GABAA Receptor M2–M3 Loop Secondary Structure and Changes in Accessibility during Channel Gating

Amal K Bera,Maya Chatav,Myles H Akabas

doi:10.1074/jbc.m206321200

Abstract

The gamma-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 alpha(1)Arg-273 to alpha(1)Ile-289 were mutated to cysteine, one at a time. MTSET(+) or MTSES(-) reacted with all mutants from alpha(1)R273C to alpha(1)Y281C, except alpha(1)P277C, in the absence and presence of GABA. The MTSET(+) closed-state reaction rate was >1000 liters/mol-s at alpha(1)N274C, alpha(1)S275C, alpha(1)K278C, and alpha(1)Y281C and was <300 liters/mol-s at alpha(1)R273C, alpha(1)L276C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C. These two groups of residues lie on opposite sides of an alpha-helix. The fast reacting group lies on a continuation of the M2 segment channel-lining helix face. This suggests that the M2 segment alpha-helix extends about two helical turns beyond alpha(1)N274 (20'), aligned with the extracellular ring of charge. At alpha(1)S275C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C the reaction rate was faster in the presence of GABA. The reagents had no functional effect on the mutants from alpha(1)A282C to alpha(1)I289C, except alpha(1)A284C. Access may be sterically hindered possibly by close interaction with the extracellular domain. We suggest that the M2 segment alpha-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.

Highlights

The GABAA1 receptors mediate inhibitory neurotransmission in the central nervous system [1]
We suggest that the M2 segment ␣-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
Mutations of three residues in the GABAA receptor ␥2 subunit, two in the M2 segment and one in the M2–M3 loop aligned with ␣1Ala280, uncouples benzodiazepine binding in the extracellular domain from benzodiazepine potentiation of GABA-evoked currents [19]

Summary

EXPERIMENTAL PROCEDURES

Molecular Biology—The cDNAs encoding the rat GABAA ␣1, ␤1, and ␥2S subunits in the pGEMHE vector were used [30]. 1 mM MTSETϩ or 10 mM MTSESϪ Ϯ near-saturating GABA concentration (5 times EC50) was applied for 1 min This was followed by two submaximal GABA test pulses. For some mutants the average effect following MTS reagent application was small Whether such an effect was statistically significant depended, in part, on the stringency of the one-way ANOVA post hoc test used. If complete reaction caused 100% inhibition of the GABA-induced current, with a detection threshold of 30% effect and the MTSETϩ reaction conditions of 1 mM applied for 1 min, the slowest reaction rate that we can detect must have a second order reaction rate constant of Ͼ6 liters/mol-s. Similar second order rates were obtained in all cases consistent with the reaction being a simple bimolecular reaction

RESULTS

10 Ϯ 2a 12 Ϯ 1

DISCUSSION