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

Jeffrey Horenstein, Paul Riegelhaupt, Myles H. Akabas

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21 Scopus citations


The γ-aminobutyric acid, type A (GABAA), 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.

Original languageEnglish (US)
Pages (from-to)1573-1581
Number of pages9
JournalJournal of Biological Chemistry
Issue number2
StatePublished - Jan 14 2005

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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