title: Synthetic peptides and four-helix bundle proteins as model systems for the pore-forming structure of channel proteins. II. Transmembrane segment M2 of the brain glycine receptor is a plausible candidate for the pore-lining structure.
abstract: A synthetic 23-mer peptide (M2GlyR) with the amino acid sequence of the putative transmembrane segment M2 of the strychnine-binding alpha subunit of the inhibitory glycine receptor forms anion-selective channels in phospholipid bilayers. The most frequent events show single-channel conductances, gamma, of 25 pS and 49 pS in symmetric 0.5 M KCl with channel open lifetimes, tau o, in the millisecond time range. These properties match those of authentic glycine receptors studied in inside-out patches of cultured rat spinal cord neurons, namely gamma = 27 pS and gamma = 45 pS, and tau o in the millisecond time range. The channel activity of M2GlyR is sequence-specific: 1) a synthetic peptide with the sequence of putative transmembrane segment M1 (M1GlyR), not considered to contribute to the channel lining, does not form channels; 2) an analog of M2GlyR with site-specific substitutions displays distinct channel properties: 2 arginine residues at the N and C termini of M2, postulated to contribute to the anion selectivity of the channel, are substituted by glutamic acids, and the analog peptide ([Glu3,22]M2GlyR) forms cation-selective channels. Further, a four-helix bundle protein (T4M2GlyR) formed by tethering four identical M2GlyR modules to a carrier template forms homogeneous anion-selective channels with gamma = 25 pS in 0.5 M KCl. These channels are blocked by picrotoxin and by the anion channel blockers 9-anthracene carboxylic acid and niflumic acid, but not by an analog of the local anesthetic lidocaine (QX-222), a cation channel blocker. Observed single-channel properties suggest that a pentameric assembly of alpha and beta subunits with a central pore lined by M2 segments would account for conductance properties of the authentic glycine receptor and the 2 arginines at either end of M2 could confer anion specificity to the receptor channel.
authors.affiliation: Department of Biology, University of California, San Diego, La Jolla 92093-0319.
authors.forename: G L
authors.initials: GL
authors.lastname: Reddy
authors.forename: T
authors.initials: T
authors.lastname: Iwamoto
authors.forename: J M
authors.initials: JM
authors.lastname: Tomich
authors.forename: M
authors.initials: M
authors.lastname: Montal
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions; D001923:Brain Chemistry; D002412:Cations; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D004563:Electrochemistry; D005998:Glycine / Q000378:metabolism; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry* / Q000187:drug effects; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D051381:Rats; D018009:Receptors, Glycine; D017981:Receptors, Neurotransmitter / Q000737:chemistry* / Q000378:metabolism; D014101:Torpedo
chemical_list: D000838:Anions; D002412:Cations; D007473:Ion Channels; D010455:Peptides; D018009:Receptors, Glycine; D017981:Receptors, Neurotransmitter; D005998:Glycine