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 nAChRα1 ECD, we generated a model of the human GlyRα1 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 GlyRα1 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)(6)(7)(8). Accordingly, each subunit of a Cys-loop receptor comprises a large amino-terminal ECD adopting a twisted ␤-sandwich structure that precedes four ␣-helical transmembrane segments (TM1-TM4), each connected to its neighbors via intra-and extracellular loops. TM2 is an amphipathic helix that forms the inner lining of the channel pore. The ligand-binding pocket is located at the interface between adjacent ECDs and is covered by the flexible loop C (9). Consequently, channel gating is mediated through an interaction of the ECD with the TMs.
The Cys-loop, a loop within the ECD harboring two conserved cysteines, is eponymous for the superfamily of these ligand-gated ion channels. The Cys-loop extends into the interface between the ECD and TM domains, suggesting an important role in channel gating (10). It is generally assumed that these cysteine residues form a family-specific disulfide bond stabilizing the Cys-loop. Their functional role for the nAChR was explored using reducing agents and site-directed mutagenesis, which demonstrated influences on receptor expression and physiology (11,12). Direct experimental evidence for the formation of the disulfide bond in the Cys-loop comes from chemical modification of informative peptides of purified Torpedo nAChR and from crystallography of the ECD of the murine nAChR␣1 subunit (7,13).
In the GlyR ECD, in addition to the Cys-loop, loop C is also thought to be stabilized by a second, unique disulfide bond. This assumption is based on recombinant expression studies with mutant receptor constructs (14). In the human GlyR␣1 ECD, putative formation of the Cys-loop disulfide bond (Cys 138 -Cys 152 ) and the GlyR-specific disulfide bond (Cys 198 -Cys 209 ) would leave one cysteine residue (Cys 41 ) unpaired. To our knowledge, there is no direct experimental evidence for disulfide bond formation in the GlyR ECD.
Starting from a multiple sequence alignment, we generated a model for the GlyR␣1 ECD, consistent with specific formation of both the Cys-loop and the GlyR-specific disulfide bond. Making use of isolated GlyR␣1 ECD obtained from Escherichia coli expression and functional refolding (15), we identified tryptic fragments corresponding to the predicted disulfide bonds by matrix-assisted laser-desorption-ionization time-offlight (MALDI-TOF) mass spectrometry. To elucidate their functional relevance, we generated serine mutants for each cysteine residue, preventing the formation of the respective disulfide bonds in isolated ECD and full-length receptor constructs. Our results suggest that the cysteine residues play important roles in receptor maturation.

EXPERIMENTAL PROCEDURES
Sequence Alignment and Homology Modeling-For sequence alignment, amino acid sequences of mature human nAChR␣2, ␣3, ␣5, ␣7, and ␤2 subunits, human GlyR␣1, ␣2, and ␣3 subunits, the murine nAChR␣1 subunit (mm-ECD), and the L. stagnalis AChBP were obtained from the Swiss Prot data base (16). For transmembrane proteins, ECD sequences were used as annotated. A multiple sequence alignment was performed with ClustalW (17) followed by minimal manual corrections. Based on the crystal structure of the murine nAChR␣1 ECD (9), the homology model of the human GlyR␣1 ECD was generated using Swiss Model (18). Appropriate cysteine bonds were manually added followed by 100 steps of conjugate gradient energy minimization in Sybyl 7.3 (Tripos Inc., Munich, Germany).
Constructs and Mutagenesis-For bacterial expression, the pET30a vector (EMD, San Diego, CA) containing the isolated human GlyR␣1 ECD was used. The full-length human GlyR␣1 receptor subunit in pRK5 was used for expression in HEK293 cells. Single amino acid substitutions were introduced using PCR-based site-directed mutagenesis. All plasmids were sequence verified.
Bacterial Protein Expression and Refolding-Recombinant expression of the isolated GlyR␣1 ECD constructs and refolding under oxidative conditions (using CuCl 2 ) was carried out essentially as described (15). Deviating from this protocol, RosettaGami2 cells (EMD) were used for expression and affinity purification was omitted.
