Differential protein mobility of the γ-aminobutyric acid, type A, receptor α and β subunit channel-lining segments

The gamma-aminobutyric acid, type A (GABA(A)), receptor ion channel is lined by the second membrane-spanning (M2) segments from each of five homologous subunits that assemble to form the receptor. Gating presumably involves movement of the M2 segments. We assayed protein mobility near the M2 segment extracellular ends by measuring the ability of engineered cysteines to form disulfide bonds and high affinity Zn2+-binding sites. Disulfide bonds formed in alpha(1)beta(1)E270Cgamma(2) but not in alpha(1)N275Cbeta(1)gamma(2) or alpha(1)beta(1)gamma(2)K285C. Diazepam potentiation and Zn2+ inhibition demonstrated that expressed receptors contained a gamma subunit. Therefore, the disulfide bond in alpha(1)beta(1)E270Cgamma(2) formed between non-adjacent subunits. In the homologous acetylcholine receptor 4-Angstrom resolution structure, the distance between alpha carbon atoms of 20' aligned positions in non-adjacent subunits is similar to19 Angstrom. Because disulfide trapping involves covalent bond formation, it indicates the extent of movement but does not provide an indication of the energetics of protein deformation. Pairs of cysteines can form high affinity Zn2+-binding sites whose affinity depends on the energetics of forming a bidentate-binding site. The Zn2+ inhibition IC50 for alpha(1)beta(1)E270Cgamma(2) was 34 nM. In contrast, it was greater than 100 muM in alpha(1)N275Cbeta(1)gamma(2) and alpha(1)beta(1)gamma(2)K285C receptors. The high Zn2+ affinity in alpha(1)beta(1)E270Cgamma(2) implies that this region in the beta subunit has a high protein mobility with a low energy barrier to translational motions that bring the positions into close proximity. The differential mobility of the extracellular ends of the beta and alphaM2 segments may have important implications for GABA-induced conformational changes during channel gating.