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Departments of Physiology & Biophysics and Ophthalmology, School of Medicine, State University of New York at Buffalo, 124 Sherman Hall, Buffalo, NY 14214, USA
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Abstract |
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1, 2 or 2* glycine subunits were in vitro expressed in human embryonic kidney cells (HEK 293). In cells expressing the 1 subunit, responses to 200 µM glycine were blocked by 1 µM strychnine but not by 500 µM DCKA. In cells expressing the 2 subunit, both 1 µM strychnine and 500 µM DCKA were effective antagonists of 200 µM glycine. In cells expressing 2* subunits, which are much less glycine-sensitive, 10 mM glycine was inhibited by 500 µM DCKA but not by 1 µM strychnine. A single amino acid mutation in the 1 subunit (R196G), converted this subunit from DCKA-insensitive to DCKA-sensitive. In conclusion, the comparative effectiveness of strychnine and DCKA can be used to distinguish between the 1, 2 and 2* receptor responses. Furthermore, a single amino acid near the glycine receptor's putative agonist binding site may account for differences in DCKA sensitivity amongst the subunits.
(Received 7 October 2003;
accepted after revision 27 November 2003;
first published online 28 November 2003)
Corresponding author M. M. Slaughter: University at Buffalo School of Medicine, Department of Physiology & Biophysics, 124 Sherman Hall, 3435 Main Street, Buffalo, NY 14214, USA. Email: mslaught{at}buffalo.edu
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Introduction |
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The rodent spinal cord has been used as a model system for studies of the glycine receptor, leading to the conclusion that subunit composition is an important developmental switch. The receptor in the prenatal spinal cord is a pentamer of subunits, while the glycine receptor in the adult is composed of and ß subunits in a 3 : 2 stoichiometry. During development there is a switch from 2 subunit predominance in the fetal spinal cord to 1 in the adult (Becker et al. 1988; Langosch et al. 1988; Akagi et al. 1991, 1994; Takahashi et al. 1992). However, various glycine subunits have been shown to have uneven regional distributions in the adult central nervous system (Malosio et al. 1991; Betz, 1991). This implies that, in addition to their significance in development, the permutations permitted by the expression of multiple subunits can add dimensions to the information processing capacity of the adult nervous system. In this context, anatomical studies have demonstrated that 1, 2, and 3 as well as beta subunits are expressed in retinal ganglion cells of the adult rat (Greferath et al. 1994). Thus, glycine receptors on a single neurone might produce different responses based on their subunit composition.
This is intriguing because we recently observed that retinal ganglion cells manifest two kinetically distinct glycine currents that serve as low and high pass filters of information into retinal ganglion cells (Han et al. 1997). One glycine current is characterized by fast onset and desensitization and is blocked by nanomolar concentrations of strychnine. The other current has a slow onset and very slow desensitization. It is less sensitive to strychnine but is selectively inhibited by 5,7-dichlorokynurenic acid (DCKA).
These responses may relate to subunit composition. This possibility was explored by correlating the pharmacology of in vitro expressed glycine receptor subunits with the native glycine responses in retinal ganglion cells. Multiple GlyR alpha isoforms have been cloned from rat, mouse and human. Of particular interest is the unique 2* clone, isolated from newborn rat spinal cord (Kuhse et al. 1990), which has unusually low strychnine sensitivity. Since the DCKA-sensitive glycine current in ganglion cells was relatively strychnine-insensitive, we determined if DCKA and strychnine sensitivity could be related to subunit composition. We found that 1, 2, and 2* each has a unique profile of strychnine/DCKA sensitivity.
While the 2* isoform has low strychnine sensitivity, another neonatal isoform, 2 is very sensitive to strychnine inhibition (Akagi et al. 1991). These two isoforms differ in one key amino acid residue. Switching glutamate-167 residue in 2* to the corresponding glycine in 2 changes the subunit from strychnine-insensitive to strychnine-sensitive (Kuhse et al. 1990). In this paper, we report a correspondingly important amino acid that appears to account, at least in part, for the DCKA sensitivity of alpha subunits.