Tryptic Proteolysis and Chemical Modification-For tryptic proteolysis of the refolded ECD, the proteins were dialyzed against cleavage buffer (25 mM NH 4 HCO 3 ). The protease-toprotein ratio was adjusted to 1:100 and cleavage was carried out at 37°C overnight to obtain complete digestion. For limited proteolysis, protease-to-protein ratios of 1:500 or less were FIGURE 1. Alignment and modeling of the GlyR␣1 ECD. A, human nAChR␣2, ␣3, ␣5, ␣7, and ␤2 subunits, human GlyR␣1, ␣2, and ␣3 subunits, the murine nAChR␣1 subunit (mm-ECD), and the L. stagnalis AChBP are used for multiple sequence alignments with ClustalW (17). Cysteine residues are boxed in gray. Numbers correspond to the respective position in the processed human GlyR␣1. Asterisks indicate identical, colons conserved, and dots semi-conserved substitutions. B, model of the human GlyR␣1 ECD based on the crystal structure of the murine nAChR␣1 ECD (9) after manual bonding of adjacent cysteines and energy minimization. Details covering the positions of the cysteine residues are enlarged (I and II). The model is consistent with the conjunction of four cysteines to form two distinct disulfide bonds. Cysteines 138 and 152 form the Cys-loop disulfide bond (I), whereas Cys 198 and Cys 209 form the GlyR-specific disulfide bond (II). One cysteine (C41) remains unpaired (I).
used. Where needed, dithiothreitol (5 mM final concentration) was added and reduction of the disulfide bonds was performed at 56°C for 30 min. Modification of cysteine residues was performed for 1 h with a 40-fold molar excess of N-maleoyl-␤alanine (Sigma) over the concentration of dithiothreitol used. Lysine modification was performed with O-methylisourea using the proteomass guanidination kit (Sigma). All peptide samples were cleaned using Zip-Tip reversed phase microcolumns (Millipore, Billerica) prior to MALDI-TOF mass spectrometry.
MALDI-TOF Mass Spectrometry of Digested Protein Samples-Tryptically digested protein samples were dried using a vacuum concentrator (Eppendorf, Hamburg). The pellet was dissolved in 0.1% (v/v) trifluoroacetic acid. 2 l of the sample were mixed with 2 l of 2% (v/v) trifluoroacetic acid and 2 l of dihydroxyacetophenone matrix (15 mg/ml of 2,5-dihydroxyacetophenone, 5 mg/ml of diammonium hydrogen citrate in 75% ethanol). An aliquot of 0.5 l was spotted on a stainless steel target and allowed to dry at ambient temperature. MALDI-TOF mass spectrometry was performed on a Bruker Autoflex (Bruker Daltonik, Bremen, Germany), equipped with a nitrogen laser ( ϭ 337 nm). Positive ions were analyzed in the reflector mode after acceleration by 20 kV. External calibration was performed using the peptide calibration standard (Bruker Daltonik, Bremen, Germany). For each displayed mass spectrum, at least 250 individual spectra obtained from several positions on a spot were averaged. Spectrum analysis was performed using Flex Analysis software (Bruker Daltonik).
CD Spectroscopy-Circular dichroism spectra were recorded on a Jasco J810 spectropolarimeter (Jasco, Groß-Umstadt) equipped with a Peltier element. Data were recorded from wild-type as well as cysteinemutant GlyR␣1 ECDs in 10 mM sodium phosphate buffer, pH 7.4, at 20°C from 260 to 190 nm with a step size of 0.2 nm and an integration time of 2 s in a quartz cell with 0.1-cm path length. Concentrations were determined by measuring the absorbance at 280 nm and ranged from 2.0 to 5.6 M. A blank spectrum with buffer only was recorded under identical conditions and subtracted from the spectra. Spectra were smoothed using a Savitzky-Golay filter with a window size of 11 data points. Secondary structure contents were estimated using the DICHROWEB service (19,20) applying the CDSSTR algorithm with the SP175(190) reference data base (21).

H]Ligand Binding Assays-Binding of [ 3 H]strychnine and [ 3 H]glycine (PerkinElmer Life
Sciences) to refolded proteins was performed as described (15). To achieve retention of the refolded protein on GF/C filters, it was precipitated with an equal volume of 15% PEG400 in binding buffer (25 mM potassium P i , pH 7.4, 200 mM KCl). Filters were washed with binding buffer after application of the protein; short washing times were essential. Unlabeled strychnine (200 M) and glycine (200 mM) were used for competition and unspecific binding was subtracted. The affinity of [ 3 H]strychnine binding was determined by Scatchard analysis (15).
