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Keywords:

  • agonist affinity;
  • β-amino acid agonist;
  • human glycine receptor α1 subunit;
  • substituted cysteine accessibility scan

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

The β-amino acid, taurine, is a full agonist of the human glycine receptor α1 subunit when recombinantly expressed in a mammalian (HEK293) cell line, but a partial agonist of the same receptor when expressed in Xenopus oocytes. Several residues in the Ala101–Thr112 domain have previously been identified as determinants of β-amino acid binding and gating mechanisms in Xenopus oocyte-expressed receptors. The present study used the substituted cysteine accessibility method to investigate the role of this domain in controlling taurine-specific binding and gating mechanisms of glycine receptors recombinantly expressed in mammalian cells. Asn102 and Glu103 are identified as taurine and glycine binding sites, whereas Ala101 is eliminated as a possible binding site. The N102C mutation also abolished the antagonistic actions of taurine, indicating that this site does not discriminate between the putative agonist- and antagonist-bound conformations of β-amino acids. The effects of mutations from Lys104–Thr112 indicate that the mechanism by which this domain controls β-amino acid-specific binding and gating processes differs substantially depending on whether the receptor is expressed in mammalian cells or Xenopus oocytes. Thr112 is the only domain element in mammalian cell-expressed GlyRs which was demonstrated to discriminate between glycine and taurine.

Abbreviations used
DTT

dithiothreitol

GlyR

glycine receptor chloride channel

MTS

methanethiosulfonate

MTSES

sulfonatoethyl methanethiosulfonate

MTSET

2-trimethylammoniumethyl methanethiosulfonate

nH

Hill coefficient

WT

wild type.

The glycine receptor chloride channel (GlyR) is a member of the ligand-gated ion channel family that mediates inhibitory neurotransmission in the central nervous system. Glycine receptor chloride channels in vivo are considered to form pentameric protein complexes, containing α and β subunits in the stoichiometry 3 : 2 (Langosch et al. 1988). The α subunit forms functional homo-oligomeric receptor channels upon heterologous expression in Xenopus oocytes or mammalian cell lines (for a review see Kuhse et al. 1995; Rajendra et al. 1997).

Following the terminology of Corringer et al. (2000), four discontinuous extracellular domains (A–D) have been proposed to form the GlyR agonist binding pocket. In loop A, Ile93, Ala101 and Asn102 were found to form agonist binding sites for taurine and glycine (Vafa et al. 1999). In loop B, residues Phe159, Gly160 and Tyr161 have been proposed to be important for agonist discrimination and antagonist binding (Vandenberg et al. 1992a; Schmieden et al. 1993). The loop C residues Lys200, Tyr202 and Thr204 are involved both agonist and antagonist binding (Vandenberg et al. 1992b; Rajendra et al. 1995a). Loop D, which includes the naturally occurring murine spasmodic mutation, A52S, may be a minor determinant of agonist binding (Ryan et al. 1994; Saul et al. 1994). Mutations in the M1-M2 and M2-M3 loops also have profound effects on the efficacies and apparent affinities of agonists (Langosch et al. 1994; Rajendra et al. 1994), but these are more likely to be mediated by disruptions to the channel gating mechanism (Rajendra et al. 1995b; Lynch et al. 1997, 2001; Lewis et al. 1998).

The β-amino acid, taurine, is widely used as a probe to investigate the ligand binding properties of GlyR. Taurine behaves as a full agonist of the human α1 homomeric GlyR when recombinantly expressed in mammalian cell lines (Rajendra et al. 1995b), but as a partial agonist of the same GlyR when recombinantly expressed in Xenopus oocytes (Schmieden et al. 1992). The origin of this differential effect is not known. Like all β-amino acids, taurine flickers spontaneously between cis- and trans-conformations. Schmieden and Betz 1995) have argued that its partial agonist action in the Xenopus oocyte-expressed GlyR results from it binding as an antagonist in the trans-conformation and as an agonist in the cis-conformation. Their model predicts a specific antagonist recognition site in a common ligand binding pocket that is accessible only by trans-isomers. By binding in the trans- conformation, β-sterically prevent glycine from binding in the pocket, or both. In a subsequent study employing Xenopus oocyte-expressed GlyRs, Schmieden et al. (1999) demonstrated that mutating either Lys104, Phe108 or Thr112 to alanines increased the relative magnitude of β-amino acid-gated currents while simultaneously causing modest decreases in their EC50 values. It was concluded that these mutations selectively reduced the apparent affinity of β-amino acids bound in the antagonist configuration, by disrupting either an antagonist binding site or an inhibitory allosteric mechanism.

This model raises the following questions regarding the role of the Ala101–Thr112 domain. Does this domain also control the binding and gating processes of β-amino acid agonists in the α1 GlyR subunit when expressed in mammalian cell lines? Does this domain contain a binding site that can discriminate between glycine and β-amino acid agonists? Does the Ala101–Thr112 domain undergo different conformational changes depending on whether glycine or taurine is bound to the receptor? The aim of the present study is to resolve these questions.

