TY - JOUR
T1 - Identification of a tyrosine in the agonist binding site of the homomeric ρ1 γ-aminobutyric acid (GABA) receptor that, when mutated, produces spontaneous opening
AU - Torres, Viviana I.
AU - Weiss, David S.
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2002/11/15
Y1 - 2002/11/15
N2 - Mutagenesis of recombinant ρ1 γ-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 ρ1 receptor is also associated with the agonist binding site. Wild-type and mutant ρ1 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 EC50 increased 31-, 214-, 664-, and 8752-fold, respectively. An increase in the IC50 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.
AB - Mutagenesis of recombinant ρ1 γ-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 ρ1 receptor is also associated with the agonist binding site. Wild-type and mutant ρ1 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 EC50 increased 31-, 214-, 664-, and 8752-fold, respectively. An increase in the IC50 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.
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U2 - 10.1074/jbc.M202007200
DO - 10.1074/jbc.M202007200
M3 - Article
C2 - 12226075
AN - SCOPUS:0037113888
VL - 277
SP - 43741
EP - 43748
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 46
ER -