Mapping of Disulfide Bonds within the Amino-terminal Extracellular Domain of the Inhibitory Glycine Receptor

Abstract

The strychnine-sensitive glycine receptor (GlyR) is a ligandgated 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 Cysloop 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 nAChR␣1 ECD, we generated a model of the human GlyR␣1 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 GlyR␣1 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.The glycine receptor (GlyR) 2 is a ligand-gated ion channel that mediates fast neuronal inhibition predominantly in the spinal cord and brain stem (1). In the mammalian central nervous system, five gene variants of homologous GlyR subunits have been identified (2), four belong to the ␣-type (␣1-␣4) and one to the ␤-type. The GlyR complex presents as a rosette-like assembly of five subunit polypeptides surrounding a central anion pore. During development of the spinal cord, embryonic ␣2-homopentamers are largely replaced by adult heteromeric isoforms (3), composed of 3 ␤-and 2 ␣-subunits (4).The GlyR belongs to the superfamily of cysteine loop (Cysloop) receptors, which also comprises GABA A/C (␥-aminobutyric acid) receptors, nicotinic acetylcholine receptors (nAChR), and 5-hydroxytryptamine type 3 receptors. Structural predictions for Cys-loop receptors rely on sequence-based predictions and mutational studies. Crystal structures of the Lymnaea stagnalis acetylcholine-binding protein (AChBP), the Torpedo marmorata nAChR, the extracellular domain (ECD) of the murine nAChR␣1, and prokaryotic proton-gated channels indicate a common, conserved protein fold (5-8). Accordingly, each subunit of a Cys-loop receptor comprises a large amino-terminal ECD adopting a twisted ␤-sandwich structure that pr...

Glycine receptor mouse mutants: model systems for human hyperekplexia

“…In addition to the eponymous Cys-loop, the ECD of the Gly receptor carries two additional cysteine residues in loop C that are conserved in the recently solved GluCl receptor structure. These cysteines have been found to form another disulphide bond, which affects both the formation of the eponymous Cys-loop and the correct membrane integration needed for channel function (Vogel et al, 2009;Hibbs and Gouaux, 2011). Several lines of evidence indicate a rigid body capping motion of loop C upon agonist binding, thereby trapping bound agonist molecules (Hansen et al, 2005).…”

Single Expressed Glycine Receptor Domains Reconstitute Functional Ion Channels without Subunit-specific Desensitization Behavior

“…Ligand application using a U-tube gave a time resolution of 20 -30 ms. The external buffer consisted of NaCl 137 mM, KCl 5.4 mM, CaCl 2 1.8 mM, MgCl 2 1.0 mM, Hepes 5.0 mM, pH adjusted to 7.2 with NaOH; the internal buffer was CsCl 120 mM, N(Et) 4 Cl 20 mM, CaCl 2 1.0 mM, MgCl 2 2.0 mM, EGTA 11 mM, Hepes 10 mM, pH adjusted to 7.2 with CsOH. Current responses were measured at a room temperature of 21-23°C, the holding potential was Ϫ60 mV.…”

“…The adult GlyR consists of two ␣ and three ␤ subunits (2). The extracellular ligand-binding domain of the glycine receptor harbors two disulfide bonds, one of which is eponymous for the receptor family and a second disulfide bond located in loop C is described for the glycine receptor and the glutamate-gated chloride channel (Glu-Cl) from Caenorhabditis elegans (3,4). The large intracellular loop between TM3 and TM4 (TM3-4 loop, also referred to as ICD) is of highest diversity among CLRs.…”

Distinct Properties of Glycine Receptor β+/α− Interface

“…For the voltage clamp fluorometry (VCF) experiments, the cysteines equivalent to the ␣1 Cys 41 and ␤ Cys 115 were mutated to alanines to minimize possible background labeling. Neither mutation affects channel function (24,25).…”