The substituted cysteine accessibility method can be used to identify residues that line the surface of a receptor protein in different functional states (Karlin and Akabas 1998). As cysteine-substituted mutations are well tolerated by proteins (Karlin and Akabas 1998), we first performed a serial cysteine mutagenesis of all residues in the Ala101–Thr112 domain and determined the effects of each mutation on the glycine and taurine EC50 values and their relative peak current magnitudes. We then assayed the surface accessibility of each introduced cysteine by comparing the reaction rates of both positively and negatively charged methanethiosulfonate (MTS) reagents in the unliganded state, in the glycine-bound state and in the taurine-bound state. Taken together, the results indicate that residues in this domain are involved in both binding and gating of taurine in GlyRs expressed in mammalian cell lines. However, the domain does not appear to function in the same way as it does when the same GlyR is expressed in Xenopus oocytes.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Mutagenesis and expression of human GlyR α1 subunit cDNAs

Site-directed mutations were incorporated into the human GlyR α1 subunit cDNA (Grenningloh et al. 1990) in the pCIS2 expression vector using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA, USA) and the successful incorporation of mutations was confirmed by sequencing the clones. The wild type (WT) and all mutant GlyRs investigated in this study also incorporated the C41A mutation. Plasmid vectors incorporating cDNAs for the WT and mutant GlyRs were transiently transfected into HEK293 cells (ATCC CRL 1573) using a modified calcium phosphate precipitation method. After 24 h incubation in a 37°C incubator under a 3% CO2 atmosphere, cells were washed with culture medium (Eagle's minimum essential medium supplemented with 2 mm glutamine and 10% fetal calf serum), and patch-clamp studies were conducted over the following 24–72 h.

Electrophysiology

Glycine- and taurine-gated currents were measured by whole-cell recording at a holding potential of −50 mV. Currents were recorded using an Axopatch 1D patch-clamp amplifier (Axon Instruments, Foster City, CA, USA). Data were filtered at 1 kHz and digitized directly onto an IBM Pentium PC. A Digidata 1200 interface controlled by PClamp6 software (Axon Instruments, Foster City, CA, USA) was used to control pipette potential and data acquisition parameters. Coverslips containing transfected cells were placed into a recording chamber with a volume of around 2 mL. Cells were continually perfused at ∼ 2 mL/min with the standard bathing solution containing (in mm): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.4. Patch pipettes were heat polished and had tip resistances of 2–4 MΩ when filled with the standard intracellular solution containing (in mm): 145 CsCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 10 EGTA, pH 7.4. Glycine and taurine were applied to cells by a manually controlled parallel tube perfusion system, which effected solution exchange with a time constant of ∼ 100 ms. Agonist applications were separated by > 1 min intervals to minimize the effects of receptor desensitization. At least 50% of maximum series resistance compensation was applied in all recordings. Experiments were performed at room temperature (18–23°C).

Methanethiosulfonate-ethyltrimethylammonium (MTSET) and methanethiosulfonate-ethylsulfonate (MTSES), obtained from Toronto Research Chemicals (Toronto, Canada), were prepared as stock solutions of 20 and 200 mm, respectively, in distilled water and maintained on ice for up to 3 h. These compounds were applied to the cells immediately after being dissolved into bathing solution at room temperature (18–23°C). The disulfide reducing agent, dithiothreitol (DTT), was prepared daily as a 1 mm solution in the standard bathing solution.

The protocol for the assessing the steady-state effects of MTSET and MTSES was as follows. First, the stability of glycine-gated currents was ascertained before addition of MTS reagents, using both saturating and EC50 agonist concentrations. The criterion for acceptable stability was that the peak currents at the tested concentration varied < 5% over a 3-min period. Then the glycine and taurine dose–responses were measured by applying increasing concentrations of either agonist at 1 min intervals. After application of the MTS-containing solution, cells were washed in control solution for at least 2 min before the maximum current magnitudes and agonist dose–response were measured again. In long-term patch recordings, both parameters were measured continually and were observed to remain constant in all mutant GlyRs for period as long as 40 min. This strongly suggests that irreversible changes in both parameters were caused by covalent modification of exposed cysteines. It is estimated that a 10% irreversible change in current over 1 min would have been reliably detected. The current amplitude changes by MTS reagents modification were calculated as (Iafter/Ibefore– 1) × 100%. If an irreversible effect was observed, the concentration of MTS reagents were adjusted so that the time constant of the current response was between 0.5 and 50 s. The receptor desensitization rate was < 0.005 s−1 for all WT and mutant GlyRs used in this study. Thus, desensitization did not impact significantly on the measurement of MTS reactivity rates.

The MTS modification rates were measured as follows. Each MTS compound was applied at a single concentration to each mutant GlyR. The concentrations of MTSET and MTSES applied to each mutant are given in Table 1. The reaction rates in the glycine- and taurine-bound states were determined at a constant agonist EC value (between EC20 and EC50) for each mutant GlyR. The glycine and taurine concentrations used at each mutant GlyR are also given in Table 1. Examples of the method of measuring the time course of the MTS reaction in both the unliganded and liganded states are shown in Fig. 2, below. The time course of the MTS reaction was fitted by a single exponential, yielding a time constant, τ. The MTS reaction rate was calculated as 1/(τ*[MTS]).

Table 1.  Concentrations of MTSET, MTSES, glycine and taurine used to investigate the reactivity of introduced cysteines
GlyR[MTSET] µm[MTSES] mm[glycine] µm[taurine] µm
  1. *200 and 400 µm were used for glycine- and taurine-gated currents, respectively.

WT200510100
A101C200, 400*510100
N102C4005200050 000
E103C10055003000
K104C2005520
G105C100520100
A106C200510200
H107C200510100
F108C20022050
H109C200520100
E110C200520100
I111C200520100
T112C20022050

The GlyR contains an uncrosslinked external cysteine at position 41, which could potentially complicate the interpretation of results obtained with the cysteine-modifying agents (Karlin and Akabas 1998). Hence, the WT and all mutant GlyRs examined in this study also incorporated the C41A mutation. The C41A mutation had no significant effect on GlyR agonist sensitivity, or on the peak magnitude of glycine-activated currents (Lynch et al. 2001).

