title: Functional expression and purification of a homomeric human alpha 1 glycine receptor in baculovirus-infected insect cells.
abstract: The human alpha 1 glycine receptor (GlyR) was expressed in Sf9 insect cells infected with a recombinant Autographa californica nuclear polyhedrosis baculovirus. Previous studies had indicated that transient expression of this subunit in Xenopus oocytes or human kidney cell lines is sufficient to form active agonist-gated chloride channels. Expression of the alpha 1 GlyR protein resulted in functional channels present on the cell surface of infected Sf9 cells as evidenced by whole-cell patch-clamping and single-channel recordings. These channels were gated by glycine, but not in the presence of strychnine. An immunoreactive 48-kDa protein could be easily visualized on Coomassie-stained sodium dodecyl sulfate-polyacrylamide gels of whole-cell lysates with maximal expression 3 days postinfection. The alpha 1 GlyR protein was solubilized from a membrane fraction of infected Sf9 cells in 1% digitonin and 0.1% deoxycholate and purified by affinity chromatography using aminostrychnine agarose, yielding 0.33 mg/liter of cells. Given the low natural abundance of the native channel, the development of this expression system now provides sufficient purified channel protein for future biochemical and biophysical characterization. Since the glycine receptor shares sequence and structural homology with other members of a ligand-gated channel superfamily, further characterization may establish general rules governing the structure and mechanism of these membrane protein channels.
authors.affiliation: Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06511.
authors.forename: M
authors.initials: M
authors.lastname: Cascio
authors.forename: N E
authors.initials: NE
authors.lastname: Schoppa
authors.forename: R L
authors.initials: RL
authors.lastname: Grodzicki
authors.forename: F J
authors.initials: FJ
authors.lastname: Sigworth
authors.forename: R O
authors.initials: RO
authors.lastname: Fox
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D009036:Moths; D018009:Receptors, Glycine / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology
chemical_list: D018076:DNA, Complementary; D018009:Receptors, Glycine