Expression in HEK293 Cells, Membrane Preparation, and Western Blot-HEK293 cells were transfected according to a modified calcium phosphate protocol. Where necessary, cells were cotransfected with enhanced green fluorescent protein.
For Western blot analysis, cells were harvested and homogenized in PBS at 4°C. After centrifugation for 30 min at 100,000 ϫ g, the pellet was resuspended in 20 mM potassium phosphate buffer containing protease inhibitors (Complete EDTA-free; Roche Applied Science). The protein concentration was determined and equal amounts were subjected to SDS-PAGE and Western blot. For detection of GlyR␣1, the mouse monoclonal antibody mAb4a (supernatant of hybridoma cells) was used. Goat anti-mouse antibody coupled to Cy5 (1:250) or horseradish peroxidase (1:10,000) was used for visualization (Dianova, Hamburg).

Immunocytochemistry and Electrophysiology of HEK293
Cells-For immunocytochemistry, transfected HEK293 cells were washed 3 times in PBS and fixed for 30 min with paraformaldehyde (3% w/v in PBS) followed by quenching with 50 mM ammonium chloride in PBS. Blocking was performed with 5% (v/v) sheep serum in PBS for 30 min at room temperature. To perform staining of intracellular GlyRs, 0.1% (v/v) Triton X-100 was added to the blocking solution to permeabilize the plasma membrane. For staining of the ECD of GlyR␣1, purified mouse monoclonal antibody mAb2b was used in blocking solution (1:200). Transfected cells were incubated with the primary antibody for 1 h at room temperature and washed 3 times with PBS. This procedure was repeated with goat anti-mouse antibody coupled to Alexa 488 (1:400 in blocking solution; Invitrogen). Finally, cells were embedded in Mowiol and subjected to confocal microscopy on a DMIRE2 confocal microscope (Leica, Wetzlar). Electrophysiology was carried out essentially as described (22). Under the conditions used, recombinant GlyRs showed little desensitization, consistent with earlier reports (23).

RESULTS
Modeling of the GlyR␣1 ECD-The human GlyR␣1 ECD contains five cysteine residues (Cys 41 , Cys 138 , Cys 152 , Cys 198 , and Cys 209 ). We generated a multiple sequence alignment for ECDs of several nAChR and GlyR subunit variants as well as the AChBP (Fig. 1A). This confirmed that only the cysteines within the Cys-loop (C138, C152) were conserved among the proteins analyzed, which is in agreement with previous alignments (4,6).
Based on the crystal structure of the murine nAChR␣1 (7), a model of the human GlyR␣1 ECD was generated. This model indicated the opposition of both conserved cysteine residues in the Cys-loop as well as two GlyR-specific cysteines in the loop C (Cys 198 and Cys 209 ), allowing disulfide bond formation. The model also predicted that cysteine residue Cys 41 is buried, suggesting that it is not involved in disulfide bond formation (Fig. 1B).
Probing for Disulfide Bonds in the GlyR␣1 ECD with MALDI-TOF Mass Spectrometry-To analyze the status of the GlyR extracellular cysteines under in vitro conditions, the isolated GlyR␣1 ECD was expressed in E. coli and refolded from inclusion bodies under oxidative conditions as described (15), obtaining native-like ligand-binding capability. Then, the ECD was digested with trypsin and analyzed by MALDI-TOF mass spectrometry, yielding a signal of 6728.1 Da matching the calculated mass of the tryptic fragments holding Cys 138 and Cys 152 linked by a disulfide bond ( Fig. 2A). When the peptide mixture was chemically reduced to break disulfide bonds, this molecular mass disappeared, leaving signals (1318.9 and 5414.3 Da), which matched the calculated masses of the respective reduced fragments ( Fig. 2A). Apparently, the pseudo-ion observed in the non-reduced sample contained a bond cleavable under mild reducing conditions, as expected for a disulfide bond. Respective single fragments, however, were still detectable in the nonreduced samples, suggesting that cysteine bonding was not fully completed during in vitro refolding.
In some experiments, the tryptic peptides were chemically modified using side chain reactive agents. When the single fragments were modified with N-maleoyl-␤-alanine, a mass shift of ϩ169 Da occurred, consistent with the presence of a single sulfhydryl group in each fragment (Fig. 2, B and C). Finally, the reduced fragment holding Cys 138 was modified once with O-methylisourea (ϩ42 Da), which is in agreement with the presence of a single lysine residue, further corroborating the identity of this fragment (Fig. 2D).