Data analysis

The empirical Hill equation, fitted by a non-linear least squares algorithm (Origin 4.0, Microcal Software, MA, USA), was used to calculate the half-maximal effective agonist concentration (EC50) and Hill coefficient (nH) values. Exponential curve fits were performed by the same method. All results are expressed as mean ± SEM of n independent experiments. Statistical significance was determined by one-way anova using the Student-Newman-Keuls post hoc test for unpaired data (Sigmastat 3.0, Jandel, CA, USA), with p < 0.05 representing significance.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Glycine and taurine sensitivity of mutant GlyRs

The aim of this study is to investigate the differential involvement of the Ala101–Thr112 domain in contributing to the binding and signal transduction mechanisms of glycine and taurine. The known roles of residues in this domain are summarized in Table 2.

Table 2.  Known roles of residues in the Ala101 – Thr112 domain of the human GlyR α1 subunit
Residue RoleMutationGlyR subunit and expression systemRef.
101Aglycine and taurine agonist binding siteA101Hhuman GlyR α1, HEK293 cellsVafa et al. 1999
Nglycine and taurine agonist binding siteN102Ahuman GlyR α1, HEK293 cellsVafa et al. 1999
E    
Kdeterminant of taurine antagonist affinityK104Ahuman GlyR α1, Xenopus oocytesSchmieden et al. 1999
G    
A    
Hzinc inhibitory binding siteH107Ahuman GlyR α1, HEK293 cellsHarvey et al. 1999
Fdeterminant of taurine antagonist affinityF108Ahuman GlyR α1, Xenopus oocytesSchmieden et al. 1999
Hzinc inhibitory binding siteH109Ahuman GlyR α1, HEK293 cellsHarvey et al. 1999
E    
Ideterminant of taurine agonist efficacyI111Vhuman GlyR α1 & α2, Xenopus oocytesSchmieden et al. 1992
112Tdeterminant of taurine antagonist affinityT112Ahuman GlyR α1, Xenopus oocytesSchmieden et al. 1999
determinant of strychnine inhibitory potency  Vafa et al. 1999
determinant of zinc inhibitory potency  Laube et al. 2000
determinant of tropeine inhibitory potency  Maksay et al. 1999

The first step was to determine the glycine- and taurine-sensitivity of the cysteine-substituted mutant GlyRs. Sample glycine dose–responses for the WT and representative mutant GlyRs are displayed in the left panel of Fig. 1(a), and averaged glycine dose–response relationships for the same receptors are shown in Fig. 1(b). Similarly, sample taurine dose–responses are shown in the right panel of Fig. 1(a) and averaged taurine dose–response relationships are shown in Fig. 1(c). The mean saturating current magnitude (Imax) values, together with the mean EC50 and nH values for the WT and all mutant GlyRs examined in this study are summarized in Table 3. As shown in this table, the N102C, E103C and G105C mutations significantly increased the glycine EC50 value, whereas the K104C and F108C mutations significantly decreased the glycine EC50 value. The N102C and E103C mutations significantly reduced the peak glycine-activated current, whereas the I111C mutation significantly increased the peak glycine-activated current.

image

Figure 1. Activation of WT and mutant GlyRs by glycine and taurine. (a) The left side shows examples of glycine-activated currents in the WT, N102C, E103C and K104C mutant GlyRs. The right side shows examples of taurine-activated currents in the WT, N102C, E103C and K104C mutant GlyRs. All numbers represent agonist concentration in mM and all traces corresponding to a given GlyR were recorded from the same cell. (b) Examples of averaged glycine dose–responses for the WT (●), N102C (○), E103C (▵) and K104C (□) mutant GlyRs. Curves represent Hill equation fits through averaged points. In this and all subsequent figures, error bars represent the SEM and are shown when larger than symbol size. All n-values are given in Table 3. (c) Examples of averaged taurine dose–responses for the WT (●), N102C (○), E103C (▵) and K104C (□) mutant GlyRs. Curves represent Hill equation fits through averaged points. All n-values are given in Table 3.

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Table 3.  Functional properties of WT and mutant GlyRs
 Glycine activationTaurine activation
GlyREC50m)Hill coeff't (nH)Imax (nA)nEC50m)Hill coeff't (nH)Max Itau/Iglyan
  • *

    Significantly different with respect to WT GlyR values.

  • a

    a In determining this ratio, taurine- and glycine-gated currents were both measured in the same cell.

