Extrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors

doi:10.1186/1471-2210-4-16

BMC Pharmacol. 2004; 4: 16.

Published online 2004 August 9. doi: 10.1186/1471-2210-4-16

PMCID: PMC514606

Extrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors

Jeffrey S Farroni^1,² and Brian A McCool^1,³

¹Department of Medical Pharmacology & Toxicology, Texas A&M University System Health Science Center, College Station TX 77843, USA

²Department of Marine Biology, Texas A&M University at Galveston, Galveston TX 77553, USA

³Department of Physiology & Pharmacology, Wake Forest University School of Medicine, Winston-Salem NC 27157, USA

Corresponding author.

Jeffrey S Farroni: farronij/at/tamug.edu; Brian A McCool: bmccool/at/wfubmc.edu

Received June 3, 2004; Accepted August 9, 2004.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

This article has been cited by other articles in PMC.

Abstract

Background

Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha₂+ beta heteromeric channels. This subunit composition is distinct from the alpha₁+ beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha₂beta receptors are poorly defined compared to 'neonatal' alpha₂homomeric channels or 'spinal' alpha₁beta heteromers. In addition, the pharmacologic properties of native alpha₂beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha₂beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels.

Results

While exploring pharmacological properties of alpha₂beta glycine receptors compared to alpha₂-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The β-amino acid taurine possessed 30–50% efficacy for alpha₂-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for β-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion.

Conclusions

Our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology.

Background

It has been well established that the amygdala is important in the acquisition and maintenance of fear/anxiety-related behaviors [1]. Strychnine-sensitive glycine receptors have recently been found in the adult rat basolateral amygdala (BLA) using whole cell and intracellular electrophysiology [2,3]. Reverse transcription polymerase chain reaction on whole BLA tissue and single cells revealed a prominent expression of α₂mRNA; and these receptors are likely to be α₂β heteromers due to their low picrotoxin sensitivity [4]. This finding is consistent with prominent BLA 'general' immunoreactivity for α/β subunit protein but no apparent α₁-specific protein expression [3]. A similar enrichment of α₂/β heteromers is also evident in striatal cholinergic interneurons [5]. It is quite possible then that the α₂β strychnine-sensitive glycine receptors present in the adult BLA and other forebrain areas represents a receptor population that could be functionally distinguished from those found in the spinal cord. Because the BLA regulates a number of anxiety- or fear-related behaviors [6], it is possible that this population of strychnine-sensitive glycine receptors may represent a novel therapeutic target for anxiety disorders. To insure that novel α₂β compounds possess an appropriate therapeutic index, the pharmacology of these forebrain glycine receptors must be elucidated and extensively compared with the spinal isoform.

There have been conflicting reports regarding the details of glycine receptor pharmacology when expressed in heterologous systems. For example, taurine acts as a partial agonists (ca. 50% efficacy compared to glycine) for GlyRα₁expressed in Xenopus ooctyes [7] whereas it shows nearly full agonist efficacy for GlyRα₁expressed in HEK 293 cells [8]. Compared to GlyRα₁, taurine efficacy is even weaker for GlyRα₂(ca. 5–10% efficacy) when expressed in Xenopus oocytes [7]. However, native GlyRα₂β receptors expressed by BLA neurons possess >50% efficacy for taurine and almost full efficacy for β-alanine [2]. While these results might initially be dismissed as expression system-dependent phenomena, brain region-specific effects are also evident in the literature. Taurine has markedly different efficacies at glycine receptors expressed by isolated adult lateral/basolateral amygdala neurons [2], adult hypothalamic magnocellular neurons [9], and juvenile spinal cord neurons [10]. It is therefore possible that the mechanisms regulating brain region-specific effects are related to those governing the divergence among heterologous expression systems. However, such mechanisms have not been systematically investigated, despite their potential usefulness in understanding region-to-region pharmacologic heterogeneity evident for some native receptors.

This study utilizes whole-cell patch clamp electrophysiology to examine the influence of distinct heterologous expression systems on the β-amino acid pharmacology of glycine receptors composed of distinct subunit combinations. We have focused on the α₂and α₂β receptors since these appear to be the predominate isoforms found in the embryonic and adult forebrain, respectively. Our results provide potentially important insight into the types of mechanisms that may govern brain region-to-brain region variation in glycine receptor pharmacology. Several aspects of this work have appeared in abstract form [11,12].

