Abstract

The large intracellular loop (IL) of the glycine receptor (GlyR) interacts with various signaling proteins and plays a fundamental role in trafficking and regulation of several receptor properties, including a direct interaction with Gß?. In the present study, we found that mutation of basic residues in the N-terminal region of the IL reduced the binding of Gß? to 21 ± 10% of control. Two basic residues in the C-terminal region, on the other hand, contributed to a smaller extent to Gß? binding. Using docking analysis, we found that both basic regions of the IL bind in nearby regions to the Gß? dimer, within an area of high density of amino acids having an electronegative character. Thereafter, we generated a 17-amino acid peptide with the N-terminal sequence of the wild-type IL (RQH) that was able to inhibit the in vitro binding of Gß? to GlyRs to 57 ± 5% of control in glutathione S-transferase pull-down assays using purified proteins. More interestingly, when the peptide was intracellularly applied to human embryonic kidney 293 cells, it inhibited the Gß?-mediated modulations of G protein-coupled inwardly rectifying potassium channel by baclofen (24 ± 14% of control) and attenuated the GlyR potentiation by ethanol (51 ± 10% versus 10 ± 3%). Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics.