Naturally expressed nicotinic acetylcholine receptors composed of ␣4 and ␤2 subunits (␣4␤2-nAChR) are the predominant form of high affinity nicotine binding site in the brain implicated in nicotine reward, mediation of nicotinic cholinergic transmission, modulation of signaling through other chemical messages, and a number of neuropsychiatric disorders. To develop a model system for studies of human ␣4␤2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human ␣4 and ␤2 subunits were stably introduced into the native nAChR-null human epithelial cell line SH-EP1. Heterologously expressed ␣4␤2-nAChR engage in high-affinity, specific binding of Rbϩ efflux assays indicate full efficacy of epibatidine, nicotine, and acetylcholine; partial efficacy for 1,1-dimethyl-4-phenyl-piperazinium, cytisine, and suberyldicholine; competitive antagonism by dihydro-␤-erythroidine, decamethonium, and methyllycaconitine; noncompetitive antagonism by mecamylamine and eserine; and mixed antagonism by pancuronium, hexamethonium, and d-tubocurarine. These results demonstrate utility of transfected SH-EP1 cells as models for studies of human ␣4␤2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human ␣4␤2-nAChR.

Dopamine (DA) neurons located in the mammalian midbrain have been generally implicated in reward and drug reinforcement and more specifically in nicotine dependence. However, roles played by nicotinic acetylcholine receptors, including those composed of ␣7-subunits [␣7-nicotinic acetylcholine receptors (nAChRs)], in modulation of DA signaling and in nicotine dependence are not clearly understood. Although midbrain slice recording has been used previously to identify functional ␣7-nAChRs, these preparations are not optimally designed for extremely rapid and reproducible drug application, and rapidly desensitized, ␣7-nAChRmediated currents may have been underestimated or not detected. Here, we use patch-clamp, whole-cell current recordings from single neurons acutely dissociated from midbrain nuclei and having features of DA neurons to characterize acetylcholineinduced, inward currents that rapidly activate and desensitize, are mimicked by the ␣7-nAChR-selective agonist, choline, blocked by the ␣7-nAChR-selective antagonists, methyllycaconitine and ␣-bungarotoxin, and are similar to those of heterologously expressed, human ␣7-nAChRs. We also use reverse transcriptasepolymerase chain reaction, in situ hybridization, and immunocytochemical staining to demonstrate nAChR ␣7 subunit gene expression as message and protein in the rat substantia nigra pars compacta and ventral tegmental area. Expression of ␣7 subunit message and of ␣7-nAChR-mediated responses is developmentally regulated, with both being absent in samples taken from rats at postnatal day 7, but later becoming present and increasing over the next 2 weeks. Collectively, this electrophysiological, pharmacological, and molecular evidence indicates that nAChR ␣7 subunits and functional ␣7-nAChRs are expressed somatodendritically by midbrain DA neurons, where they may play important physiological roles and contribute to nicotine reinforcement and dependence.

␣7-Nicotinic acetylcholine receptors (␣7-nAChRs) are broadly distributed in the central nervous system, where they play important roles in chemical and electrical signaling, and perhaps in neurite outgrowth, synaptic plasticity, and neuronal death/survival. To help elucidate their normal and pathophysiological roles, we have heterologously expressed human ␣7-nAChR in transfected SH-EP1 human epithelial cells. Reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses demonstrate expression of human ␣7 subunits as messenger RNA. Patch-clamp recordings exploiting a novel strategy to prevent functional rundown of whole-cell peak current responses to repeated acute challenges with nicotinic agonists show successful expression of functional ␣7-nAChR that mediate inward currents characterized by rapid phases of activation and inactivation. Concentration-response curves show that nicotine, acetylcholine, and choline are efficacious agonists at human ␣7-nAChRs. Currentvoltage relationships show inward rectification for agonist-induced currents. Human ␣7-nAChRs exhibit some sensitivity to ␣7-nAChR antagonists ␣-bungarotoxin (Bgt) or methyllycaconitine (MLA) when applied coincidentally with agonist, but much higher affinity block occurs when cells and ␣7-nAChRs are pre-exposed to antagonists for 2 min before challenge with agonist. Both Bgt and MLA are competitive inhibitors of ␣7-nAChR function. Whole-cell current peak amplitudes and halftimes for inactivation of ␣7-nAChR functional responses to nicotine are dramatically reduced in the absence of extracellular Ca 2ϩ , suggestive of high Ca 2ϩ permeability of the ␣7-nAChR channel. Thus, heterologously expressed human ␣7-nAChR in mammalian cells have properties of native ␣7-nAChR or of ␣7-nAChR heterologously expressed in other systems and serve as excellent models for studies of molecular bases of ␣7-nAChR function.

Level IV-economic and decision analysis.