Naturally expressed nicotinic acetylcholine receptors (nAChR) containing α4 subunits (α4 * -nAChR) in combination with β2 subunits (α4β2-nAChR) are among the most abundant, high-affinity nicotine binding sites in the mammalian brain. β4 subunits are also richly expressed and colocalize with α4 subunits in several brain regions implicated in behavioural responses to nicotine and nicotine dependence. Thus, α4β4-nAChR also may exist and play important functional roles. In this study, properties were determined of human α4β2-and α4β4-nAChR heterologously expressed de novo in human SH-EP1 epithelial cells. Whole-cell currents mediated via human α4β4-nAChR have ∼4-fold higher amplitude than those mediated via human α4β2-nAChR and exhibit much slower acute desensitization and functional rundown. Nicotinic agonists induce peak whole-cell current responses typically with higher functional potency at α4β4-nAChR than at α4β2-nAChR. Cytisine and lobeline serve as full agonists at α4β4-nAChR but are only partial agonists at α4β2-nAChR. However, nicotinic antagonists, except hexamethonium, have comparable affinities for functional α4β2-and α4β4-nAChR. Whole-cell current responses show stronger inward rectification for α4β2-nAChR than for α4β4-nAChR at a positive holding potential. Collectively, these findings demonstrate that human nAChR β2 or β4 subunits can combine with α4 subunits to generate two forms of α4 * -nAChR with distinctive physiological and pharmacological features. Diversity in α4 * -nAChR is of potential relevance to nervous system function, disease, and nicotine dependence.

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Aim: To characterize the functional and pharmacological features of agonist-induced hump currents in human α4β2-nicotinic acetylcholine receptors (nAChR). Methods: Whole-cell and outside-out patch recordings were performed using human α4β2-nAChR heterologously expressed in stably-transfected, native nAChR-null subclonal human epithelial 1 (SH-EP1) cells. RT-PCR was used to test the mRNA expression of transfected nAChR. Homology modeling and acetylcholine (ACh) docking were applied to show the possible ACh-binding site in the channel pore. Results: The rapid exposure of 10 mmol/L ACh induced an inward current with a decline from peak to steady-state. However, after the removal of ACh, an additional inward current, called "hump" current, reoccurred. The ability of agonists to produce these hump currents cannot be easily explained based on drug size, charge, acute potency, or actions as full or partial agonists. Hump currents were associated with a rebound increase in whole-cell conductance, and they had voltage dependence-like peak currents induced by agonist action. Hump currents blocked by the α4β2-nAChR antagonist dihydro-β-erythroidine were reduced when α4β2-nAChR were desensitized, and were more pronounced in the absence of external Ca 2+. Outside-out single-channel recordings demonstrated that compared to 1 µmol/L nicotine, 100 µmol/L nicotine reduced channel current amplitude, shortened the channel mean open time, and prolonged the channel mean closed time, supporting an agonist-induced open-channel block before hump current production. A docking model also simulated the agonist-binding site in the channel pore. Conclusion: These results support the hypothesis that hump currents reflect a rapid release of agonists from the α4β2-nAChR channel pore and a rapid recovery from desensitized α4β2-nAChR. Key wordsacetylcholine; dihyd r o -β-er y th r o id in e; dimethyl-phenyl-piperazinium; epibatidine; nicotinic acetylcholine receptor