Loose Protein Packing around the Extracellular Half of the GABA A Receptor β1 Subunit M2 Channel-lining Segment

Eric N. Goren, David C. Reeves, Myles H. Akabas

Research output: Contribution to journalArticle

40 Scopus citations

Abstract

GABAA receptors are ligand-gated ion channels formed by the pseudosymmetrical assembly of five homologous subunits around the central channel axis. The five M2 membrane-spanning segments largely line the channel. In the present work we probed the water surface accessibility of the β 1 subunit M2 segment using the substituted cysteine accessibility method. We assayed the reaction of the negatively charged sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS-), by its effect on subsequent currents elicited by EC50 and saturating GABA concentrations. PCMES-, applied with GABA, reacted with 14 of the 19 residues tested. At the M2 cytoplasmic end from 2′ to 6′ only β1LA252C (2′) and β1PT256C (6′) were pCMBS--reactive in the presence of GABA. We infer that the M2 segments are tightly packed in this region. Toward the extracellular half of M2 all residues from β1T262C (12′) through β 1E270C (20′) reacted with pCMBS- applied with GABA. We infer that this region is highly mobile and loosely packed against the rest of the protein. Based on differences in pCMBS- reaction rates two domains can be distinguished on the putative channel-lining side of M2. A faster reacting domain includes the 2′, 9′, 12′, 13′, and 16′ residues. The slower reacting face contains the 6′, 10′, and 14′ residues. We hypothesize that these may represent the channel-lining faces in the closed and open states and that gating involves an 80-100° rotation of the M2 segments. These results are consistent with the loose packing of the M2 segments inferred from the structure of the homologous Torpedo nicotinic acetylcholine receptor.

Original languageEnglish (US)
Pages (from-to)11198-11205
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number12
DOIs
StatePublished - Mar 19 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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