WT19.2 ± 3.01.88 ± 0.1013.0 ± 3.110120 ± 311.64 ± 0.131.03 ± 0.066
A101C19.3 ± 1.71.89 ± 0.1514.3 ± 3.11184.8 ± 8.8*1.48 ± 0.110.95 ± 0.0711
N102C2730 ± 310*1.15 ± 0.102.6 ± 0.2*1041500 ± 4800*2.44 ± 0.290.29 ± 0.02*12
E103C850 ± 110*1.33 ± 0.157.2 ± 1.7*114200 ± 670*1.10 ± 0.110.90 ± 0.078
K104C7.4 ± 1.0*2.32 ± 0.1712.6 ± 3.6537.2 ± 6.2*2.52 ± 0.340.95 ± 0.08*10
G105C53.4 ± 7.32.80 ± 0.2915.7 ± 2.49260 ± 8*1.42 ± 0.090.95 ± 0.057
A106C14.3 ± 1.41.77 ± 0.3810.5 ± 2.16193 ± 35.71.49 ± 0.190.92 ± 0.056
H107C12.8 ± 2.42.30 ± 0.3015.3 ± 2.9788.5 ± 12.32.15 ± 0.351.03 ± 0.076
F108C7.4 ± 1.0*2.23 ± 0.2216.4 ± 6.19157 ± 652.78 ± 0.371.15 ± 0.084
H109C17.8 ± 1.71.76 ± 0.2214.7 ± 2.211161 ± 291.64 ± 0.241.07 ± 0.036
E110C25.7 ± 4.21.68 ± 0.1821.6 ± 5.4783.9 ± 14.91.68 ± 0.181.02 ± 0.059
I111C21.2 ± 4.11.62 ± 0.2440.5 ± 9.4*878.0 ± 13.11.55 ± 0.221.02 ± 0.037
T112C20.6 ± 3.01.50 ± 0.2911.3 ± 2.0732.8 ± 5.1*1.78 ± 0.141.03 ± 0.067

The N102C mutation caused a greater proportionate increase (346-fold) in the taurine EC50 value than in the glycine EC50 value (142-fold; Fig. 1; Table 3). This mutation also strongly reduced the peak magnitude of taurine-gated currents relative to those activated by glycine in the same cell (Table 3). The E103C and G105C mutations also significantly increased the mean taurine EC50 value, whereas the K104C and T112C mutations significantly decreased the mean taurine EC50 value. With the exception of N102C, the peak current amplitudes of the taurine-gated currents were not significantly different to those activated by glycine in the same cells (Table 3).

As the N102A mutation has previously been shown to form part of the glycine and taurine binding sites (Vafa et al. 1999), it was expected that the N102C mutation would have a significant effect on the agonist EC50 values. However, in contrast to N102A which disrupted the glycine sensitivity to a greater extent than the taurine sensitivity (Vafa et al. 1999), N102C had a proportionately much greater disruptive effect on taurine sensitivity (Table 3). This observation enabled us to determine whether N102C selectively abolished taurine agonist activity or whether it abolished both its agonist and antagonist activities. A simple experiment designed to investigate this is shown in Fig. 2. All currents displayed in this figure were recorded from the same cell. Figure 2(a) shows examples of currents activated by glycine at both half-saturating (2 mm) and saturating (50 mm) concentrations, and a current activated by a saturating (150 mm) concentration of taurine. As shown in the example in Fig 2(b) and 150 mm taurine appeared to cause no inhibition of glycine-activated currents. Results pooled from four such experiments, summarized in Fig. 2(c), confirmed that N102C abolished the antagonistic actions of taurine. This provides strong evidence that Asn102 is a crucial element of the taurine binding site, irrespective of whether it binds as an agonist or as an antagonist.

image

Figure 2. Both the agonist and antagonist actions of taurine are disrupted by the N102C mutation. (a) Examples of currents activated in the same cell by a half-saturating (2 mm) glycine concentration, a saturating (50 mm) glycine concentration and a saturating (150 mm) taurine concentration. (b) The left and right panels show the effect of 150 mm taurine on currents activated by 2 mm glycine and 50 mm glycine, respectively. Currents displayed in this panel were recorded from the same cell as (a). (c) Currents activated by 2 mm and 50 mm glycine are normalised in the left and right panels, respectively. Results averaged from 4 cells indicate that 150 mm taurine has no significant effect on currents activated by 2 or 50 mm glycine.

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The effects of MTSET on mutant GlyRs

The substituted cysteine accessibility method (Karlin and Akabas 1998) was used to investigate the pattern of water accessibility of the introduced cysteines in the unbound state, in the glycine-bound state and in the taurine-bound state. It has previously been demonstrated that positively charged MTSET has no irreversible effect on the WT GlyR (Lynch et al. 2001). Examples of MTSET effects on glycine- and taurine-gated currents in the A101C mutant GlyR are shown in Fig. 3. To assess the MTSET reactivity in the unliganded state, cells were alternately exposed to an agonist-only solution and an MTSET-only solution (Fig. 3a). In the displayed experiment, glycine was applied at 10 µm (EC50) and MTSET was applied at 400 µm. Under these conditions, MTSET caused a strong inhibition of the glycine-gated current that did not reverse following a 2 min wash in control solution (Fig. 3a). This irreversible inhibition strongly suggests that MTSET was acting by covalent modification of A101C. The relationship between the current magnitude change and the cumulative exposure time to MTSET for this cell is shown in Fig. 3(b). In the displayed example, the reaction was well described by a single exponential with a time constant of 56 s, which corresponds to a rate constant of 44.6 m−1 s−1. The mean unliganded state MTSET rate constant averaged from several cells where glycine was used as a probe of the reactivity rate was 44 ± 9 m−1 s−1 (n = 4). The corresponding rate constant measured using a 100-µm (EC50) concentration of taurine as the reactivity rate probe was 56 ± 10 m−1 s−1 (n = 4). As expected, the values obtained with taurine and glycine were not significantly different. Thus, these agonists can be used interchangeably as probes for monitoring the MTS modification rate in the unliganded state. In the remainder of this study, unless otherwise indicated, the MTS modification rates in the unliganded state were measured using glycine as the reaction rate probe.