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Methods |
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The cDNAs encoding the 1 and 2 subunits of the glycine receptor were gifts from Dr Akagi of The Tokyo Metropolitan Institute of Medical Science. They were subcloned from pSPT 19 and pBluescript SK(–) vectors separately into pcDNA3 mammalian vector. The 2 cDNA was mutated at glycine-167 to replicate the strychnine insensitivity of the 2* subunit. This mutated subunit is referred to as 2* in Results. All mutations were made using a QuikChange site-directed mutagenesis kit (Stratagene, CA, USA). All mutations were sequence-confirmed before further experimentation.
Expression of glycine receptors in HEK 293 cells
One day before transfection, HEK 293 cells were plated out on glass coverslips in culture dishes. The culture medium was Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum. Plasmid DNA containing the cDNA encoding the glycine receptor subunits was added to subconfluent cell layers using the calcium phosphate transfection technique (Ausubel et al. 1992) or FuGENE-6 (Roche Inc). Plasmid pGREEN LANTERN-1 (Gibco, Grand Island, NY, USA), containing green fluorescent protein (GFP), was cotransfected as an expression marker. A total of 3.5 µg DNA, with the ratio 3 : 1 (GlyR : GFP), was used per 35 mm culture dish. The medium was changed about 20 h after the start of transfection. Cells were incubated for another 24 h before electrophysiological experiments. Plasmid pGREEN LANTERN-1 was used as a positive control for monitoring expression. The fluorescent cells were used to study the properties of GlyRs; non-fluorescing cells were used as a non-transfected control. Normal HEK 293 cells were unaffected by glycine. The cells with green fluorescence after transfection generated glycine responses (except for one mutant, as mentioned in Results).
Electrophysiological recordings
Patch clamp recordings were made 1–3 days after transfection. The whole-cell voltage clamp mode was used. During recording, HEK 293 cells were bathed at room temperature with Dulbecco's phosphate-buffered saline (PBS, Gibco) containing (mM): 137 NaCl, 2.7 KCl, 0.9 CaCl2, 0.5 MgCl2, 1.5 KH2PO4 and 6.6 Na2HPO4, pH 7.3. Glycine and DCKA (RBI, Natick, MA, USA) were dissolved in PBS and were applied with a local superfusion system (DAD-12, ALA Scientific Instrument, Long Island, NY, USA). The recording pipettes contained (mM): 140 potassium gluconate or KCl, 5.4 NaCl, 2.0 MgCl2, 1.0 CaCl2, 11 EGTA, buffered with 10 mM Hepes and adjusted to pH 7.4 with KOH. With this internal solution, electrodes had resistances of 5–10 M
. Data were recorded with an Axopatch-2B amplifier in combination with a Pentium computer and pCLAMP software (Axon Instruments, Union City, CA, USA). Cumulative data are expressed as means ±S.E.M. Two particular issues arose in electrophysiological recordings. One was that the currents were sometimes large, which could lead to voltage clamp errors due to currents through the access resistance. This was addressed by resistance compensation of up to 80% and by sometimes clamping cells at potentials to reduce the chloride driving force to 20 mV instead of 60 mV. Consequently, the estimated access resistance error did not exceed 10 mV and was generally much less. Secondly, there are a number of studies showing that the glycine EC50 measurements change with the amount of receptor expression. Legendre et al. (2002) has shown that the increased receptor expression results in faster desensitization, compressing the dose–response curve and making it appear that the EC50 is reduced. The experiments described in this paper were done in two stages, the first using calcium phosphate for transfection, the second using FuGENE 6 (Roche, Inc.). Experiments in the second stage exhibited larger whole-cell currents and lower EC50 values. When using the calcium phosphate transfection technique the glycine-induced currents were generally below 1 nA and rarely exceeded 2 nA in 1 or 2 homo-oligomeric receptors. But when using FuGENE-6 transfection the glycine-induced currents averaged slightly above 2 nA and sometimes approached 10 nA (with a 60 mV chloride driving force) in 1 or 2 homo-oligomeric receptors. This reflects the correlation between receptor expression and EC50 measurements. However, as seen in Results, there was not much variation in dose–response measurements within each transfection protocol. Qualitatively the results were the same with either protocol. But quantitative comparisons between different drugs were only made within the experimental group using the same transfection protocol.
