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J. Biol. Chem., Vol. 279, Issue 19, 19559-19565, May 7, 2004
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**
From the
School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072 and ¶Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia
Received for publication, January 18, 2004 , and in revised form, February 19, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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1 GlyR. Here we compare the surface accessibility changes induced by the full agonist, glycine, and the partial agonist, taurine. In GlyRs incorporating the A272C, S273C, L274C, or P275C mutation, the reaction rate of the cysteine-specific compound, methanethiosulfonate ethyltrimethylammonium, depended on how strongly the receptors were activated but was agonist-independent. Reaction rates could not be compared in the R271C and K276C mutant GlyRs because methanethiosulfonate ethyltrimethylammonium did not modify the extremely small currents induced by saturating taurine or equivalent low glycine concentrations. The results indicate that bound taurine and glycine molecules impose identical conformational changes to the M2-M3 domain. We therefore conclude that the higher efficacy of glycine is due to an increased ability to stabilize a common activated configuration. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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-helical membrane-spanning domains. The second membrane-spanning (M2) domains curve radially so as to form a tapering, water-filled pore with a hydrophobic barrier (or channel gate) at either its mid-point (4) or intracellular boundary (5). The N-terminal domains contain the agonist-binding sites and a disulfide loop that is an invariant feature of LGIC receptors (6). Agonists binding in the N-terminal domain initiate conformational changes that propagate as a wave toward the channel gate (7). Different agonists induce these conformational changes with different efficacies (where efficacy is the ability of an agonist to open the channel once bound to the receptor). If the efficacy of an agonist is sufficiently low, it will behave as a partial agonist (8). The structural basis of differential agonist efficacy is not yet understood for any member of the LGIC family.
Partial agonism could be caused by one, or a combination, of the following two sharply contrasting mechanisms. First, it is possible that different agonists induce different structural changes throughout the protein. A clear example of this has been characterized recently (9–11) in the ionotropic glutamate receptor cation channel. These studies show that low efficacy agonists induce a smaller degree of binding site core closure than do high efficacy agonists, and show that this smaller degree of closure is translated to the activation gate as a smaller degree of channel opening (11). An alternative possibility is that high and low efficacy agonists induce identical global conformational changes but that the activated state is more stable for the more efficacious agonist.
Glycine and taurine activate the GlyR with different efficacies; taurine has a low efficacy and generally behaves as a partial agonist at this receptor, whereas glycine is a full agonist (12). The present study seeks to establish whether or not taurine and glycine induce identical conformational changes in a domain that links the ligand-binding site to the activation gate. This may provide insights into the structural basis of partial agonist action at the GlyR.
The substituted cysteine accessibility method can quantitate changes in protein conformation. The method entails introducing cysteine residues one at a time into a protein domain of interest. The reactivity of these cysteines is then assayed by exposure to highly soluble, sulfhydryl-specific reagents, generally methanethiosulfonate derivatives (13). If a functional property of the channel is irreversibly modified upon exposure to such a reagent, the cysteine is assumed to be exposed at the water-accessible protein surface. Differences in the cysteine modification rate between the closed and open states may provide information about the associated structural changes. Our laboratory previously used this approach to demonstrate a glycine-induced increase in the surface exposure of six continuous residues (Arg271–Lys276) in the GlyR M2-M3 linker domain (14). These residues lie mid-way between the binding site and the activation gate (7), and it is now well established that they experience a conformational change that is crucial for the activation of GlyRs (14–18), 
-aminobutyric acid, type A receptors (19–24), and nAChRs (7, 25–27). A structural study of the Torpedo nAChR has shown recently (4) that these residues form an extramembranous extension to the M2 -helix. This domain is likely to interact closely with loops 2 and 5 of the ligand-binding domain (4, 17, 18, 23, 24). A particular advantage of investigating this domain is that it lies outside the ligand-binding pocket (6), so changes in methanethiosulfonate reaction rates are unlikely to be influenced by steric effects of ligand binding. The aim of the present study is to employ substituted cysteine accessibility method to determine whether or not glycine and taurine induce identical changes in the surface accessibility of residues Arg271–Lys276.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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1 Subunit cDNAs—Site-directed mutations were incorporated into the human GlyR 1 subunit cDNA in the pCIS2 vector using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA). The successful incorporation of the mutations was verified by cDNA sequencing. The WT and all cysteine mutant GlyRs investigated in this study incorporated the C41A mutation, which eliminated the only free external sulfhydryl group. This mutation had no significant effect on GlyR EC50 values or peak current magnitudes (14). The WT and mutant plasmid constructs were transiently transfected into HEK293 cells using a modified calcium phosphate precipitation method. After transfection for 24 h, the cells were washed twice with cell culture medium, and electrophysiological studies were conducted over the following 24–72 h.
