title: GABA(A) receptor M2-M3 loop secondary structure and changes in accessibility during channel gating.
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.
authors.affiliation: Department of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Amal K
authors.initials: AK
authors.lastname: Bera
authors.forename: Maya
authors.initials: M
authors.lastname: Chatav
authors.forename: Myles H
authors.initials: MH
authors.lastname: Akabas
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine; D003545:Cysteine; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007532:Isoleucine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000502:physiology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D021122:Protein Subunits; D000644:Quaternary Ammonium Compounds; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D007532:Isoleucine; C039795:trimethylethylammonium; D005680:gamma-Aminobutyric Acid; D001120:Arginine; D003545:Cysteine