Identification of a tyrosine in the agonist binding site of the homomeric rho1 gamma-aminobutyric acid (GABA) receptor that, when mutated, produces spontaneous opening

Mutagenesis of recombinant rho1 gamma-aminobutyric acid (GABA) receptors has previously identified five residues in the amino terminal extracellular domain that play an important role in GABA binding. Here, we present evidence that the tyrosine at position 102 of the rho1 receptor is also associated...

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Bibliographic Details
Published in:The Journal of biological chemistry 2002-11, Vol.277 (46), p.43741
Main Authors: Torres, Viviana I, Weiss, David S
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Binding Sites
Binding, Competitive
Crotonates - pharmacology
Dimerization
Dose-Response Relationship, Drug
Electrophysiology
Ethyl Methanesulfonate - analogs & derivatives
Ethyl Methanesulfonate - pharmacology
GABA Antagonists - pharmacology
Glycine - metabolism
Humans
Imidazoles - pharmacology
Mesylates - pharmacology
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation
Oocytes - metabolism
Organophosphorus Compounds - pharmacology
Picrotoxin - pharmacology
Protein Binding
Receptors, GABA-B - metabolism
RNA, Complementary - metabolism
Serine - metabolism
Transcription, Genetic
Tryptophan - metabolism
Tyrosine - metabolism
Xenopus laevis - metabolism
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Summary:Mutagenesis of recombinant rho1 gamma-aminobutyric acid (GABA) receptors has previously identified five residues in the amino terminal extracellular domain that play an important role in GABA binding. Here, we present evidence that the tyrosine at position 102 of the rho1 receptor is also associated with the agonist binding site. Wild-type and mutant rho1 receptors were expressed in Xenopus laevis oocytes and examined using the two-electrode voltage clamp. When Tyr-102 was mutated to cysteine, serine, tryptophan, or glycine the EC(50) increased 31-, 214-, 664-, and 8752-fold, respectively. An increase in the IC(50) was also observed for the competitive antagonist 3-APMPA, but not for the non-competitive antagonist picrotoxin. Y102C was accessible to modification by methanethiosulfonate, and this modification was prevented by both GABA and 3-APMPA. An interesting characteristic of the Y102S mutant receptor was that, in the absence of GABA, there was an unusually high oocyte resting conductance that was blocked by both 3-APMPA and picrotoxin, indicating spontaneously opening GABA receptors. It appears that mutation of Tyr-102 perturbs the binding site and gates the pore. We conclude that Tyr-102 is a component of the GABA binding domain and speculate that Tyr-102 might be important for coupling agonist binding to channel opening.
ISSN:0021-9258