Alcohol is a Positive Allosteric Modulator on Alpha6-Containing Nicotinic Acetylcholine Receptors

Objectives: Alcohol use disorder (AUD), a serious public health problem results in tremendous social, legal and medical costs to the society. Unlike other addictive drugs, there is no clear molecular target for ethanol (EtOH). In most previous investigations, EtOH concentrations typically used are high (e.g., >50 mM), which may produce numerous non-specific effects. Neuronal nicotinic acetylcholine receptors containing α6 subunits (α6*-nAChRs) show a highly restricted distribution in midbrain neurons Human genetic study shows the single nucleotide polymorphisms in nAChR α6 subunit are associated with human alcohol dependence phenotypes. Animal studies using either α6 gained or α6 knockout (KO) mice have demonstrated an association between α6*-nAChRs and alcohol-induced drinking behavioral changes. However, whether and how EtOH modulates α6*-nAChR function are unknown. Methods: Patch-clamp whole-cell recording were performed in heterologously expressed α6*-nAChRs in human epithelia cells (SH-EP1 cells), and the effects of low dose alcohol were examined. Fast Scan Cyclic Voltammetry Recordings were performed in mouse nucleus accumbens slices to measure dopamine transient. Two-electrode voltage-clamp recordings were used to measure α6*-nAChR-mediated currents expressed in the Xenopus oocytes. Results: We find that 0.1 mM EtOH significantly enhances α6*-nAChR-mediated currents in a concentration-dependent manner of either EtOH or nicotine. EtOH increases both whole-cell current rising slope and decay constant. This EtOH modulation is selective to α6*-nAChR since it is not sensitive to α3β 4, α4β 2, or α7-nAChRs. In addition, 5 mM EtOH also increases the frequency and amplitude of DA transients, which was blocked by conotoxin MII, suggesting a role for α6-nAChRs in low-dose ethanol effects. Conclusions: Collectively, our data suggest that α6*-nAChR is a sensitive target to mediate low dose EtOH's effects through a positively allosteric mechanism, which provides new insights into the understanding of a molecular target and mechanism for low dose alcohol effect in the brain. Funding: Work toward this project was supported by NIH R01 DA035958 to SCS and NIH R21 DA026627 to PW, Barrow Neuroscience Foundation, and Philips Morris External Research Grant to JW, Department of Education of Guangdong Province (2017KTSCX069), China to FG. Production of the cell line was sponsored by Targacept. Declaration of Interest: None.