In smoker's brain, rodent brain, and in cultured cells expressing nicotinic receptors, chronic nicotine treatment induces an increase in the total number of high affinity receptors for acetylcholine and nicotine, a process referred to as up-regulation. Up-regulation induced by 1 mM nicotine reaches 6-fold for ␣3␤2 nicotinic receptors transiently expressed in HEK 293 cells, whereas it is much smaller for ␣3␤4 receptors, offering a rationale to investigate the molecular mechanism underlying upregulation. In this expression system binding sites are mainly intracellular, as shown by [ 3 H]epibatidine binding experiments and competition with the impermeant ligand carbamylcholine. Systematic analysis of ␤2/␤4 chimeras demonstrates the following. (i) The extracellular domain critically contributes to up-regulation. (ii) Only residues belonging to two ␤2 segments, 74 -89 and 106 -115, confer up-regulation to ␤4, mainly by decreasing the amount of binding sites in the absence of nicotine; on an atomic three-dimensional model of the ␣3␤2 receptor these amino acids form a compact microdomain that mainly contributes to the subunit interface and also faces the acetylcholine binding site. (iii) The ␤4 microdomain is sufficient to confer to ␤2 a ␤4-like upregulation. (iv) This microdomain makes an equivalent contribution to the up-regulation differences between ␣4␤2 and ␣4␤4. We propose that nicotine, by binding to immature oligomers, elicits a conformational reorganization of the microdomain, strengthening the interaction between adjacent subunits and, thus, facilitating maturation processes toward high affinity receptors. This mechanism may be central to nicotine addiction, since ␣4␤2 is the subtype exhibiting the highest degree of up-regulation in the brain.Nicotine is the primary substance responsible for tobacco addiction, a major cause of death in western societies (1, 2). Chronic exposure to nicotine causes a strong addiction, which is mediated by the interaction of nicotine with neuronal nicotinic acetylcholine receptors (nAChRs), 1 a class of pentameric allosteric ligand-gated ion channels engaged in cholinergic nicotinic transmission in the brain (3, 4).Post-mortem analysis of brain slices from smokers (5, 6) and from rats or mice chronically treated with nicotine (7-10) reveals large increases in the number of high affinity nicotinic binding sites, a phenomenon termed up-regulation. Nicotine up-regulates the ␣3␤2 (11, 12), ␣4␤2 (13-18), ␣7 (11, 19), and (␣1)2␤1␥␦ (19) nAChR subtypes reconstituted in cell lines or in Xenopus oocytes.In addition to the increased number of sites, modulation of the magnitude of the nicotine-elicited electrophysiological response is also observed upon chronic nicotine incubation of cell lines expressing recombinant nAChRs. This functional potentiation of the response is observed with nAChR oligomers ␣4␤2 and ␣3␤2 reconstituted in mammalian cell lines (12,20). But functional depression almost systematically takes place with the same combination of subunits reconstituted in Xenopus oocytes...

To identify the molecular determinants underlying the pharmacological diversity of neuronal nicotinic acetylcholine receptors, we compared the alpha7 homo-oligomeric and alpha4beta2 hetero-oligomeric receptors. Sets of residues from the regions initially identified within the agonist binding site of the alpha4 subunit were introduced into the alpha7 agonist binding site, carried by the homo-oligomeric alpha7-V201-5HT3 chimera. Introduction of the alpha4 residues 183-191 into alpha7 subunit sequence (chimera C2) selectively increased the apparent affinities for equilibrium binding and for ion channel activation by acetylcholine, resulting in a receptor that no longer displays differences in the responses to acetylcholine and nicotine. Introduction of the alpha4 residues 151-155 (chimera B) produced a approximately 100-fold increase in the apparent affinity for both acetylcholine and nicotine in equilibrium binding measurements. In both cases electrophysiological recordings revealed a much smaller increase (three- to sevenfold) in the apparent affinity for activation, but the concentrations required to desensitize the mutant chimeras parallel the shifts in apparent binding affinity. The data were fitted by a two-state concerted model, and an alteration of the conformational isomerization constant leading to the desensitized state accounts for the chimera B phenotype, whereas alteration of the ligand binding site accounts for the chimera C2 phenotype. Point mutation analysis revealed that several residues in both fragments contribute to the phenotypes, with a critical effect of the G152K and T183N mutations. Transfer of alpha4 amino acids 151-155 and 183-191 into the alpha7-V201-5HT3 chimera thus confers physiological and pharmacological properties typical of the alpha4beta2 receptor.

Desensitization is a general property of ligand-gated ion channels. Because of a wide array of available subunit combinations, it generates different time constants for channel closure, thereby modulating the processing of information in the brain. Within the family of neuronal nicotinic acetylcholine receptors (nAChRs), alpha 3 beta 2 and alpha 3 beta 4 receptors display contrasting properties of desensitization. When measured using two-electrode voltage-clamp in Xenopus oocytes, desensitization results in current decreases 2 s after initiation of acetylcholine application by 94% for alpha 3 beta 2 receptors, but only by 6% in the case of alpha 3 beta 4 receptors. Desensitization was analyzed by inserting different portions of the beta2 into the beta 4 subunit. Residues 1--212 of the beta2 subunit were able to confer 78% desensitization in 2 s, while smaller chimeras revealed desensitization in 2 s conferred by residues 1--42 alone to a level of 50%, by residues 72--89 to a level of 74%, and by residues 96--212 to a level of 77%. Some long-term (25 min) effects of desensitization driven by acetylcholine were found to rely partially on the same elements, including an enhancement mediated by residues 1--95 and 96--212 of the beta 2 subunit individually. Our results reveal that desensitization relies independently on diverse portions of the extracellular domain of the beta 2 subunit. Phenotype of alpha 3 beta 4 involves, in contrast, complex structural requirements involving residues dispersed throughout the entire N-terminal domain of the beta 4 subunit.