Data analysis
The peak currents produced by different concentrations of glycine were plotted and fitted to the Hill equation:
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| (1) |
Dose–inhibition curves of DCKA were constructed for glycine (200 µM for 1 and 2; 10 mM for 2*)-activated currents obtained in the absence (I) and presence (IDCKA) of various concentrations of DCKA, and fitted with the equation:
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| (2) |
The inhibition produced by DCKA at two concentrations of glycine was fit to the equation of the Schild plot:
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| (3) |
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Results |
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The native glycine receptor is a pentamer composed of two homologous glycosylated membrane polypeptides, (48 kDa) and ß (58 kDa) (Betz et al. 1987), but the subunit alone can form a functional glycine receptor in vitro (Hoch et al. 1989). Changes in homomeric receptor complexes are more easily interpreted, especially in mutagenesis experiments. Thus homomeric expression was used in this study. The clones we were interested in were GlyR 1, 2 and 2* because of the variation in their sensitivity to the classic glycine receptor antagonist, strychnine (Becker et al. 1988; Kuhse et al. 1990). HEK 293 cells expressing the 1 or 2 subunit produced large currents in response to 100–200 µM glycine application, while HEK 293 cells expressing 2* subunits required 5–10 mM glycine and produced smaller whole-cell currents (Fig. 1). The glycine dose–response relationship was determined for cells expressing each of the subunits using the calcium phosphate transfection technique (see Methods). The EC50 at the 1 subunit was 108 ± 9 µM(n= 8), at the 2 receptor it was 143 ± 45 µM(n= 9), and at the 2* it was 4.8 ± 0.2 mM(n= 5) (Fig. 1). It was previously reported that the 2* is less sensitive to glycine than the other subunits (Kuhse et al. 1990). Because of this difference in agonist potency, 200 µM glycine was used to stimulate 1 and 2, while 10 mM glycine was used to activate the 2* clone. Based on the dose–response relationships shown in Fig. 1, these glycine concentrations were of similar potency, activating approximately 80% of the maximum current (vertical lines in Fig. 1). We confirmed that the glycine currents were due to chloride flux by stepping the cell to various potentials and then applying glycine. The response to glycine reversed close to the calculated chloride reversal potential in HEK 293 cells expressing the 1, 2 or 2* subunit. Untransfected cells did not produce a current in response to glycine.
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1 subunits have a strychnine IC50 of 16 nM in the presence of 290 µM glycine; 2 subunits have an IC50 of 18 nM against 310 µM glycine; 2* subunits have an IC50 of 18 µM against 12 mM glycine (Grenningloh et al. 1990; Kuhse et al. 1990). All glycine concentrations in these inhibition experiments were the EC50 values at the respective receptors. We tested the strychnine sensitivity in our expression system by comparing the response of transfected HEK 293 cells to glycine alone (Fig. 2, trace a in each panel) or after pretreatment and in the continued presence of 1 µM strychnine (trace b). In cells expressing 1 or 2 subunits, strychnine blocked the response to 200 µM glycine. But in cells expressing 2* subunits, the current produced by 10 mM glycine was only partially reduced.
