Protein mobility and GABA-induced conformational changes in GABAA receptor pore-lining M2 segment

Protein movements underlying ligand-gated ion channel activation are poorly understood. Here we used disulfide bond trapping to examine the proximity and mobility of cysteines substituted for aligned GABA(A) receptor alpha (1) and beta (1) M2 segment channel-lining residues in resting and activated receptors. With or without GABA, disulfide bonds formed at alpha (1)N275C/beta (1)E270C (20') and alpha (1)S272C/beta (1)H267C (17'), near the extracellular end, suggesting that this end is more mobile and/or flexible than the rest of the segment. Near the middle of M2, at alpha (1)T261C/beta (1)T256C (6'), a disulfide bond formed only in the presence of GABA and locked the channels open. Channel activation must involve an asymmetric rotation of two adjacent subunits toward each other. This would move aligned engineered cysteines on different subunits into proximity and allow disulfide bond formation without blocking conduction. Asymmetric rotation of M2 segments is probably a common gating mechanism in other ligand-gated ion channels.