A residue in loop 9 of the beta2-subunit stabilizes the closed state of the GABAA receptor.

In gamma-aminobutyric acid type A (GABA(A)) receptors, the structural elements that couple ligand binding to channel opening remain poorly defined. Here, site-directed mutagenesis was used to determine if Loop 9 on the non-GABA binding site interface of the beta2-subunit may be involved in GABA(A) receptor activation. Specifically, residues Gly(170)-Gln(185) of the beta2-subunit were mutated to alanine, co-expressed with wild-type alpha1- and gamma2S-subunits in human embryonic kidney (HEK) 293 cells and assayed for their activation by GABA, the intravenous anesthetic propofol and the endogenous neurosteroid pregnanolone using whole cell macroscopic recordings. Three mutants, G170A, V175A, and G177A, produced 2.5-, 6.7-, and 5.6-fold increases in GABA EC(50) whereas one mutant, Q185A, produced a 5.2-fold decrease in GABA EC(50). None of the mutations affected the ability of propofol or pregnanolone to potentiate a submaximal GABA response, but the Q185A mutant exhibited 8.3- and 3.5-fold increases in the percent direct activation by propofol and pregnanolone, respectively. Mutant Q185A receptors also had an increased leak current that was sensitive to picrotoxin, indicating an increased gating efficiency. Further Q185E, Q185L, and Q185W substitutions revealed a strong correlation between the hydropathy of the amino acid at this position and the GABA EC(50). Taken together, these results indicate that beta2 Loop 9 is involved in receptor activation by GABA, propofol, and pregnanolone and that beta2(Q185) participates in hydrophilic interactions that are important for stabilizing the closed state of the GABA(A) receptor.