Dataset Information

Pharmacological and functional comparisons of ?6/?3?2?3-nAChRs and ?4?2-nAChRs heterologously expressed in the human epithelial SH-EP1 cell line.

ABSTRACT: Neuronal nicotinic acetylcholine receptors containing ?6 subunits (?6*-nAChRs) show highly restricted distribution in midbrain neurons associated with pleasure, reward, and mood control, suggesting an important impact of ?6*-nAChRs in modulating mesolimbic functions. However, the function and pharmacology of ?6*-nAChRs remain poorly understood because of the lack of selective agonists for ?6*-nAChRs and the challenging heterologous expression of functional ?6*-nAChRs in mammalian cell lines. In particular, the ?6 subunit is commonly co-expressed with ?4*-nAChRs in the midbrain, which masks ?6*-nAChR (without ?4) function and pharmacology. In this study, we systematically profiled the pharmacology and function of ?6*-nAChRs and compared these properties with those of ?4?2 nAChRs expressed in the same cell line. Heterologously expressed human ?6/?3 chimeric subunits (?6 N-terminal domain joined with ?3 trans-membrane domains and intracellular loops) with ?2 and ?3 subunits in the human SH-EP1 cell line (?6*-nAChRs) were used. Patch-clamp whole-cell recordings were performed to measure these receptor-mediated currents. Functionally, the heterologously expressed ?6*-nAChRs exhibited excellent function and showed distinct nicotine-induced current responses, such as kinetics, inward rectification and recovery from desensitization, compared with ?4?2-nAChRs. Pharmacologically, ?6*-nAChR was highly sensitive to the ?6 subunit-selective antagonist ?-conotoxin MII but had lower sensitivity to mecamylamine and dihydro-?-erythroidine. Nicotine and acetylcholine were found to be full agonists for ?6*-nAChRs, whereas epibatidine and cytisine were determined to be partial agonists. Heterologously expressed ?6*-nAChRs exhibited pharmacology and function distinct from those of ?4?2-nAChRs, suggesting that ?6*-nAChRs may mediate different cholinergic signals. Our ?6*-nAChR expression system can be used as an excellent cell model for future investigations of ?6*-nAChR function and pharmacology.

SUBMITTER: Chen DJ

PROVIDER: S-EPMC6289390 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Pharmacological and functional comparisons of α6/α3β2β3-nAChRs and α4β2-nAChRs heterologously expressed in the human epithelial SH-EP1 cell line.

Chen De-Jie DJ Gao Fen-Fei FF Ma Xiao-Kuang XK Shi Gang-Gang GG Huang Yuan-Bing YB Su Quang-Xi QX Sudweeks Sterling S Gao Ming M Dharshaun Turner T Eaton Jason Brek JB Chang Yong-Chang YC Mcintosh J Michael JM Lukas Ronald J RJ Whiteaker Paul P Steffensen Scott C SC Wu Jie J

Acta pharmacologica Sinica 20180524 10

Neuronal nicotinic acetylcholine receptors containing α6 subunits (α6*-nAChRs) show highly restricted distribution in midbrain neurons associated with pleasure, reward, and mood control, suggesting an important impact of α6*-nAChRs in modulating mesolimbic functions. However, the function and pharmacology of α6*-nAChRs remain poorly understood because of the lack of selective agonists for α6*-nAChRs and the challenging heterologous expression of functi ...[more]