Patch Clamp Electrophysiology—Glycine- and taurine-gated currents were measured using whole-cell recording at a holding potential of –50 mV using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA), and data were directly recorded to disk using pCLAMP6 software (Axon Instruments). 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 megohms when filled with the standard intracellular solution containing (in mM): 145 CsCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 10 EGTA, pH 7.4. At least 50% of full series resistance compensation was applied in all recordings.
MTSET, obtained from Toronto Research Chemicals (Toronto, Canada), was prepared as a stock solution of 10 mM in distilled water and maintained on ice for up to 3 h until used. It was applied to cells within 30 s of being dissolved into room temperature bathing solution. The disulfide-reducing agent, dithiothreitol (DTT), was prepared daily as a 1 mM solution in the standard bathing solution. This DTT-containing solution had no irreversible effect on the magnitude of currents in the WT GlyR. Solutions were applied to cells via a parallel system of gravity-fed tubes, and solution exchange was effected with a time constant of about 100 ms. Experiments were performed at room temperature (19–22 °C).
The effects of MTSET on glycine- and taurine-gated currents were tested using the following procedure. Prior to MTSET application, cells were bathed in 1 mM DTT for 1 min to ensure that exposed sulfhydryl groups were fully reduced. Then the agonist dose response was measured by applying increasing agonist concentrations at 1-min intervals. The relative magnitude of currents activated by saturating concentrations of taurine and glycine was also measured. Following this, three consecutive brief applications of a constant glycine concentration were applied at 1-min intervals to establish that the current magnitude was invariant (±5%) prior to the application of MTSET. Following application of the MTSET-containing solution, cells were washed in control solution for at least 2 min before the maximum current magnitudes and taurine EC50 values were measured again. If an irreversible effect was observed, the MTSET concentration was adjusted so that the time constant of the current response was between 0.3 and 20 s. The receptor desensitization rate was low (<0.005 s–1) for all mutant GlyRs used in this study and as such did not impact significantly on the measurement of MTSET reactivity rates. It is estimated that a 10% irreversible change in current over 1 min would have been reliably detected.
Data Analysis—Results are expressed as means ± S.E. of four or more independent experiments. The empirical Hill equation, fitted by a non-linear least squares algorithm (Origin, Microcal, Northampton, MA), was used to calculate the 50% effective concentrations for activation (EC50) and inhibition (IC50) and the Hill coefficient (nH) values. Exponential fits were performed using the same non-linear least squares algorithm. Statistical significance was determined by one-way ANOVA using the Student-Newman-Keuls post hoc test for unpaired or paired data as appropriate (Sigmastat, Jandel, San Rafael, CA), with p < 0.05 representing significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The taurine agonist EC50 and nH values for the WT and all cysteine-substituted mutant GlyRs examined in this study are summarized in Table I. The taurine EC50 values for the A272C, S273C, L274C, and P275C mutant GlyRs are significantly increased relative to the WT value. Averaged glycine and taurine dose responses for the WT and L274C mutant GlyRs are shown in Fig. 1A. The peak magnitudes of taurine-gated currents were too small to permit reliable EC50 measurement in the R271C and K276C mutant GlyRs. The corresponding glycine EC50 values for the WT and all mutant GlyRs, reproduced from Lynch et al. (14), are included for comparison. The ratio of the maximum current activated by taurine relative to that activated by glycine (Imax, tau/Imax, gly) for each mutant GlyR is also shown (Table I). In calculating these ratios, the glycine- and taurine-gated currents were both recorded from the same cell. The cysteine substitution mutations invariably caused a significant reduction in this ratio. Thus, taurine behaves as a full agonist in the WT GlyR but as a partial agonist relative to glycine in all mutant receptors examined.
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Steady-state Effects of MTSET—It has been demonstrated previously that MTSET covalently modifies all introduced cysteines from R271C to K276C (14). This modification increased the glycine EC50 of the P275C GlyR but decreased the glycine EC50 of the remaining mutant GlyRs, all without affecting the saturating glycine-gated current magnitude (14).
Fig. 2A shows examples of currents activated by saturating concentrations of taurine and glycine in the R271C and L274C GlyRs both before and after a 1-min MTSET application. In these and all subsequent experiments, MTSET was applied at a concentration of 100 µM at the A272C, S273C, and L274C GlyRs and at 200 µM at the R271C, P275C, and K276C GlyRs. These concentrations were chosen to yield reaction time constants in an appropriate range (1–10 s) that was neither so rapid as to be influenced by the solution exchange rate nor so slow as to be influenced by receptor desensitization. In the experiments summarized in Fig. 2, MTSET was applied in the presence of a saturating (50 mM) concentration of taurine. MTSET had no significant effect on the magnitude of currents activated by a saturating glycine concentration in any of these mutant GlyRs, consistent with previous findings (14). However, MTSET modification increased the peak magnitude of taurine-gated currents in the L274C mutant GlyR but not in the R271C GlyR (Fig. 2A). The effects of MTSET on the ratio of saturating taurine- to glycine-gated currents (Imaxtau/Imaxgly) were investigated in a similar manner for all mutant GlyRs, and averaged results are summarized in Fig. 2B. MTSET significantly increased this ratio in the A272C, S273C, and L274C GlyRs, significantly decreased it in the P275C GlyR, and had no effect on Imaxtau/Imaxgly in the R271C and K276C GlyRs.