Furthermore, we found a second informative proton adduct of 1350.8 Da, matching the theoretical mass of the tryptic fragments harboring Cys 198 and Cys 209 linked by a disulfide bond. This bonded fragment, again, disappeared after chemical reduction, leaving the corresponding single fragments (514.1  DECEMBER 25, 2009 • VOLUME 284 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 36131 and 839.5 Da) (Fig. 3A). Under oxidative conditions, signals representing single fragments were low in MALDI-TOF mass spectrometry.

Disulfide Bonds of the Glycine Receptor
Mass shifts upon modification with N-maleoyl-␤-alanine and O-methylisourea indicated the presence of cysteine residues in both single fragments as well as the presence of a lysine residue in the bonded fragment (Fig. 3, B-D). After limited tryptic proteolysis, we also found a proton adduct corresponding to the mass of a single fragment with 3 missed cleavages harboring both Cys 198 and Cys 209 (Fig. 4). The mass of this fragment was increased by 2 Da upon reduction, indicating cleavage of an intrapeptide disulfide bond. This observation confirmed our previous results.
In all of our spectra, we exclusively found signals indicating bonds formed between Cys 138 and Cys 152 , as well as Cys 198 and Cys 209 , respectively. In particular, no alternative bond between these cysteines or to Cys 41 was observed in any of the experiments. A fragment harboring Cys 41 could be readily detected and modified (Fig. 5). The identities of all detected lysine and cysteine harboring fragments were confirmed by chemical modification. Taken together, identifiable fragments covered more than 95% of the protein sequence (Fig. 6). These data indicate selective formation of the predicted Cys-loop and GlyR-specific disulfide bond in the GlyR␣1 ECD.
Mutagenesis of the GlyR␣1 ECD Construct-To analyze the process of disulfide formation in vitro, we generated single cysteine-to-serine substitutions of the ECD constructs. The mutant ECDs were expressed in E. coli, forming inclusion bodies, and subjected to refolding under oxidative conditions. Surprisingly, all mutants appeared to readily refold, yielding considerable concentrations of soluble protein. None of the mutants displayed increased precipitation, essentially resembling the wild-type situation (data not shown). This suggested that the overall fold of the ECD may be independent of disulfide bond formation. Subsequently, the mutant ECDs were subjected to tryptic digestion and analysis by MALDI-TOF mass spectrometry.
The single fragments holding mutant residues showed the expected mass shifts resulting from mutagenesis. The pseudoion corresponding to the bonding of Cys 198 and Cys 209 was observed for wild-type and mutant constructs C41S, C138S, and C152S, but not for C198S and C209S (Fig. 7). The bonding of Cys 138 and Cys 152 , however, was only observed for wild-type and C41S, indicating that, in vitro, formation of the Cys-loop disulfide bond may require the presence of the GlyR-specific bond.
Secondary Structure Analysis of Wild-type and Mutant GlyR␣1 ECD-To analyze secondary structure elements of GlyR␣1 ECDs, the refolded proteins were subjected to circular dichroism spectroscopy. The resulting spectrum of wild-type ECD was consistent with previous observations (15) and very well superimposed with spectra obtained from each of the mutants (Fig. 8A), indicating an overall comparable secondary structure content (Table 1). This result is in line with an equally efficient in vitro refolding of all ECD variants. Furthermore, the  composition of secondary structure elements is in good agreement with our proposed model of the GlyR␣1 ECD.
Ligand Binding of Refolded GlyR␣1 ECD-In a filtration assay, the refolded wild-type GlyR␣1 ECD showed high affinity [ 3 H]strychnine binding (Fig. 8B) with K D values ranging from 25 to 70 nM, similar to our earlier report (15). In addition, [ 3 H]strychnine binding was partially displaceable by an excess of unlabeled glycine. Conversely, we observed strychnine-displaceable [ 3 H]glycine binding (Fig. 8C). With the filtration assay used (15), saturation of [ 3 H]glycine binding was not achieved, consistent with the low affinity of the glycine site (2,4). This precluded a determination of the corresponding K D value. Taken together, this indicates that a mutually exclusive binding site for glycine and strychnine had formed in the refolded ECD. Apparently, the ECD folded into its native-like, functional conformation.