PMID: 29795357

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Project description:Nicotine, a major component of tobacco, is highly addictive and acts on nicotinic acetylcholine receptors (nAChRs) to stimulate reward-associated circuits in the brain. It is well known that nAChRs play critical roles in mediating nicotine reward and addiction. Current FDA-approved medications for smoking cessation are the antidepressant bupropion and the nicotinic partial agonist varenicline, yet both are limited by adverse side effects and moderate efficacy. Thus, development of more efficacious medications with fewer side effects for nicotine addiction and smoking cessation is urgently needed. l-Tetrahydropalmatine (l-THP) is an active ingredient of the Chinese medicinal herb Corydalis ambigua that possesses rich neuropharmacological actions on dopamine (DA) receptors in the mesocorticolimbic dopaminergic reward pathway. L-THP has been explored as anti-addiction treatments for drug abuse including nicotine. However, the targets and mechanisms of l-THP-caused anti-nicotine effects are largely unknown. In this study we address this question by elucidating the effects of l-THP on human neuronal nAChRs using patch-clamp recordings. Human neuronal α4β2-nAChRs were heterologously expressed in SH-EP1 human epithelial cells. Bath application of nicotine (0.1-100 μM) induced inward currents, co-application of l-THP (3 μM) inhibited nicotine-induced currents in the transfected cells. L-THP-caused inhibition was concentration-dependent (the EC50 values for inhibiting the peak and steady-state current were 18 and 2.1 μM, respectively) and non-competitive. Kinetic analysis of the whole-cell currents showed that l-THP slowed rising time and accelerated decay time constants. L-THP specifically modulated α4β2-nAChRs, as it did not affect α7-nAChRs or α1*-nAChRs (muscle type). Interestingly, two putative α4β2-nAChR isoforms, namely sazetidine A-activated, high-sensitive one (α42β23-nAChR) and cytisine-activated, low-sensitive one (α43β22-nAChR) were pharmacologically separated, and the low-sensitive one was more susceptible to l-THP inhibition than the high-sensitive one. In conclusion, we demonstrate that l-THP blocks neuronal α4β2-nAChR function, which may underlie its inhibition on nicotine addiction.

Project description:Alpha6-containing nicotinic acetylcholine receptors are primarily found in neurons of the midbrain dopaminergic (DA) system, suggesting these receptors are potentially involved in drug reward and dependence. Here, we report a novel effect that cocaine directly inhibits α6N/α3Cβ2β3-nAChR (α6*-nAChRs) function. Human α6*-nAChRs were heterologously expressed within cells of the SH-EP1 cell line for functional characterization. Mechanically dissociated DA neurons from mouse ventral tegmental area (VTA) were used as a model of presynaptic α6*-nAChR activation since this method preserves terminal boutons. Patch-clamp recordings in whole-cell configuration were used to measure α6*-nAChR function as well as evaluate the effects of cocaine. In SH-EP1 cells containing heterologously expressed human α6*-nAChRs, cocaine inhibits nicotine-induced inward currents in a concentration-dependent manner with an IC50 value of 30 μM. Interestingly, in the presence of 30 μM cocaine, the maximal current response of the nicotine concentration-response curve is reduced without changing nicotine's EC50 value, suggesting a noncompetitive mechanism. Furthermore, analysis of whole-cell current kinetics demonstrated that cocaine slows nAChR channel activation but accelerates whole-cell current decay time. Our findings demonstrate that cocaine-induced inhibition occurs solely with bath application, but not during intracellular administration, and this inhibition is not use-dependent. Additionally, in Xenopus oocytes, cocaine inhibits both α6N/α3Cβ2β3-nAChRs and α6M211L/α3ICβ2β3-nCAhRs similarly, suggesting that cocaine may not act on the α3 transmembrane domain of chimeric α6N/α3Cβ2β3-nAChR. In mechanically isolated VTA DA neurons, cocaine abolishes α6*-nAChR-mediated enhancement of spontaneous inhibitory postsynaptic currents (sIPSCs). Collectively, these studies provide the first evidence that cocaine directly inhibits the function of both heterologously and naturally expressed α6*-nAChRs. These findings suggest that α6*-nAChRs may provide a novel pharmacological target mediating the effects of cocaine and may underlie a novel mechanism of cocaine reward and dependence.

Dataset Information

Pharmacological and functional comparisons of ?6/?3?2?3-nAChRs and ?4?2-nAChRs heterologously expressed in the human epithelial SH-EP1 cell line.

Publications

Pharmacological and functional comparisons of α6/α3β2β3-nAChRs and α4β2-nAChRs heterologously expressed in the human epithelial SH-EP1 cell line.

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