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1, 2 and 2*
Since we found that those retinal glycine responses that were relatively insensitive to strychnine were selectively suppressed by DCKA, we tested the ability of DCKA to suppress glycine responses in HEK 293 cells expressing the 1, 2 or 2* homo-oligomers. Glycine alone elicited an outward current (Fig. 3, curve a in each panel). Then cells were pretreated with 500 µM DCKA and glycine was reapplied in the continued presence of DCKA. The glycine-induced current was blocked by DCKA in HEK 293 cells transfected with GlyR 2 or 2* but not cells transfected with GlyR 1. Figure 4 shows the inhibitory dose–response curves for DCKA inhibition of receptors expressed by each of the three subunits. These were obtained using the fixed concentrations of glycine (200 µM for 1 and 2; 10 mM for 2*) and increasing concentrations of DCKA. The DCKA IC50 value for 2 was 188 ± 13 µM(n= 6) and for 2* it was 243 ± 61 µM(n= 10). DCKA was an ineffective antagonist, at concentrations up to 1 mM, at 1 homo-oligomeric glycine receptors (n= 5). Comparing Figs 2–4 discloses that sensitivity to strychnine and DCKA can distinguish between the three alpha subunits. The 2 subunit is sensitive to both antagonists, the 1 is inhibited selectively by strychnine, and the 2* selectively by DCKA.
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1 and 2*
Sequence comparisons between GlyRs 1, 2 and 2* indicate that they are highly homologous. Each subunit is composed of a large extracellular glycosylated N-terminal domain, followed by four hydrophobic, putative transmembrane regions that form the ion channel. Site-directed mutagenesis has led to the suggestion that the ligand binding sites of the GlyR are composed of discontinuous extracellular regions of the N-terminal domain, involving three loops created by two disulphide bonds (Rajendra et al. 1995). When comparing these putative binding regions of the three glycine alpha subunits, there are pronounced differences at three residues: G160/A175/R196 in 1, the corresponding G167/P182/G203 in 2, and E167/P182/G203 in 2* (Fig. 5, the three sites enclosed in boxes). In the DCKA-insensitive 1 and the DCKA-sensitive 2 and 2*, each of these residues either incorporates or replaces a glycine or proline. Both of these amino acids are known to place kinks in an amino acid chain. Since DCKA is a much larger molecule than glycine, steric hindrance might be a determinant of DCKA action. If true, then a bend in the amino acid chain could be critical to DCKA antagonism. Therefore, the influence of each of the three amino acid residues on the receptor's sensitivity to DCKA was evaluated. In each case, the amino acid in one alpha subunit was compared to the homologous amino acid in another subunit.
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1, glycine-167 in 2, glutamate-167 in 2*) is critical for strychnine sensitivity (Kuhse et al. 1990) but does not appear to affect DCKA sensitivity. This conclusion is based on experiments indicating that 1 is not DCKA-sensitive and that 2 is, even though they both have a glycine at this site. Furthermore, 2* is DCKA-sensitive though it has a glutamate at this site.
The second site (alanine-175 in 1, proline-182 in 2 and 2*) had a slight effect on the efficacy of DCKA, but was not critical to its action. Thus, when the alanine in the 1 subunit was replaced with proline (A175P), these mutated 1 homo-oligomers were still insensitive to DCKA (Fig. 6, left panel) while the glycine EC50 was not significantly different from the wild-type 1 subunit receptor. Interestingly, the reverse mutation, in which the proline in 2* was replaced by alanine found at this position in 1 subunits, resulted in an 2*(P182A) GlyR with reduced DCKA sensitivity (Fig. 6, right panel). The DCKA IC50 shifted from 243 ± 61 µM(n= 10) for wild-type 2* to 724 ± 52 µM(n= 6) after the 2*(P182A) mutation. The glycine EC50 remained essentially unchanged. This indicates that this proline, although not a critical determinant of DCKA action, may nevertheless be a factor in the activity of this antagonist at the different alpha subunits.
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1 was converted to the glycine that is normally found at the corresponding location in 2 and 2*, then the 1(R196G) subunit became susceptible to DCKA inhibition (Fig. 6, left panel). The mutated 1(R196G) homo-oligomer was still activated by comparatively low concentrations of glycine and was blocked by strychnine, indicating that the mutation had a specific effect on DCKA activity.
