Mutation of the GABAA receptor M1 transmembrane proline increases GABA affinity and reduces barbiturate enhancement.

All GABA(A) receptor (GABAR) subunits include an invariant proline in a consensus motif in the first transmembrane segment (M1). In receptors containing bovine alpha1, beta1 and gamma2 subunits, we analyzed the effect of mutating this M1 proline to alanine in the alpha1 or beta1 subunit using 3 different expression systems. The beta1 subunit mutant, beta1(P228A), reduced the EC(50) for GABA about 10-fold in whole cell recordings in HEK293 cells and L929 fibroblasts. The corresponding alpha1 subunit mutant (alpha1(P233A)) also reduced the GABA EC(50) when expressed in Xenopus oocytes; alpha1(P233A)beta1gamma2S receptors failed to assemble in HEK293 cells. Binding of [(3)H]flumazenil and [(3)H]muscimol to transfected HEK293 cell membranes showed similar levels of receptor expression with GABARs containing beta1 or beta1(P228A) subunits and no change in the affinity for [(3)H]flumazenil; however, the affinity for [(3)H]muscimol was increased 6-fold in GABARs containing beta1(P228A) subunits. In L929 cells, presence of the beta1(P228A) subunit reduced enhancement by barbiturates without affecting enhancement by diazepam or alfaxalone. Single channel recordings from alpha1beta1gamma2S and alpha1beta1(P228A)gamma2L GABARs showed similar channel kinetics, but beta-mutant containing receptors opened at lower GABA concentrations. We conclude that the beta1 subunit M1 segment proline affects the linkage between GABA binding and channel gating and is critical for barbiturate enhancement. Mutation of the M1 proline in the alpha1 subunit also inhibited receptor assembly.