image

Figure 3. The effects of MTSET on the A101C mutant GlyR. (a) To assess MTSET reactivity in the closed state, the cell was rapidly switched between a solution that contained only 10 µm glycine and a solution that contained only 0.4 mm MTSET. The right panel shows examples of currents recorded following a 2-min wash in control solution to show that the effect was irreversible. (b) Measurement of reaction time constant for the cell in (a). The solid bars (▪) represent the proportionate decrease in current magnitude plotted against cumulative exposure time to MTSET in the unbound state. The final steady-state current amplitude has been normalized to 0 and the initial current magnitude normalized to 1. The line represents an exponential fit obtained using a non-linear least squares fitting routine with time constant of best fit as shown. (c) An example of the effect of 0.2 mm MTSET applied in the presence of 10 µm glycine. (d) Measurement of reaction time constant for the cell in (c). The current trace has been replotted from (c), with the final steady-state current amplitude normalized to 0 and the initial current magnitude normalized to 1. The line represents an exponential fit with time constant of best fit as shown. (e) An example of the effect of 0.4 mm MTSET applied in the presence of 100 µm taurine. (f) Measurement of reaction time constant for the cell in (e). The current trace has been replotted from (e), with the final steady-state current amplitude normalized to 0 and the initial current magnitude normalized to 1. The line represents an exponential fit with time constant of best fit as shown.

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Figure 3(c) displays an example of the effect of 200 µm MTSET applied in the presence of 10 µm glycine. Again, MTSET induced strong irreversible inhibition. The reaction for the cell displayed in Fig. 3(c) proceeded with a time constant of 26 s (Fig. 3d), which corresponds to a rate constant of 192 m−1s−1. The mean rate constant in the glycine-activated state was 206 ± 20 m−1 s−1 (n = 6), which was significantly faster than that measured in the unliganded state. An example of the effect of 400 µm MTSET on currents activated by a 100-µm (EC50) concentration of taurine is shown in Fig. 4(e). The reaction time constant was 52 s (Fig. 4f), which corresponds to a rate constant of 48.1 m−1 s−1. The mean rate constant in the taurine-bound state thus determined for several cells was 43 ± 11 m−1 s−1 (n = 4), which is significantly slower than that measured in the glycine-bound state, but not significantly different from that measured in the unliganded state. A partial reversible inhibition of taurine-gated currents was also caused by MTSET in the trace shown in Fig. 4(e). Similar effects were sporadically observed in a range of mutant GlyRs in response to either glycine or taurine activation. The origin of this effect was not investigated. However, as its onset and recovery were rapid, it did not affect the measurement of the slow, irreversible MTSET modification.

image

Figure 4.  Summary of the effects of MTSET in the WT and mutant GlyRs. (a) Steady-state effect of MTSET on current magnitude. In each case MTSET was applied alone, in the presence of glycine and in the presence of taurine, as indicated in the panel inset. MTSET was applied for a sufficient time for current changes to reach a steady-state value, or for a period of 1 min, which ever came first. The percentage current change was calculated as (Iafter/I,before– 1) × 100%. All points were averaged from at least 4 cells and asterisks represent significantly difference with respect to WT GlyR values. In no case was the current magnitude change significantly dependent on the receptor state of a given mutant GlyR. In all experiments, glycine and taurine concentrations were chosen to lie between the EC20 and EC50 values for each mutant GlyR. The MTSET, glycine and taurine concentrations used at each mutant GlyR are given in Table 1. (b) MTSET reaction rates in the unbound state (▵), glycine-bound state (●) and taurine-bound state (○) for those mutant GlyRs which responded to MTSET. In the A101C mutant GlyR, the reactivity rate was significantly faster in the glycine-bound state than in either the unbound or taurine-bound states.

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A similar experimental protocol was used to investigate all other cysteine-substituted mutant GlyRs and averaged results are presented in Fig. 4. The steady-state effects of MTSET on current magnitude in the unliganded, glycine-bound and taurine-bound states summarised in Fig. 4(a). The respective concentrations of MTSET, glycine and taurine used at each mutant GlyR are given in Table 1. As summarized in Fig. 4, MTSET induced irreversible inhibition in the A101C and E103C mutant GlyRs and irreversible potentiation in the G105C mutant GlyR, but had no effect on any other mutant GlyR. The steady-state effects of MTSET in the unliganded, glycine-bound and taurine-bound states were not significantly different for any mutant GlyR (Fig. 4a). It is important to note that glycine and taurine were applied at EC20–EC50 concentrations in these experiments. When saturating concentrations of these agonists were used, no effects of MTSET were observed, indicating that MTSET acts by modulating the GlyR agonist sensitivity. A 1-min application of 1 mm DTT reversed the effects of MTSET in the E103C and G105C mutant GlyRs, but had no effect in the A101C mutant GlyR (n ≥ 5 for each mutant GlyR).

The MTSET modification rate constants for the A101C, E103C and G105C mutant GlyRs measured in the unliganded, glycine-bound and taurine-bound states are summarized in Fig. 4(b). In the E103C and G105C mutant GlyRs, the MTSET modification rate exhibited no significant state-dependence. However, as outlined above, the A101C mutant GlyR reacted more rapidly with MTSET in the glycine-bound state, relative to both the taurine-bound state and the unliganded state.