Because this experiment indicated that glycine-196 could induce DCKA sensitivity in 1 subunits, we also performed the reverse experiment to determine if removing this glycine from 2* (at position 203) would remove DCKA inhibition. Unfortunately, when we replaced the glycine in the 2* with arginine normally found in 1(G203R), we were unable to observe a glycine response, even at a glycine concentration of 20 mM. This mutation either disrupted expression or the receptor was not functional.
DCKA is a competitive antagonist
Since DCKA inhibits glycine at the 1(R196G) GlyR, we compared the potency and mechanism of action at this mutated receptor with the 2 GlyR, which is also DCKA-sensitive. As opposed to the experiments described above which used calcium phosphate transfection, the following series of experiments were performed on HEK 293 cells transfected using FuGENE 6. This resulted in higher levels of receptor expression and lower values for glycine EC50 (see Methods). Under these conditions the glycine EC50 at the 2 GlyR was 62 ± 0.89 µM(n= 8). Pretreatment with 250 µM or 500 µM DCKA shifted the glycine dose–response relationship to the right (Fig. 7). The EC50 of glycine was 95 ± 5.86 µM in the presence of 250 µM DCKA (n= 5), and it was 128 ± 5.62 µM in the presence of 500 µM DCKA (n= 6). DCKA inhibition did not produce a statistically significant change in the Hill coefficient (1.49 ± 0.03 without DCKA, 1.37 ± 0.10 in 250 µM DCKA, and 1.43 ± 0.08 in 500 µM DCKA).
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1 GlyR (R196G) revealed that the glycine EC50 was 56 ± 2.14 µM(n= 8). This is slightly less than the EC50 for either wild-type 1 or 2 GlyR. Therefore, the DCKA inhibition at the mutated receptor was not due to a reduced responsiveness to glycine. DCKA still appeared to be a competitive antagonist. The EC50 was 73 ± 0.83 µM in the presence of 250 µM DCKA (n= 6) and was 90 ± 4.10 µM in the presence of 500 µM DCKA (n= 6). The Hill coefficient was not altered significantly (1.61 ± 0.07 in control, 1.72 ± 0.03 in 250 µM DCKA, and 1.72 ± 0.11 in 500 µM DCKA).
DCKA was a competitive antagonist at both the 2 GlyR and at the mutated 1(R196G). This suggests that the R196G mutation alters the 1 GlyR so that it behaves like the 2 GlyR. Schild plots of these two subunits gave slopes very close to 1, indicative of a competitive mechanism of inhibition. The plots indicated that the Kd of DCKA was 467 µM at the 2 GlyR and 802 µM at the 1(R196G) GlyR. Thus, DCKA is a weak competitive antagonist at a subset of glycine receptors.
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Discussion |
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There have been only a few efforts to distinguish subtypes of the native glycine receptor. One potentially valuable method is to define the pharmacology of glycine subunits and use this to probe native receptors. An example of this approach is the evaluation of cyanotriphenylborate (CTB), a glycine receptor antagonist that was found to block 1 homo-oligomers and 1/ß hetero-oligomers (IC50= 2–3 µM) but was less effective at 2 homo-oligomers (IC50 > 20 µM) and had intermediate effectiveness at 2/ß hetero-oligomers (IC50= 7.5 µM) (Rundstrom et al. 1994). Glycine receptors in rat retinal rod bipolar cells contain 1 subunits and CTB blocks their glycine responses (Enz & Bormann, 1995).
We followed this approach in reverse. DCKA selectively suppressed one type of glycine response in retinal ganglion cells. This response could be distinguished from a second glycine response based on its kinetics (Han et al. 1997), effects of phosphorylation (Han & Slaughter, 1998), and zinc inhibition (Han & Wu, 1999). All these factors suggest that DCKA is inhibiting one subtype of glycine receptor. The current study provides additional evidence that DCKA may distinguish a subtype of glycine receptor. The relative effectiveness of strychnine and DCKA can also be used to distinguish between 1, 2 and 2* subunits.
