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

The γ-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 α₁β₁E270Cγ₂ but not in α₁N275Cβ₁γ₂ or α₁β₁γ₂K285C. Diazepam potentiation and Zn²⁺ inhibition demonstrated that expressed receptors contained a γ subunit. Therefore, the disulfide bond in α₁β₁E270Cγ₂ formed between non-adjacent subunits. In the homologous acetylcholine receptor 4-Å resolution structure, the distance between α carbon atoms of 20′ aligned positions in non-adjacent subunits is ≃19 Å. 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 α ₁β₁E270Cγ₂ was 34 nM. In contrast, it was greater than 100 μM in α₁N275Cβ ₁γ₂ and α₁β₁γ ₂K285C receptors. The high Zn²⁺ affinity in α₁β₁E270Cγ₂ implies that this region in the β 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 β and α M2 segments may have important implications for GABA-induced conformational changes during channel gating.