Abstract
The α1-subunit containing glycine receptors (GlyRs) is potentiated by ethanol, in part, by intracellular Gβγ actions. Previous studies have suggested that molecular requirements in the large intracellular domain are involved; however, the lack of structural data about this region has made it difficult to describe a detailed mechanism. Using circular dichroism and molecular modeling, we generated a full model of the α1-GlyR, which includes the large intracellular domain and provides new information on structural requirements for allosteric modulation by ethanol and Gβγ. The data strongly suggest the existence of an α-helical conformation in the regions near transmembrane (TM)-3 and TM4 of the large intracellular domain. The secondary structure in the N-terminal region of the large intracellular domain near TM3 appeared critical for ethanol action, and this was tested using the homologous domain of the γ2-subunit of the GABAA receptor predicted to have little helical conformation. This region of γ2 was able to bind Gβγ and form a functional channel when combined with α1-GlyR, but it was not sensitive to ethanol. Mutations in the N- and C-terminal regions introduced to replace corresponding amino acids of the α1-GlyR sequence restored the ability to be modulated by ethanol and Gβγ. Recovery of the sensitivity to ethanol was associated with the existence of a helical conformation similar to α1-GlyR, thus being an essential secondary structural requirement for GlyR modulation by ethanol and G protein.
Footnotes
- Received June 29, 2014.
- Accepted October 21, 2014.
↵1 Current affiliation: Department of Physiology & Membrane Biology & Shriners Hospital for Children Northern California, University of California, Davis School of Medicine, Sacramento, California.
C.F.B. and P.A.C. contributed equally to this work.
This work was supported by the National Institutes of Health National Institute on Alcohol Abuse and Alcoholism [Grant R01-AA15150].
↵This article has supplemental material available at jpet.aspetjournals.org.
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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