Cysteine Mutagenesis in Full-length GlyR Constructs-To address the function of the disulfide bonds, we performed single cysteine-to-serine substitutions in the ECD of full-length GlyR␣1 constructs. The constructs were expressed in HEK293 cells and immunostained with mAb2b recognizing the extracellular domain of GlyR␣1. Discrimination between surface expression and intracellular retention of the GlyR protein was achieved through facultative permeabilization of the cells prior to immunostaining. In confocal immunocytochemistry of transfected, non-permeabilized HEK293 cells, mutant GlyRs C41S and C138S showed reduced surface expression. In contrast, mutants C152S, C198S, and C209S showed no detectable surface expression (Fig. 9A). Accordingly, we found pronounced perinuclear staining for all mutants, indicating an increased intracellular retention. This finding contrasts the situation in vitro, where refolding appeared to be independent of the cysteine residues. The number of cells positive for intracellular GlyR stains was markedly reduced for C152S, C198S, and C209S (data not shown). Consequently, we found low GlyR immunoreactivity for these mutants in membrane preparations analyzed by Western blots of transfected HEK293 cells (Fig. 9B).
Finally, the physiological activity of the mutant receptors was measured in whole cell voltage-clamp recordings, applying saturating glycine concentrations (Fig. 9C). As expected from total and surface expression, mutant constructs C41S and C138S, but not C152S, C198S, and C209S were active in electrophysiology. Partially conserved maturation of mutant C41S is in agreement with the in vitro data, which shows that Cys 41 is neither involved in disulfide bond formation nor in folding sta-  bility of the ECD. Surprisingly, the differences observed between Cys-loop mutants C138S and C152S indicate a functional asymmetry of the single cysteines during maturation of the protein, as substitution of Cys 152 exerts a stronger impact on surface accumulation and channel function.
To address whether the observed maturation defects resulted from exposure of unpaired cysteines, we generated double mutants C138S/C152S and C198S/C209S. The double mutant C198S/C209S neither showed surface expression nor functionality in electrophysiology, essentially resem-bling the respective single mutants (Fig. 10). This indicates that intracellular retention did not result from exposed, unpaired cysteines 198 or 209. Likewise, the double mutant C138S/C152S was also retained, exhibiting neither surface expression nor ion channel activity, as observed for C152S but not for C138S. Hence, for surface expression, the mutation C152S is dominant over C138S. These data confirm that intracellular retention did not result from exposure of single unpaired cysteine residues.

DISCUSSION
Based on primary sequence analysis, GlyRs have been predicted to carry both a Cys-loop and a GlyR-specific disulfide bond (24). Here, we present experimental evidence for the formation of these disulfide bonds in the recombinant ECD of GlyR␣1 and for their role in GlyR biogenesis.
Homology modeling of the human GlyR␣1 ECD strongly suggested formation of both bonds, as the respective cysteines were directly opposed to each other. To test this hypothesis, we used the refolded ECD as a model for the receptor. Upon refolding under oxidative conditions, the ECD construct gains a native-like fold and attains high affinity strychnine binding (15). As evident from glycine displaceable [ 3 H]strychnine binding and strychnine-displaceable [ 3 H]glycine binding, the GlyR␣1 ECD carries determinants for both agonist and antagonist recognition (25).
The purity of the ECD constructs allowed for reproducible detection of tryptic fragments covering nearly the entire sequence of the protein. Upon random disulfide bond formation between the five cysteine residues present, 15 different bonds (10 inter-or intrasubunit and 5 intersubunit bonds) are possible. Of these, only the predicted two disulfide bonds were found. This strongly indicates specificity in the formation of the disulfide bonds.
