U18666A, a cholesterol-inhibition agent, modulates human neuronal nicotinic acetylcholine receptors heterologously expressed in SH-EP1 cell line

In this study, we evaluate the effects of (3β)-3-[2-(diethylamino) ethoxy]androst-5-en-17-one dihydrochloride (U18666A), a cholesterol synthesis/transporter inhibitor, on selected human neuronal nicotinic acetylcholine receptors (nAChRs) heterologously expressed in the SH-EP1 cell line using whole-cell patch-clamp recordings. The results indicate that with 2-min pretreatment, U18666A inhibited different nAChR subtypes with a rank-order of potency (IC₅₀ of whole-cell peak current): α4β2 (8.0 ± 3.0 nM) > α3β2 (1.7 ± 0.4 μM) > α4β4 (26 ± 7.2 μM) > α7 (> 100 μM), suggesting this compound is more selective to α4β2-nAChRs. Thus, the pharmacological profiles and mechanisms of U18666A acting on α4β2-nAChRs were investigated in detail. U18666A suppresses both peak and steady state components of whole-cell currents mediated by human α4β2-nAChRs in response to nicotine. In nicotine-induced concentration-response curves, U18666A reduces nicotine-induced current at maximally effective agonist concentrations without influencing nicotine's EC₅₀ value, suggesting a non-competitive inhibition. U18666A-induced inhibition of nAChR function is concentration-, voltage-, and use-dependent, suggesting an open channel block. Taken into consideration of ∼10 000-fold enhancement of the potency of U18666A after 2-min pre-treatment, this compound also likely inhibits α4β2-nAChRs through a close channel block. In addition, the U18666A-induced inhibition in α4β2-nAChRs is not mediated by either increased receptor endocytosis or altered cell cholesterol. These data indicate that U18666A is a potent antagonist of α4β2- nAChRs and may be useful as a tool in the functional characterization and pharmacological profiling of nAChRs, as well as a potential candidate for smoking cessation.