Abstract
A critical obstacle to developing effective medications to prevent and/or treat alcohol use disorders is the lack of specific knowledge regarding the plethora of molecular targets and mechanisms underlying alcohol (ethanol) action in the brain. To identify the role of individual receptor subunits in ethanol-induced behaviors, 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 four 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, demonstrated an increase in ethanol sensitivity of 100-fold over wild-type receptors by significantly decreasing the threshold 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. Collectively, our studies set the stage for using USER technology in genetically engineered animals as a unique tool to increase understanding of the neurobiological basis of the behavioral effects of ethanol.
Footnotes
- Received May 13, 2014.
- Accepted September 19, 2014.
A.N. and K.H.M. contributed equally to this work.
This work was supported in whole or in part by the National Institutes of Health National Institute on Alcohol Abuse and Alcoholism [Grants R01-AA13378, R01-AA13992, K01-AA17243, and R01-AA022448], the American Foundation for Pharmaceutical Education, and the University of Southern California School of Pharmacy.
The authors declare no conflict of interest.
↵This work was conducted as partial fulfillment of the requirements for the Ph.D. degree in Molecular Pharmacology and Toxicology (A.N.) and Master’s degree in Pharmaceutical Sciences (K.H.M.), University of Southern California.
Naito A, Muchhala K, Asatryan L, Trudell JR, Homanics GE, Davies DL, and Alkana RL (2014) Mutations in loop 2 of glycine and GABAA receptor subunits create ultra-sensitive ethanol receptors (USERs) with potential application as brain mapping tools. Gordon Research Conference, Alcohol and the Nervous System; Feb. 16–21, 2014; Galveston, TX.
Naito A, Muchhala K, Asatryan L, Trudell JR, Homanics GE, Davies DL, and Alkana RL (2014) Loop 2 mutations of glycine and GABAA receptor subunits create ultra-sensitive ethanol receptors (USERs) with potential application in brain mapping. 37th Annual Meeting of the Research Society on Alcoholism, and 17th Congress of ISBRA; Jun. 21–25, 2014; Bellevue, WA.
Naito A, Muchhala K, Asatryan L, Homanics GE, Trudell JR, Perkins DI, Davies DL, and Alkana RL (2013) Developing ultrasensitive ethanol receptors (USERS) as novel tools for alcohol research. Society for Neuroscience Annual Meeting; Nov. 9–13, 2013; San Diego, CA.
This work has previously been presented at the following meetings in the form of abstracts: Muchhala K, Naito A, Asatryan L, Homanics GE, Trudell JR, Perkins DI, Davies DL, and Alkana RL (2013) Developing ultrasensitive ethanol receptors (USERs) as novel tools for alcohol research. Moving Targets: Neurodegenerative Diseases; Aug. 23, 2013; Los Angeles, CA.
Naito A, Muchhala K, Asatryan L, Trudell JR, Homanics GE, Davies DL, Alkana RL, and Perkins DI (2013) Mutations in GABAA receptor loop 2 create ultrasensitive ethanol receptors (USERs) sensitive to micromolar ethanol concentrations. 36th Annual Meeting of the Research Society on Alcoholism, Alcohol: Clin Exp Res 37:P199; Jun. 22–26, 2013; Orlando, FL.
Naito A, Muchhala K, Asatryan L, Davies DL, Perkins DI, and Alkana RL (2013) Flare in the night receptors: development of ultra-sensitive ethanol receptors (USERs) in glycine receptors to elucidate the molecular mechanism of alcohol addiction. USC Graduate Research Symposium; Apr. 2, 2013; Los Angeles, CA.
Alkana RL, Naito A, Muchhala K, Asatryan L, Li K, Homanics GE, Trudell JR, Davies DL, and Perkins DI (2012) Development of loop 2 mutant GlyRs that are sensitive to micromolar ethanol concentrations. Society for Neuroscience Annual Meeting; Oct. 13–17, 2012; New Orleans, LA.
Alkana RL, Trudell JR, Asatryan L, Naito A, Muchhala K, Li K, Davies DL, and Perkins DI (2012) Functional insights into the mechanisms of ethanol action in GlyRs and GABAARs revealed by manipulation of extracellular domain loop 2. International Society for Biomedical Research on Alcoholism. International Congress. Sept. 9–12, 2012; Sapporo, Japan.
Alkana RL, Perkins DP, Li K, Homanics GE, Naito A, Trudell JR, and Davies DL (2012) Loop 2 sequence profoundly affects the sensitivity of alpha 1 glycine receptors (GlyRs) to ethanol. Research Society on Alcoholism Annual Meeting; Jun. 23–27, 2012; San Francisco, CA.
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- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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