Glycine receptor structure
The binding pocket of the glycine receptor is thought to be formed by three loops made from the two disulphide bonds in the extracellular, N-terminal domain of the alpha subunit (Fig. 5). A discontinuous binding sequence formed by amino acids in the three loops may be a common property of binding pockets in this family of receptors, shared by the nicotinic acetylcholine and the GABAA receptors (Grenningloh et al. 1987; Deviller-Thiery et al. 1993; Rajendra et al. 1995). Two discontinuous regions in these loops on the 1 subunit are known to be involved in agonist and antagonist binding. The F-G-Y region in the second loop is essential for strychnine binding. For example, altering the glycine (G167) in 2 to a glutamate (E167), found in 2*, converts the receptor from strychnine- sensitive to -insensitive. The third loop region is also important for both strychnine and glycine binding. This loop is predicted to form a ß sheet with a ß turn produced by glycine (G205). Furthermore, the region around glycine-205 (T-G-X-F/Y) is conserved in the and ß subunits of the glycine receptor and in the 1, ß1, and 
subunits of the GABA receptor. In the glycine receptor, residues 200–204 form part of the putative ß sheet that may be terminated by a ß turn at the invariant residue glycine-205. Mutations of lysine-200, tyrosine-202 and threonine-204 reduced agonist binding, while lysine-200 and tyrosine-202 mutations eliminated strychnine binding. The opposite side of the putative ß-sheet does not appear to be involved in ligand binding (Vandenberg et al. 1992a, b; Rajendra et al. 1995). Here we show that a mutation of arginine-196 in 1 to glycine induced DCKA sensitivity in 1 homo-oligomer receptors. Arginine-196 is just outside the postulated third loop (near the putative glycine site) and this mutation would be predicted to cause a bend in the protein structure. The fact that 2 and 2* subunits contain a glycine at this position and are sensitive to DCKA, coupled with the observation that a single mutation to incorporate a glycine at this site makes the 1 subunit DCKA-sensitive, could indicate that a repositioning of the third loop is a determinant of DCKA inhibition. Unfortunately, the structure of the glycine receptor has not been determined so the link between mutational analysis and receptor binding sites is speculative.
The glycine binding site on the inhibitory and NMDA receptors
Glycine can bind to two different transmitter receptor proteins: the inhibitory glycine receptor and the NMDA subtype of the glutamate receptor. DCKA is a potent blocker of the glycine site of the NMDA receptor. Primary structure reveals little homology between the two proteins. The L-form of glycine analogues acts as an agonist at the inhibitory glycine receptor while D-amino acids such as D-serine are preferred agonists at the NMDA receptor. But there are suggestions that the pharmacophores of the two proteins share significant similarities. For example, Kuryatov et al. (1994) point out a F-x-Y motif is important for agonist and antagonist action near the putative glycine binding site in both receptors. Also, closely related quinolinic acid derivatives act as antagonists at the two receptors. Schmieden et al. (1996) suggest that both receptors form hydrogen bonds with the amino nitrogen moiety in position 1 and the hydroxyl group at position 4 of quinolinic acid and form charge–charge interactions with the carboxyl group of quinolinic acid. Quinolinic acid derivatives with a carboxyl group at the 3 position are selective antagonists to GlyR 1 and inactive at the NMDA receptor. DCKA, which is a quinolinic acid derivative with the carboxylic acid in the 2 position, does not affect the 1 receptor but blocks GlyR 2 and 2* (as well as the glycine binding site on the NMDA receptor). It may indicate that a key difference between the 1 and the 2 subunit is the position of the portion of the protein engaged in this charge–charge interaction with the carboxyl group of quinolinic acids.
