GABA_A receptor M2-M3 loop secondary structure and changes in accessibility during channel gating

The γ-aminobutyric acid type A (GABA_A) receptor M2-M3 loop structure and its role in gating were investigated using the substituted cysteine accessibility method. Residues from α₁Arg-273 to α₁aIle-289 were mutated to cysteine, one at a time. MTSET⁺ or MTSES^- reacted with all mutants from α₁R273C to α₁Y281C, except α₁P277C, in the absence and presence of GABA. The MTSET⁺ closed-state reaction rate was >1000 liters/ mol-s at α₁N274C, α₁S275C, α₁K278C, and α₁Y281C and was <300 liters/mol-s at <₁R273C, α₁L276C, α₁V279C, α₁A280C, and α₁A284C. These two groups of residues lie on opposite sides of an α-helix. The fast reacting group lies on a continuation of the M2 segment channel-lining helix face. This suggests that the M2 segment α-helix extends about two helical turns beyond α₁N274 (20′), aligned with the extracellular ring of charge. At α₁S275C, α₁V279C, α₁A280C, and α₁A284C the reaction rate was faster in the presence of GABA. The reagents had no functional effect on the mutants from α₁A282C to α₁I289C, except α₁A284C. Access may be sterically hindered possibly by close interaction with the extracellular domain. We suggest that the M2 segment α-helix extends beyond the predicted extracellular end of the M2 segment and that gating induces a conformational change in and/or around the N-terminal half of the M2-M3 loop. Implications for coupling ligand-evoked conformational changes in the extracellular domain to channel gating in the membrane-spanning domain are discussed.