Mapping of disulfide bonds within the amino-terminal extracellular domain of the inhibitory glycine receptor.
The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel and a member of the superfamily of cysteine loop (Cys-loop) neurotransmitter receptors, which also comprises the nicotinic acetylcholine receptor (nAChR). Within the extracellular domain (ECD), the eponymous Cys-loop harbors two conserved cysteines, assumed to be linked by a superfamily-specific disulfide bond. The GlyR ECD carries three additional cysteine residues, two are predicted to form a second, GlyR-specific bond. The configuration of none of the cysteines of GlyR, however, had been determined directly. Based on a crystal structure of the nAChRalpha1 ECD, we generated a model of the human GlyRalpha1 where close proximity of the respective cysteines was consistent with the formation of both the Cys-loop and the GlyR-specific disulfide bonds. To identify native disulfide bonds, the GlyRalpha1 ECD was heterologously expressed and refolded under oxidative conditions. By matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we detected tryptic fragments of the ECD indicative of disulfide bond formation for both pairs of cysteines, as proposed by modeling. The identity of tryptic fragments was confirmed using chemical modification of cysteine and lysine residues. As evident from circular dichroism spectroscopy, mutagenesis of single cysteines did not impair refolding of the ECD in vitro, whereas it led to partial or complete intracellular retention and consequently to a loss of function of full-length GlyR subunits in human embryonic kidney 293 cells. Our results indicate that the GlyR ECD forms both a Cys-loop and a GlyR-specific disulfide bond. In addition, cysteine residues appear to be important for protein maturation in vivo.
Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Erlangen 91054.
Nicolas
N
Vogel
Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Erlangen 91054.
Christoph J
CJ
Kluck
Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Erlangen 91054.
Nima
N
Melzer
Lehrstuhl für Biopolymere, Universität Bayreuth, Bayreuth 95440, Germany.
Stephan
S
Schwarzinger
Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Erlangen 91054.
Ulrike
U
Breitinger
Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Erlangen 91054.
Silke
S
Seeber
Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Erlangen 91054. Electronic address: cmb@biochem.uni-erlangen.de.
Cord-Michael
CM
Becker
D000818:Animals; D002460:Cell Line; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D010449:Peptide Mapping; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000502:physiology; D018009:Receptors, Glycine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018118:Chloride Channels; D004220:Disulfides; D018009:Receptors, Glycine