title: Characterization of the effects of charged residues in the intracellular loop on ion permeation in alpha1 glycine receptor channels.
abstract: The Cys loop receptor channels mediate fast synaptic transmission in the nervous system. The M2-demarcated transmembrane pore is an important determinant of their ion permeation properties. Portals within the intracellular domain are also part of the permeation pathway in cationic Cys loop receptors, with charged residues in a helical MA stretch partially lining these openings profoundly affecting channel conductance. It is unknown whether analogous portals contribute to the permeation pathway in anionic Cys loop receptors. We therefore investigated the influence of charged residues within the proposed MA stretch on functional properties of the homomeric glycine alpha1 receptor. Up to eight basic residues in the MA stretch were concurrently mutated to a negatively charged glutamate, and wild-type and mutant subunits were expressed in HEK-293 cells. Mutation of all eight residues produced a non-functional receptor. The greatest reduction in conductance at negative membrane potentials (from 92.2+/-2.8 to 60.0+/-2.2 picosiemens) was observed with glutamate present at the 377, 378, 385, and 386 positions (the 4E subunit). Inclusion of additional glutamate residues within this subunit did not decrease conductance further. Neutralizing these residues (the 4A subunit) caused a modest decrease in conductance (80.5+/-2.3 picosiemens). Outward conductance at positive potentials was not markedly affected. Anion to cation selectivity and concentration-response relationships were unaffected by the 4A or 4E mutations. Our results identify basic residues affecting conductance in the glycine receptor, suggesting that portals are part of the extended permeation pathway but that the M2-demarcated channel pore is the dominant determinant of permeation properties in glycine receptors.
authors.affiliation: School of Medical Sciences, University of New South Wales, Sydney, 2052, Australia.
authors.forename: Jane E
authors.initials: JE
authors.lastname: Carland
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Cooper
authors.forename: Silas
authors.initials: S
authors.lastname: Sugiharto
authors.forename: Hyo-Jin
authors.initials: HJ
authors.lastname: Jeong
authors.forename: Trevor M
authors.initials: TM
authors.lastname: Lewis
authors.forename: Peter H
authors.initials: PH
authors.lastname: Barry
authors.forename: John A
authors.initials: JA
authors.lastname: Peters
authors.forename: Jeremy J
authors.initials: JJ
authors.lastname: Lambert
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Moorhouse
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004594:Electrophysiology; D006801:Humans; D007477:Ions / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018009:Receptors, Glycine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D007477:Ions; D021122:Protein Subunits; D018009:Receptors, Glycine