Blockade of ethanol-induced potentiation of glycine receptors by a peptide that interferes with Gbetagamma binding.
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 Gbetagamma. In the present study, we found that mutation of basic residues in the N-terminal region of the IL reduced the binding of Gbetagamma to 21 +/- 10% of control. Two basic residues in the C-terminal region, on the other hand, contributed to a smaller extent to Gbetagamma binding. Using docking analysis, we found that both basic regions of the IL bind in nearby regions to the Gbetagamma 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 Gbetagamma 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 Gbetagamma-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%).
Guzman
Leonardo
L
Laboratory of Neurophysiology, Department of Physiology, University of Concepción, Concepción, Chile.
Moraga-Cid
Gustavo
G
Avila
Ariel
A
Figueroa
Maximiliano
M
Yevenes
Gonzalo E
GE
Fuentealba
Jorge
J
Aguayo
Luis G
LG
D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D004594:Electrophysiology; D004926:Escherichia coli / Q000235:genetics; D000431:Ethanol / Q000494:pharmacology*; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits; D018080:Receptors, GABA-B / Q000235:genetics; D018009:Receptors, Glycine / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction / Q000187:drug effects
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D010446:Peptide Fragments; D021122:Protein Subunits; D018080:Receptors, GABA-B; D018009:Receptors, Glycine; D011993:Recombinant Fusion Proteins; D000431:Ethanol; D005982:Glutathione Transferase