Abstract
Glycine receptors (GlyRs) are inhibitory ligand-gated ion channels. Ethanol potentiates glycine activation of the GlyR, and putative binding sites for alcohol are located in the transmembrane (TM) domains between and within subunits. To alter alcohol sensitivity of GlyR, we introduced two mutations in the GlyR α1 subunit, M287L (TM3) and Q266I (TM2). After expression in Xenopus laevis oocytes, both mutants showed a reduction in glycine sensitivity and glycine-induced maximal currents. Activation by taurine, another endogenous agonist, was almost abolished in the M287L GlyR. The ethanol potentiation of glycine currents was reduced in the M287L GlyR and eliminated in Q266I. Physiological levels of zinc (100 nM) potentiate glycine responses in wild-type GlyR and also enhance the ethanol potentiation of glycine responses. Although zinc potentiation of glycine responses was unchanged in both mutants, zinc enhancement of ethanol potentiation of glycine responses was absent in M287L GlyRs. The Q266I mutation decreased conductance but increased mean open time (effects not seen in M287L). Two lines of knockin mice bearing these mutations were developed. Survival of homozygous knockin mice was impaired, probably as a consequence of impaired glycinergic transmission. Glycine showed a decreased capacity for displacing strychnine binding in heterozygous knockin mice. Electrophysiology in isolated neurons of brain stem showed decreased glycine-mediated currents and decreased ethanol potentiation in homozygous knockin mice. Molecular models of the wild-type and mutant GlyRs show a smaller water-filled cavity within the TM domains of the Q266I α1 subunit. The behavioral characterization of these knockin mice is presented in a companion article (J Pharmacol Exp Ther 340:317–329, 2012).
Footnotes
This work was supported by the National Institutes of Health National Institute of Alcohol Abuse and Alcoholism [Grants AA06399, AA11525, AA10422]; and the National Institutes of Health National Institute of General Medical Sciences [Grant GM47818].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- GlyR
- glycine receptor
- TM
- transmembrane
- WT
- wild type
- HEK
- human embryonic kidney
- ES
- embryonic stem
- ANOVA
- analysis of variance
- PCR
- polymerase chain reaction
- GLIC
- proton-gated ion channel
- CRC
- concentration-response curve
- bp
- base pairs
- kb
- kilobases
- aCSF
- artificial cerebrospinal fluid
- MBS
- Modified Barth's solution.
- Received August 10, 2011.
- Accepted October 27, 2011.
- U.S. Government work not protected by U.S. copyright
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