Comparisons of glycine responses of retinal neurones and glycine subunits
Experiments on retinal ganglion cells have revealed two, temporally distinct glycine currents (Han et al. 1997). Synaptic glycinergic inputs to ganglion cells consist of a fast, strychnine-sensitive IPSP and a slow, relatively strychnine-insensitive IPSP. Exogenous glycine application elicited a current that could be separated into fast and slow components based on their selective inhibition by 1 µM strychnine and 500 µM DCKA. The present study demonstrates that there is a correlation between the pharmacology of the native glycine responses and that of individual glycine receptor subunits. The 1 subunit, like the fast native glycine current, is strychnine-sensitive and DCKA-insensitive. In contrast, like the slow native glycine current, the 2* subunit is less sensitive to strychnine and suppressed by DCKA. The 2 subunit is inhibited by both strychnine and DCKA. Thus, the relative sensitivity to strychnine and DCKA can be used to distinguish between three subunits of the glycine receptor as well as glycine currents in ganglion cells. DCKA may be a useful tool in analysing inhibitory glycinergic synapses.
But there are also many differences between the properties of each alpha subunit and that of the native retinal glycine receptors. The 2 subunit is inhibited by DCKA, but unlike the retinal current it is also sensitive to strychnine. The 2* is very insensitive to glycine, but the DCKA-sensitive current in ganglion cells has the same glycine affinity as the strychnine-sensitive glycine current (Han et al. 1997). Nor did the clones show the distinct differences in desensitization that were observed in native receptors. It is possible that combinations of alpha subunits or the addition of beta subunits are required to explain the properties of the native glycine receptors. Temporal differences may depend on the presence of beta subunits in the native glycine receptor, similar to the influence of 
2 subunits in the GABA receptor (Dominguez-Perrot et al. 1996).
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Footnotes |
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Akagi H, Majima T & Uchiyama M (1994). Function and modulation of the cloned glycine receptor channels expressed in Xenopus oocytes. Jap J Physiol 44, S91–S96.[Medline]
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Enz R & Bormann J (1995). Expression of glycine receptor subunits and gephyrin in single bipolar cells of the rat retina. Vis Neurosci 12, 501–507.[Medline]
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Han Y & Slaughter MM (1998). Protein kinases modulate two glycine currents in salamander retinal ganglion cells. J Physiol 508, 681–690.
Han Y & Wu SM (1999). Modulation of glycine receptors in retinal ganglion cells by zinc. Proc Natl Acad Sci U S A 96, 3234–3238.
Han Y, Zhang J & Slaughter MM (1997). Partition of transient and sustained inhibitory glycinergic input to retinal ganglion cells. J Neurosci 17, 3392–3400.
Hoch W, Betz H & Becker CM (1989). Primary cultures of mouse spinal cord express the neonatal isoform of the inhibitory glycine receptor. Neuron 3, 339–348.[CrossRef][Medline]
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Malosio ML, Marqueze-Pouey B, Kuhse J & Betz H (1991). Widespread expression of glycine receptor subunit mRNAs in the adult and developing rat brain. EMBO J 10, 2401–2409.[Medline]
Rajendra S, Vandenberg RJ, Pierce KD, Cunningham AM, French PW, Barry PH & Schofield PR (1995). The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element. EMBO J 14, 2987–2998.[Medline]
Rundstrom N, Schmieden V, Betz H, Bormann J & Langosch D (1994). Cyanotriphenylborate: subtype-specific blocker of glycine receptor chloride channels. Proc Natl Acad Sci U S A 91, 8950–8954.
Schmieden V, Jezequel S & Betz H (1996). Novel antagonists of the inhibitory glycine receptor derived from quinolinic acid compounds. Mol Pharmacol 50, 1200–1206.[Abstract]
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Vandenberg RJ, French CR, Barry PH, Shine J & Schofield PR (1992b). Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site. Proc Natl Acad Sci U S A 89, 1765–1769.
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Acknowledgements |
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Author's present address
Y. Han: The University of Texas-Houston, School of Medicine, Department of Ophthalmology & Visual Science, 6431 Fannin Street, MSB 7.024, Houston, TX 77030, USA.
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20 mV and 
) subunit or two mutations of this subunit (A175P (
) and R196G (
)). Mutation R196G in GlyR 