Several lines of evidence confirm the identity of the detected proton adducts. (i) The deviation of the informative signals from the calculated m/z values was below 1 Da. (ii) The signals were detectable under oxidative conditions only, as expected for ions holding a disulfide bond. (iii) The signals were absent from tryptic digests of the respective cysteine mutants. For the GlyR-specific bond, further evidence comes (iv) from the chemical modification of a lysine residue and (v) from the detection of a signal corresponding to a fragment with 3 missed cleavages. This fragment carried an intrapeptide disulfide bond that was readily reduced to obtain the predicted mass shift of ϩ2 Da. We conclude that  the GlyR holds both the conserved bond in the Cys-loop between Cys 138 and Cys 152 , as well as the GlyR-specific bond in loop C between Cys 198 and Cys 209 . Single-cysteine mutagenesis did not disturb refolding in vitro, indicating that the overall native fold is not strongly dependent on disulfide bond formation. Circular dichroism spectroscopy revealed a comparable secondary structure content of both wild-type (15) and mutant ECDs, indicating that disulfide bond disruption remained without major structural changes. A similar secondary structure distribution was also observed for a chimeric glycine-binding protein, derived from human GlyR␣1 and AChBP (26). Thus, achieving the overall native-like fold of the isolated GlyR␣1 ECD during in vitro refolding appeared to be independent of disulfide bond formation. Curiously, however, the formation of its Cys-loop disulfide bond was dependent on the pres-ence of Cys 198 and Cys 209 forming the GlyR-specific bond. This suggests that the formation of the GlyR-specific bond leads to a minor conformational shift allowing for the eponymous Cys-loop bond to form.
The effect of disulfide bond formation on the function of the GlyR has been studied using reducing and oxidizing agents. The oxidative status of the receptor has no effect on the affinity to strychnine and glycine, but modifies the Hill coefficient of binding (27). Furthermore, oxidation and reduction have opposing effects on whole cell currents in rat retinal ganglion cells (28). This finding is contrasted by a recent study detecting no effect of redox agents on glycinergic currents in mouse hippocampal neurons (29). Taken together, no gross reproducible functional defect of surface-expressed receptors was observed with reducing agents. None of these studies, however, determined the oxidative status of the single cysteines after manipulation.
By site-directed mutagenesis, we tested the influence of the cysteine residues on receptor expression and function. For mutant C41S, we observed reduced surface expression and activity, indicating that beyond disulfide bond formation, cysteines play important roles in the process of receptor maturation. In contrast to C152S, C138S is to some extent surface-expressed and functional. This discrepancy shows that two cysteines involved in the formation of a single disulfide bond can have different significance for receptor maturation and function. As observed for C152S, mutants C198S and C209S were not only inactive, but also lacked surface expression. This is contrasted by a previous report of a surface-expressed, but inactive, mutant C209S (14). Nevertheless, both studies sustain the hypothesis of functional asymmetry of cysteine residues. In our hands, none of the inactive mutants were surface expressed. Additionally, cysteine mutagenesis might interfere with glycine-gated chloride channel function, i.e. ligand binding or channel open probability, which cannot readily be separated from ion channel biogenesis in heterologous cell expression. Intracellular retention and dysfunctionality of double mutants C138S/C152S and C198S/C209S suggests that exposure of an unpaired cysteine was not the cause for the FIGURE 9. Characterization of full-length GlyR␣1 variants. A, immunocytochemistry of transfected HEK293 cells, surface-stained or permeabilized for intracellular staining of GlyR␣1 with mAb2b. The wild-type receptor (WT) shows intensive surface and intracellular staining. Note that surface expression is significantly reduced for mutants C41S and C138S, and abolished for C152S, C198S, and C209S. This reduction coincides with an intracellular accumulation of the GlyR␣1 antigen in perinuclear regions, implying defects in maturation. B, Western blot analysis of membrane preparations of HEK293 cells using mAb4a reflects reduced total expression of C152S, C198S, and C209S, which may correlate with disturbed maturation. Note the faint band at about 30 kDa for C209S indicating a reduced stability of this receptor variant. C, examples of whole cell patch clamp recordings of transfected HEK293 cells with saturating glycine application (2 mM) are shown (n Ն 6). In contrast to WT, C41S, and C138S, no activity is observed for receptor mutants C152S, C198S, and C209S, which correlates well with abolished surface expression. intracellular retention of the single mutants. Moreover, refolding experiments with mutated constructs showed that the formation of disulfide bonds may be interdependent, even though the overall stability of the fold is not dramatically influenced by bond formation as shown by circular dichroism spectroscopy. Hence, the cysteines seem to have important functions in the maturation of the receptor in vivo that go beyond the formation of disulfide bonds. Eventually, it is tempting to speculate that disulfide bond formation occurs as a late step in folding the ECD. Taken together, this study does not only provide experimental evidence for the formation of both the Cys-loop and the GlyR-specific disul-fide bond, but also offers a paradigm for the analysis of the folding mechanism of the ECD.