Tyrosine 62 of the gamma-aminobutyric acid type A receptor beta 2 subunit is an important determinant of high affinity agonist binding

The gamma-aminobutyric acid type A receptor (GABA(A)R) carries both high (K -D = 10-30 nM) and low (K-D = 0.1-1.0 mu M) affinity binding sites for agon ists. We have used site-directed mutagenesis to identify a specific residue in the rat beta 2 subunit that is involved in high affinity agonist bindin g. Tyrosine residues at positions 62 and 74 were mutated to either phenylal anine or serine and the effects on ligand binding and ion channel activatio n were investigated after the expression of mutant subunits with wild-type alpha 1 and gamma 2 subunits in tsA201 cells or in Xenopus oocytes. None of the mutations affected [H-3]Ro15-4513 binding or impaired allosteric inter actions between the low affinity GABA and benzodiazepine sites. Although mu tations at position 74 had little effect on [H-3]muscimol binding, the Y62F mutation decreased the affinity of the high affinity [H-3]muscimol binding sites by similar to 6-fold, and the Y62S mutation led to a loss of detecta ble high affinity binding sites. After expression in oocytes, the EC50 valu es for both muscimol and GABA-induced activation of Y62F and Y62S receptors were increased by 2- and 6-fold compared with the wild-type. We conclude t hat Tyr-62 of the beta subunit is an important determinant for high affinit y agonist binding to the GABA(A) receptor.