The effects of MTSES on mutant GlyRs

To determine the relative influence of electrostatic interactions and surface accessibility changes on sulfhydryl reactivity, it is also necessary to investigate the reactivity of a negatively charged MTS derivative (Karlin and Akabas 1998). Thus, the effects of MTSES were also examined on the WT and all mutant GlyRs. MTSES has previously been shown to have no irreversible effect on the WT GlyR (Lynch et al. 2001). Examples of the effects of MTSES on the F108C mutant GlyR are shown in Fig. 5. As summarised in Figs 5(a and b), when 2 mm MTSES was applied in the unliganded state, with a 10-µm (EC50) concentration of glycine as the reaction rate probe, no MTSES-induced current magnitude change was observed. Similarly, no irreversible MTSES-induced current change was observed in the unliganded state when a 50-µm (EC30) concentration of taurine was used (Fig. 5c and d). However, when 2 mm MTSES was applied in the presence of 10 µm glycine or 50 µm taurine, a statistically significant irreversible reduction in current magnitude was observed (Fig. 6a). However, the magnitude of this reduction did not vary between the glycine-bound and taurine-bound states. An example of the effect of 2 mm MTSES applied in the presence of 10 µm glycine is shown in Fig. 5(e). In the displayed example, the reaction was characterized by a time constant of 14.4 s,  which corresponds to a rate constant of 34.7 m−1 s−1(Fig. 5f). The mean MTSES rate constant averaged from 7 cells in the glycine-bound state was 43 ± 7 m−1 s−1. In addition, an example of the effect of 2 mm MTSES in the taurine-bound state is shown in Fig. 6(g). The displayed experiment proceed with a time constant of 10.6 s, corresponding to a rate constant of 34.7 m−1 s−1(Fig. 5h). The mean MTSES rate constant in the taurine-bound state was 38 ± 8 m−1 s−1 (n = 6). There was no significant difference between the rate constants measured in the taurine- and glycine-bound states. However, as the unliganded state reaction rate was too low to be measured, it was obviously significantly different to those measured the agonist-bound states.

image

Figure 5.  The effects of MTSES on the F108C mutant GlyR. (a) To assess MTSES reactivity in the unbound state, the cell was rapidly switched between a solution that contained only 10 µm glycine and a solution that contained only 2 mm MTSES. The right panel shows examples of currents recorded following a 2-min wash in control solution to show that the effect was irreversible. (b) Measurement of reaction rate for the cell in (a). The filled squares represent the proportionate change in current magnitude plotted against cumulative exposure time to MTSES in the unbound state. The current amplitude has been normalized to the initial current amplitude. The line represents a linear fit to the data. (c) MTSES reactivity in the closed state using 50 µm taurine as probe. (d) Measurement of reaction rate for the cell in (c). Points are plotted as described in (b). (e) An example of the effect of 2 mm MTSES applied in the presence of 10 µm glycine. (f) Measurement of reaction time constant for the cell in (e). The current trace has been replotted from (e), with the final steady-state current amplitude normalized to 0 and the initial current magnitude normalized to 1. The line represents an exponential fit with time constant of best fit as shown. (g) An example of the effect of 2 mm MTSES applied in the presence of 50 µm taurine. (h) Measurement of reaction time constant for the cell in (g). The current trace has been replotted from (g), with the final steady-state current amplitude normalized to 0 and the initial current magnitude normalized to 1. The line represents an exponential fit with time constant of best fit as shown.

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image

Figure 6.  Summary of the effects of MTSES in the WT and mutant GlyRs. (a) Steady-state effect of MTSES on current magnitude. In each case MTSES was applied alone, in the presence of glycine and in the presence of taurine, as indicated in the panel inset. The percentage current change was calculated as (Iafter/I,before – 1) × 100%. All points were averaged from at least four cells and asterisks represent significant difference with respect to WT GlyR values. In the F108C mutant GlyR, the current magnitude change in the glycine- and taurine-bound states was significantly different to that measured in the closed state. In all experiments, glycine and taurine concentrations were chosen to lie between the EC20 and EC50 values for each mutant GlyR. The MTSES, glycine and taurine concentrations used in these experiments are given in Table 1. (b) MTSES reaction rates in the unbound state (▵), glycine-bound state (●) and taurine-bound state (○) for the mutant GlyRs which responded to MTSES. In the A101C mutant GlyR, the reactivity rate was significantly faster in the glycine-bound state than in the unbound state. In the F108C mutant GlyR, the reactivity rate was faster in the glycine- and taurine-bound states than in the unbound state. The unbound state reaction rate at F108C was immeasurably slow, but for display purposes is arbitrarily shown as being equal to 1.

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The steady-state effects of MTSES applied in the unliganded, taurine-bound and glycine-bound states for all mutant GlyRs are summarized in Fig. 6(a). The MTSES, glycine and taurine concentrations used in these experiments are summarised in Table 1. It is apparent from Fig. 6(a) that MTSES induced irreversible inhibition in the A101C, F108C and T112C mutant GlyRs. As discussed above, inhibition in the F108C occurred only in the open state. However, the magnitude of steady-state inhibition observed in the A101C and T112C mutant GlyRs was not significantly state-dependent (Fig. 6a). Again, glycine and taurine were applied at EC20– EC50 concentrations in these experiments. When saturating concentrations of these agonists were used, no effects of MTSES were observed, indicating that MTSES also acts by modulating the GlyR agonist sensitivity. A 2-min application of 1 mm DTT did not reverse the effects of MTSES at either the A101C, F108C or T112C mutant GlyRs (n = 7 for each mutant GlyR).