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MTSET Reactivity Rates with A272C, S273C, L274C, and P275C—Because differences in MTSET reaction rate can be indicative of differences in receptor conformation, we compared the MTSET reaction rates in the taurine- and glycine-bound states in the A272C, S273C, L274C, and P275C mutant GlyRs. It is important to note that the MTSET reaction rate in the closed state is approximately an order of magnitude slower than it is in the presence of EC30 glycine in all mutant GlyRs tested here (10). An example of an experiment on the L274C mutant GlyR is shown in Fig. 6A. Both panels in this figure were recorded from the same cell. In Fig. 6A, taurine was applied at a saturating concentration (50 mM), and MTSET was applied at 100 µM. The MTSET-induced current increase was adequately described by a single exponential with a time constant of 2.78 s. Currents recorded 2 min later confirm that covalent modification has taken place (Fig. 6A, upper panel). Following a 1-min exposure to 1 mM DTT, the experiment was repeated using a glycine concentration (100 µM) that activated a current of similar magnitude to that activated by saturating taurine (Fig. 6A, lower panel). MTSET modification of the glycine-gated current proceeded with a time constant of 2.66 s. The averaged time constants in the taurine- and glycine-activated states were 2.26 ± 0.34 s (n = 9 cells) and 2.06 ± 0.43 s (n = 7 cells), respectively. There was no significant difference between these values, indicating that the conformational change experienced by Cys-274 depends on the fraction of peak current activated, rather than the identity of the agonist.
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The reaction rates of 100 µM MTSET with A272C and P275C were measured at saturating (50 mM) taurine concentrations and glycine concentrations of 200 (EC10) and 100 µM (EC10), respectively. The averaged MTSET reaction rates in the taurine- and glycine-bound states for all mutant GlyRs examined in this study are also shown in Fig. 6B. Together, the results indicate that the rate of the MTSET reaction with A272C, S273C, L274C, and P275C does not depend on the identity of the agonist.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The structural basis of partial agonism in the ionotropic glutamate receptor has been investigated using a combination of crystallographic and electrophysiological techniques (11). This study revealed that a series of partial agonists promoted a range of conformational changes at the ligand-binding site, with the magnitude of this structural change being directly correlated with the probability of entering higher subconductance states. Thus, increasing agonist efficacy was manifested by an increased ability to open the channel to its maximal extent. This is a classic KNF-type mechanism.
Alternatively, partial agonism could also result from an MWC-type model whereby high and low efficacy agonists induce identical structural changes throughout the receptor. In such a mechanism, higher efficacy would be manifested by an increased ability to stabilize the open state. Because extended MWC models can explain many characteristics of LGIC behavior (34), this model is the favored hypothesis for the present study.
Taurine as a Low Efficacy Agonist—By using classical receptor theory (8), it can be shown that Fmax = E/(1 + E), where Fmax is the maximum fraction of receptors that can be activated by a saturating agonist concentration, and E is the equilibrium constant for gating (or efficacy). A combination of rapid agonist application techniques and equilibrium single channel kinetic analysis was used by Lewis et al. (12) to estimate E values of 16 and 3.4 for glycine and taurine, respectively, at the WT GlyR. According to these figures, taurine should activate around 77% of the peak glycine current in the WT GlyR, although we find taurine to be a full agonist of the WT GlyR. As summarized in Table I, all mutant GlyRs examined in this study significantly reduced this percentage. The above equation indicates that variations in E have no measurable effect on peak current magnitude unless they occur within a limited range of around 0.1 to 10. This means that a moderate reduction may not significantly reduce the glycine Fmax, whereas any reduction in the E would yield a reduction in the Fmax for taurine. Thus, although the mutations examined in the present study reduced the Fmax of taurine relative to glycine, they did not necessarily exert a selective effect on taurine efficacy.
The fact that mutations converted taurine into a partial agonist was convenient as it facilitated the comparison between the taurine- and glycine-activated states. For the purposes of analyzing the structural basis of partial agonism, it does not matter whether partial agonism was achieved by appropriate choice of agonist, receptor mutagenesis, or a combination of the two.
