Structure of the M2 transmembrane segment of GLIC, a prokaryotic cys loop receptor homologue from gloeobacter violaceus, probed by substituted cysteine accessibility

Rishi B. Parikh, Moez Bali, Myles H. Akabas

Research output: Contribution to journalArticle

46 Scopus citations

Abstract

GLIC is a homopentameric proton-gated, prokaryotic homologue of the Cys loop receptor family of neurotransmitter-gated ion channels. Recently, crystal structures of GLIC hypothesized to represent an open channel state were published. To explore the channel structure in functional GLIC channels, we tested the ability of p-chloromercuribenzenesulfonate to react with 30 individual cysteine substitution mutants in and flanking the M2 channel-lining segment in the closed state (pH 7.5) and in a submaximally activated state (pH 5.0). Nine mutants did not tolerate cysteine substitution and were not functional. From positions 10' to 27', p-chloromercuribenzenesulfonate significantly modified the currents at pH 7.5 and 5.0 in all mutants except H234C (11′), I235C (12′), V241C (18′), T243C (20′), L245C (22′), and Y250C (27′), which were not functional, except for 12′. Currents for P246C (23′) and K247C (24′) were only significantly altered at pH 5.0. The reaction rates were all > 1000 m -1 s-1. The reactive residues were more accessible in the activated than in the resting state. We infer that M2 is tightly associated with the adjacent transmembrane helices at the intracellular end but is more loosely packed from 10′ to the extracellular end than the x-ray structures suggest. We infer that the charge selectivity filter is in the cytoplasmic half of the channel. We also show that below pH 5.0, GLIC desensitizes on a time scale of minutes and infer that the crystal structures may represent a desensitized state.

Original languageEnglish (US)
Pages (from-to)14098-14109
Number of pages12
JournalJournal of Biological Chemistry
Volume286
Issue number16
DOIs
StatePublished - Apr 22 2011

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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