The stereoselective pharmacology of phantasmidine is ascribed to its rigid and asymmetric shape as compared to the nearly symmetric conformations previously suggested for epibatidine enantiomers. While phantasmidine itself is too toxic for direct therapeutic use, we believe it is a useful platform for the development of potent and selective nicotinic agonists, which may have value as pharmacological tools.