Abstract
Ethanol alters the function of several members of the Cys-loop ligand-gated ion channel superfamily. Recent studies have shown that the sensitivity of the α1 glycine receptor (GlyR) to ethanol can be affected by the state of G protein activation mediated by the interaction of Gβγ with intracellular amino acids in the GlyR. Here, we evaluated the physicochemical property of Lys385 that contributes to ethanol modulation by using mutagenesis, patch-clamp, and biochemical techniques. A conserved substitution (K385R) did not affect either the apparent glycine EC50 (40 ± 1 versus 41 ± 0.5 μM) or the ethanol-induced potentiation (53 ± 5 versus 46 ± 5%) of the human α1 GlyR. On the other hand, replacement of this residue with glutamic acid (K385E), an acidic amino acid, reduced the potentiation of the GlyR to 10 ± 1%. Furthermore, mutations with a hydrophobic leucine (K385L), a hydrogen bond donor glutamine (K385Q), or a neutral residue (K385A) also reduced ethanol modulation. Finally, substitution by a large and hydrophobic residue (K385F) and deletion of 385 (Lys385_) reduced ethanol modulation to 10 ± 4 and 17 ± 0.4%, respectively. Experiments using dynamic cysteine substitution with a methanethiosulfonate reagent and homology modeling indicate that the basic property and the position of Lys385, probably because of its interaction with Gβγ, is critical for ethanol potentiation of the receptor.
Footnotes
This work was supported by the National Institutes of Health National Institute on Alcohol Abuse and Alcoholism [Grant AA15150]; and the Comisión Nacional de Investigación Científica y Tecnológica [Grant AT-24080125].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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ABBREVIATIONS:
- GlyR
- glycine receptor
- IL
- intracellular loop
- LGIC
- ligand-gated ion channel
- nAChR
- nicotinic acetylcholine receptor
- 5-HT3
- 5 hydroxytryptamine type 3
- TM
- transmembrane
- HEK
- human embryonic kidney
- PDB
- Protein Data Bank
- MTS
- 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt
- SCAM
- substituted-cysteine accessibility method
- WT
- wild type
- MTSEA
- methanethiosulfonate ethylammonium
- ESP
- electrostatic surface potential.
- Received July 11, 2011.
- Accepted October 28, 2011.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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