GABAA receptor transmembrane amino acids are critical for alcohol action: disulfide cross-linking and alkyl methanethiosulfonate labeling reveal relative location of binding sites
Article first published online: 28 OCT 2013
© 2013 International Society for Neurochemistry
Journal of Neurochemistry
Volume 128, Issue 3, pages 363–375, February 2014
How to Cite
J. Neurochem. (2014) 128, 363–375.
- Issue published online: 21 JAN 2014
- Article first published online: 28 OCT 2013
- Accepted manuscript online: 4 OCT 2013 01:28AM EST
- Manuscript Accepted: 30 SEP 2013
- Manuscript Revised: 26 SEP 2013
- Manuscript Received: 10 SEP 2013
- National Institutes of Health. Grant Number: AA06399
- binding site;
- GABAA receptor;
- homology model;
Alcohols and inhaled anesthetics modulate GABAA receptor (GABAAR) function via putative binding sites within the transmembrane regions. The relative position of the amino acids lining these sites could be either inter- or intra-subunit. We introduced cysteines in relevant TM locations and tested the proximity of cysteine pairs using oxidizing and reducing agents to induce or break disulfide bridges between cysteines, and thus change GABA-mediated currents in wild-type and mutant α1β2γ2 GABAARs expressed in Xenopus laevis oocytes. We tested for: (i) inter-subunit cross-linking: a cysteine located in α1TM1 [either α1(Q229C) or α1(L232C)] was paired with a cysteine in different positions of β2TM2 and TM3; (ii) intra-subunit cross-linking: a cysteine located either in β2TM1 [β2(T225C)] or in TM2 [β2(N265C)] was paired with a cysteine in different locations along β2TM3. Three inter-subunit cysteine pairs and four intra-subunits cross-linked. In three intra-subunit cysteine combinations, the alcohol effect was reduced by oxidizing agents, suggesting intra-subunit alcohol binding. We conclude that the structure of the alcohol binding site changes during activation and that potentiation or inhibition by binding at inter- or intra-subunit sites is determined by the specific receptor and ligand.
Alcohols modulate GABAA receptor function. The relative location of alcohol binding pockets in transmembrane domains was assessed by substituting critical amino acids with cysteines, and testing GABA responses and alcohol modulation before and after crosslinking double cysteine mutants and labeling single cysteine mutants. Lines show absence of crosslinking (red), and crosslinking that modified GABA responses (black) and alcohol modulation (grey).