Prior to the present study little, if any, experimental evidence was available concerning the structural basis of partial agonism in pentameric LGICs. A variety of biochemical and electrophysiological approaches have shown that the agonist-binding pocket of LGIC receptors is formed by six discontinuous domains, labeled A–F by Corringer et al. (28). The involvement of these domains in forming the pocket has been confirmed by the crystal structure of acetyl/choline binding protein (6). In the GlyR, taurine and glycine both bind in this cavity but appear to interact differently with at least one of the molecular groups lining its wall (29–32). However, even if the respective agonists induce different local structural changes at the binding site, this does not necessarily imply that they induce different global conformational changes (8).
Similarly, single channel kinetic analysis has not yet permitted an unequivocal resolution of this issue. In the WT GlyR, the difference in E values between glycine and taurine was explained by a difference in the channel opening rates, with the closing rates being similar for both agonists (12). Although these results suggest that glycine and taurine differ in their abilities to stabilize the open state, they provide no information about the underlying structural changes. In addition, taurine and glycine have been shown to activate similar unitary conductance levels (12). Again, this does not necessarily imply that both agonists induce identical structural changes at the gate, because the conductance of the open pore may be limited by regions other than the gate (e.g. the M3-M4 domain (33)).
Comparison of MTSET Reaction Rates—MTSET has no effect on the magnitude of current activated by a saturating glycine concentration (14), indicating that it does not alter the GlyR single channel conductance or maximum open probability. In the A272C, S273C, and L274C GlyRs, MTSET increased Imaxtau/Imaxgly (Fig. 2B). It also reduced the EC50 values of glycine and taurine to a similar extent (Fig. 4B; see also Fig. 5 in Ref. 14). MTSET had exactly the opposite effects on the P275C GlyR. The reaction rates of MTSET with A272C, S273C, L274C, and P275C were not significantly different for saturating taurine-gated currents and partially activating glycine-gated currents, provided that the same current magnitude was activated in the same cell (Fig. 6B). To interpret this result, it is useful to consider the equation: I = n x i x Po, where I is the macroscopic current, n is the total number of channels per cell; i is the single channel conductance, and Po is the open probability. Because MTSET reaction rates were compared in the same cell, n was a controlled variable in our experiments. Furthermore, because the taurine and glycine single channel conductances are identical (e.g. Ref. 12), i is also constant. Indeed, it should be noted that full and partial agonists at all pentameric LGIC members examined to date activate identical unitary conductances. Thus, because the MTSET reaction rate is proportional to the initial value of I, it must in turn be proportional to Po. Therefore, the MTSET reaction rate simply reflects the fraction of channels that are open and not the number of occupied binding sites. This agrees with our recent observation that the MTSET reaction rate with S273C did not change significantly when the number of agonist-binding sites per GlyR was reduced from 5 to 3 (36).
Because the M2-M3 domains are positioned mid-way along the conformational "wave" that proceeds from the ligand-binding site to the channel gate (7), it must therefore be concluded that binding site information is integrated prior to reaching the M2-M3 domain. Hence, the ligand-induced inter-subunit cooperative interactions take place in the ligand-binding domain, in accordance with the MWC model of receptor activation (34).
Mechanism of Taurine Inhibition—The taurine IC50 values were not affected by MTSET in the R271C, P275C, or K276C mutant GlyRs (Table II). Table I shows that taurine IC50 values are about 10 times lower than their corresponding EC50 values in those mutants where both can be measured. The latter observation is consistent with a model where first taurine binds with a high affinity (equal to the IC50 value) but is unable to activate the channel. At least one additional taurine must bind to a separate low affinity site (equal to the EC50 value) in order to open the channel. Because the homomeric GlyR is likely to contain five identical taurine-binding sites (6), this situation is most likely caused by negative cooperativity among agonist-binding sites. It is likely that the cysteine substitutions create or enhance this negative cooperativity. A high affinity for taurine inhibition would result if a single taurine molecule can prevent the channel opening efficaciously when the other binding sites are occupied by glycine. We propose that MTSET does not affect the affinity of the first taurine-binding reaction but increases the either the affinity of subsequent agonist binding steps or the receptor gating efficacy.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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FOOTNOTES |
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Recipient of an International Postgraduate Studentship from the Australian Commonwealth Department of Education, Training, and Youth Affairs. Present address: Dept. of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260.
|| Supported by an Australian Research Council Senior Research Fellowship.
** To whom correspondence should be addressed: School of Biomedical Sciences, University of Queensland, Brisbane QLD 4072, Australia. Tel.: 617-3365-3157; Fax: 617-3365-1766; E-mail: j.lynch{at}uq.edu.au.
1 The abbreviations used are: GlyR, glycine receptor chloride channel; LGICs, ligand-gated ion channels; nAChR, nicotinic acetylcholine receptor; MTSET, methanethiosulfonate ethyltrimethylammonium; WT, wild type; ANOVA, analysis of variance; DTT, dithiothreitol; MWC, Monod-Wyman-Changeux; KNF, Koshland-Nemethy-Filmer.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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