Development of glycine and GABAA ultra-sensitive ethanol receptors (USERs) as novel tools for alcohol and brain research

2017-07-15A critical obstacle to developing effective medications to prevent and/or treat an alcohol‐use disorder (AUD) is the lack of specific knowledge regarding the role of individual receptors in mediating ethanol‐induced reward, addiction, and dependence. The studies presented in this dissertation introduce a transformative tool to investigate the primary sites of ethanol action on individual receptor subunits that are expressed in the reward and addiction brain regions. Here, we developed a novel class of Ultra‐Sensitive Ethanol Receptors (USERs) that allow activation of a single receptor subunit population sensitized to extremely low ethanol concentrations. USERs were created by mutating as few as two residues in the extracellular Loop 2 region of glycine receptors (GlyRs) or γ-aminobutyric acid type A receptors (GABAARs), which are implicated in causing many behavioral effects linked to ethanol abuse. USERs, expressed in Xenopus oocytes and tested using two‐electrode voltage clamp electrophysiology, demonstrated an increase in ethanol sensitivity of 100‐fold over wild‐type receptors by significantly decreasing the threshold for ethanol sensitivity and increasing the magnitude of ethanol response, without altering general receptor properties including sensitivity to the neurosteroid, allopregnanolone. These profound changes in ethanol sensitivity were observed across multiple subunits of GlyRs and GABAARs. We also demonstrated the selectivity of these USERs by reporting that the extracellular Loop 2 region is a common site of action for anesthetics including ethanol, isoflurane, and lidocaine in α1 GlyRs. Interestingly, Loop 2 was not a site of action for the general anesthetic, propofol. We further employed USER technology in genetically engineered knock‐in mice to demonstrate proof‐of‐concept that USERs expressed in vivo can be activated by extremely low ethanol concentrations. Ultimately, USERs provide a unique tool to increase understanding of the individual role of receptors in mediating the reward and addiction pathways of ethanol. Thus, USERs could help identify key sites of ethanol action and potential drug targets that could treat and/or prevent alcohol addiction