Amino Acid Sequence; Binding Sites; Cell Line; Electrophysiology; Escherichia coli/genetics; Ethanol/pharmacology; G Protein-Coupled Inwardly-Rectifying Potassium Channels/metabolism; GTP-Binding Protein beta Subunits/metabolism; GTP-Binding Protein gamma Subunits/metabolism; Glutathione Transferase/genetics/metabolism; Humans; Models, Molecular; Molecular Sequence Data; Peptide Fragments/chemistry/pharmacology; Protein Binding; Protein Conformation; Protein Subunits; Receptors, GABA-B/genetics; Receptors, Glycine/antagonists & inhibitors/metabolism; Recombinant Fusion Proteins; Signal Transduction/drug effects
Abstract :
[en] 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%).
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Guzman, Leonardo
Moraga-Cid, Gustavo
Avila, Ariel
Figueroa, Maximiliano ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Yevenes, Gonzalo E
Fuentealba, Jorge
Aguayo, Luis G
Language :
English
Title :
Blockade of ethanol-induced potentiation of glycine receptors by a peptide that interferes with Gbetagamma binding.
Publication date :
2009
Journal title :
Journal of Pharmacology and Experimental Therapeutics
ISSN :
0022-3565
eISSN :
1521-0103
Publisher :
American Society for Pharmacology and Experimental Therapeutics, Bethesda, United States - Maryland
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