The MTSES modification rate constants for the A101C, F108C and T112C mutant GlyRs measured in the unliganded, glycine-bound and taurine-bound states are summarized in Fig. 6(b). As outlined above, the F108C mutant GlyR reacted more rapidly with MTSES in the agonist-bound states, relative to the unbound state. The A101C mutant GlyR reacted significantly faster in the glycine-bound state relative to the unliganded state. Although the mean reaction rate in the taurine-bound state was slightly faster than that of the unbound state, the difference did not reach statistical significance. The MTSES modification rate in the T112C mutant GlyR exhibited no significant state-dependence.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Interpretation of MTS modification results

The aim of this study was to investigate the differential involvement of the Ala101–Thr112 domain in contributing to the binding and signal transduction mechanisms of glycine and taurine. Conclusions are based partly on the reaction rates of charged MTS derivatives with cysteine-substituted mutant GlyRs. However, several limitations apply to the interpretation of such data. The first concerns the fact that some cysteine-substitution mutations resulted in large agonist sensitivity changes, implying that the mutations themselves may have induced local structural alterations. The implications of this for the interpretation of results are considered below for each affected mutant GlyR. A second limitation concerns the fact that each functional GlyR contains 5 introduced cysteines. This study has not attempted to ascertain how many cysteines need to be modified to give the entire effect. Thus, the true cysteine modification rates are not known, and hence, conclusions can only be drawn on the relative changes in the reaction rates, not on their absolute magnitudes. Finally, a lack of a measurable effect of an MTS reagent at a given substituted cysteine does not imply a lack of reactivity.

The reactivity of A101C with MTSET and MTSES indicates that Ala101 is accessible to the protein surface. The MTSET modification rate of A101C is significantly increased in the glycine-bound state relative to both the unbound and taurine-bound states (Fig. 4b). The MTSES modification rate is also significantly increased in the glycine-bound state relative to the unbound state (Fig. 6b). Therefore, electrostatic potential changes are eliminated as a major determinant of reactivity rate changes at this residue. Thus, the water accessibility of this residue is increased in the glycine-bound state. Possible causes of this effect are considered below. The reactivity rate of A101C with both MTSES and MTSET is lower than for any other residue measured in this study. In addition, DTT did not reverse the effects of MTSES or MTSET modification at A101C. This was most likely due to hindered DTT access to the disulfide bond. These observations suggest that Ala101 lies in a location of restricted surface access.

The MTS reactivity of the E103C, G105C, F108C and T112C mutant GlyRs indicates that these residues are also exposed at the protein aqueous interface. As none of these substituted cysteines responded to both MTSET and MTSES, it is not possible to draw definitive conclusions as to the roles of electrostatic forces and surface accessibility changes in contributing to differences in MTS reactivity rates. However, due to their close proximity, it is unlikely that the surface electrostatic potential differs significantly from Ala101 to Glu103. Therefore, as the MTSET modification rate of G103C is not state-dependent, it is unlikely that the surface accessibility of this residue is changed between the unbound, glycine-bound and taurine-bound states.

F108C is characterized by a drastically different MTSES reactivity rate in the unliganded state relative to the glycine- or taurine-bound states (Fig. 6b). A difference of this magnitude is unlikely to be solely the result of an electrostatic potential shift (cf. Pascual and Karlin 1998). Thus, the surface accessibility of Phe108 must change as the channel transitions from the closed to the open channel states, implying that either the residue itself or an overlying domain moves during channel activation.

A taurine binding site at Asn102

Asn102 has previously been identified as a glycine and taurine binding site, based on the effects of the N102A mutation (Vafa et al. 1999). The N102A mutation had a proportionately greater disruptive effect on glycine relative to taurine sensitivity. The present study demonstrates that N102C has a proportionately greater effect on taurine relative to glycine sensitivity (Fig. 1b). Indeed, as taurine-activated currents were very small, it was possible to demonstrate that the mutation disrupted both the agonist and antagonist effects of taurine (Fig. 2). This indicates that Asn102 forms a contact site for taurine regardless of whether it binds as an agonist or an antagonist.

Although A101H also disrupted agonist sensitivity (Vafa et al. 1999), the present study shows that the more conservative A101C mutation had no significant effect on the apparent affinities of glycine or taurine (Fig. 1b). Thus, Ala101 is not an agonist-binding site. As discussed above, the water accessibility of this residue is increased in the glycine-bound state, relative to the unbound and taurine-bound states. As the accessibility is not increased in the taurine-bound state, it is possible that this residue either undergoes a different allosteric conformational change following taurine binding or that the larger taurine molecule physically impedes the access of MTS agents to A101C. The later alternative is consistent with the earlier conclusion that Ala101 lies in a location of restricted surface access, possibly in an agonist binding cleft.

The E103C mutation also strongly reduced the apparent affinities of glycine and taurine (Fig. 1b). As the affinities of both agonists were disrupted to a similar extent, it is possible that Glu103 also contributes to a common glycine and taurine binding site. An alternate possibility is that the E103C mutation disrupts the adjacent binding site by a local structural disturbance. As no other mutations to residues in the Ala101–Thr112 domain resulted in large increases in the EC50 values for glycine or taurine, it is highly unlikely that any other domain residues form agonist-binding sites.

Control of taurine gating by the Lys104–Thr112 domain

When recombinantly expressed in mammalian cell lines, human homomeric α1 GlyRs exhibit a glycine EC50 of about 20 µm, a taurine EC50 of about 120 µm and taurine behaves a full agonist relative to glycine (e.g. Figure 1; Table 3). In contrast, when the same GlyR is recombinantly expressed in Xenopus oocytes, the glycine EC50 is increased to 200 µm, the taurine EC50 is around 2 mm and the ratio of the peak taurine- to glycine-gated currents is about 0.3 (Schmieden et al. 1995). Using the Xenopus oocyte expression system, Schmieden et al. (1995) found that β-amino acids, which rotate spontaneously between cis- and trans- isomers, act as partial agonists, and that molecules which are locked in a trans-like configuration act purely as antagonists. As glycine, which exists in the cis-conformation, acts as a full agonist, it was proposed that β-amino acid partial agonism results from the ligands binding as agonists in the cis- conformation and as antagonists in the trans- conformation. This model necessarily predicts the existence of a trans- conformation antagonist-binding site. Although this model reconciles many observations, it does not provide direct evidence for the existence of such a site.

In a subsequent study using the Xenopus oocyte expression system, Schmieden et al. (1999) demonstrated that the K104A, F108A or T112A mutations dramatically increased the magnitude of currents activated by β-amino acid agonists relative to glycine, while simultaneously causing modest reductions in β-amino acid EC50 values. On the basis of these results, it was proposed that the β-amino acid antagonistic potency was reduced by either a disruption to the β-amino acid inhibitory gating mechanism or a disruption to the putative β-amino acid antagonist-binding site.

The present study found that the K104C mutation caused modest (2–3 fold) increases in the apparent affinities of glycine and taurine (Table 3). These findings are in agreement with those of Schmieden et al. (1999). However, Schmieden et al. also found that the mutation increased the magnitude of taurine-gated currents relative to glycine-gated currents. Such an effect was not seen in the present study, presumably because taurine was already a full agonist of the WT GlyR. Hence, although no evidence for a β-amino acid-specific mechanism was found in the present study, the results do not necessarily refute the conclusion of Schmieden et al. (1999) that Lys104 is a candidate as a putative taurine antagonist-binding or gating element.

The present study also found that the F108C mutation has no effect on the affinity or relative peak current magnitude of taurine, although it did induce a significant (2.5-fold) increase in glycine sensitivity (Table 3). Hence, contrary to its effect on Xenopus oocyte-expressed GlyRs (Schmieden et al. 1999), this residue is unlikely to mediate β-amino acid-specific responses in mammalian cell-expressed GlyRs.

In an early study using Xenopus oocyte-expressed Schmieden et al. (1992) demonstrated that Ile111 is responsible for the difference in the ratio of taurine- to glycine-gated currents between the GlyR α1 and α2 homomers. As the present study indicates that I111C significantly increases both glycine- and taurine-gated current magnitudes, but does not affect the apparent affinity of either agonist (Table 3), Ile111 does not mediate β-amino acid-specific responses in the present system.

The T112C mutation induced a significant (four-fold) increase in taurine sensitivity, but had no effect on glycine sensitivity or on the relative magnitude of taurine- to glycine-gated currents (Table 3). This implies that Thr112 is a taurine-specific gating element. Contrary to the present study, Schmieden et al. (1999) found that the T112A mutation had little effect on the EC50 values of either agonist.

It is relevant to note that Thr112 is involved in mediating the inhibitory effects of other antagonists. Based on the effects of alanine substitution mutations, His107, His109 and Thr112 were identified as important determinants of zinc inhibition (Harvey et al. 1999; Laube et al. 2000). As a histidine-specific agent abolishes zinc inhibition, the zinc binding site probably formed by His107 and His109 (Harvey et al. 1999), with Thr112 more likely to be involved in the zinc inhibitory gating process. The T112A mutation also dramatically decreased the inhibitory potency of the competitive antagonist, strychnine (Schmieden et al. 1999) and induced relatively smaller decreases in the inhibitory potencies of the apparent competitive antagonists, nipecotic acid, isobutyric acid, tropisetron and atropine (Maksay et al. 1999; Schmieden et al. 1999). However, these studies have presented no evidence that Thr112 forms an antagonist-binding site. The results of the present and previous studies suggest that Thr112 may be a common mediator of inhibitory gating signals.

Conclusion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

In agreement with a previous report (Vafa et al. 1999), this study concludes that Asn102 forms an essential component of the taurine and glycine agonist-binding sites. As the N102C mutation also abolishes the antagonist effects of taurine, Asn102 is not responsible for discriminating between the putative agonist- (cis-) and antagonist- (trans-) bound conformations of β-amino acids. Contrary to a recent report (Vafa et al. 1999), Ala101 is unlikely to contribute directly to the agonist-binding process. Glu103 may be a minor determinant of agonist binding.

In agreement with Schmieden et al. (1999), Thr112 is likely to comprise a taurine-specific gating control element. Contrary to their roles in Xenopus oocyte-expressed GlyRs (Schmieden et al. 1992, 1999), Lys104, Phe108 and Ile111 do not mediate β-amino acid-specific responses in mammalian cell-expressed GlyRs. However, the mammalian cell expressed GlyRs may not be the ideal system to investigate this issue as taurine is a full agonist of the WT GlyR and it could be that taurine efficacy changes can only be observed in GlyRs where taurine behaves as a partial agonist. Evidence for a close involvement of this domain with receptor gating is provided by the observation that Phe108 undergoes a surface accessibility change between the closed and open channel states. Taken together, the results of this study indicate that the roles of Ala101–Thr112 domain residues differ substantially depending on whether the GlyR is expressed in mammalian cells or Xenopus oocytes. Thr112 is the only element of this domain in mammalian cell-expressed GlyRs which is crucial for discriminating between glycine and taurine.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

This research was supported by the Australian Research Council. NRH was the recipient of an International Postgraduate Research Studentship from the Australian Commonwealth Department of Education, Training and Youth Affairs.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References
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