Results

Subunit- and system-dependent effects on glycine pharmacology

Given the variation of glycine receptor partial agonist pharmacology in the literature, we specifically sought to identify any role that expression system may play in their pharmacological profiles. First, glycine concentration-response relationships were established for GlyRα₂, and GluRα₂/β in HEK-293 cells and in L-cell fibroblasts. Glycine-gated responses for each receptor isoform were elicited in a dose-dependent manner in both cell types (Fig. (Fig.1A).1A). The apparent EC₅₀of glycine HEK cells was 221 μM and 269 μM for α₂(n = 4–6) and α₂β (n = 7–8), respectively. GlyR subunits expressed in L-cells displayed a similar pharmacological profile. However, the apparent glycine EC₅₀of both GlyRα₂(446 μM, n = 5–7) and GlyRα₂β (667 μM, n = 4–8) appeared lower than apparent affinities for the same subunits when expressed in L-cells. Two-way ANOVA on the Log (EC₅₀) values (Table (Table1)1) indicated a significant effect of system (F = 20.01, P < 0.001). However, the presence of the β-subunit did not significantly affect glycine apparent affinity in either system nor was there a significant interaction between system and subunit composition. These results indicate that glycine is less potent for receptors expressed in L cells compared to HEK cells.

Figure 1

Glycine receptors expressed show expression-system dependent agonist pharmacology. (A) Glycine has a reduced potency in L-cells compared to HEK cells. Glycine current responses were plotted versus the log concentration of glycine and normalized to a maximal (more ...)

Table 1

Agonist Pharmacology in HEK and L-cells.

Subunit- and system-dependent effects on β-Alanine pharmacology

With the glycine pharmacological profile established, we next examined the pharmacology of the partial agonists, β-alanine and taurine. The efficacy and potency of these β-amino acids were compared to glycine by normalizing the current response at each concentration to a maximal glycine response in that same cell. In HEK cells (Fig. (Fig.1B),1B), the average β-alanine EC₅₀values calculated from individual cells were 770 μM (n = 6) and 570 μM (n = 7) for α₂and α₂/β receptors, respectively. In L-cells, β-alanine also elicited currents in a dose-dependent manner. And, like glycine, β-alanine appeared to be less potent in these cells compared to HEK cells. EC₅₀values for α₂and α₂/β receptors were 2.0 mM (n = 8) and 2.9 mM (n = 7), respectively. Two-way ANOVA on LogEC₅₀values from these studies indicate a significant effect of the expression system on β-alanine potency (F = 43.52, P < 0.0001). There was a trend for the presence of the β-subunit to influence potency but this was not significant nor was there any significant interaction between expression system and subunit composition.

β-alanine efficacy was also examined in these same experiments by normalizing the maximal β-alanine response as a fraction of a maximal glycine response. In HEK cells, the α₂and α₂β isoforms had efficacies of 80 ± 6% and 55 ± 7% of the maximal glycine response, respectively. A similar trend was noted in L-cells with the α₂and α₂β isoform with β-alanine efficacies being 39 ± 8% and 25 ± 5% of the maximal glycine response. Two-way ANOVA analysis of these data indicate that both expression system and subunit composition had a significant influence on β-alanine efficacy (F = 27.6, P < 0.0001 and F = 7.9, P < 0.01 respectively). There was no significant interaction between these variables. These data demonstrate that the presence of the β subunit reduced β-alanine efficacy of α₂-containing receptors and that this efficacy was substantially smaller L-cells compared to HEK cells.

Subunit- and system-dependent effects on taurine pharmacology

Similar analysis of taurine pharmacology in HEK and L-cells revealed more dramatic effects of system and subunit on this partial agonist (Fig. (Fig.1C).1C). In HEK cells, the apparent EC₅₀for taurine was 501 μM for GlyRα₂(n = 9) and 2 mM for GlyRα₂β (n = 7). Because of its remarkably low efficacy in L-cells (see below), we can only provide estimates of taurine potency in this expression system. Regardless, apparent taurine affinity for both GlyRα₂and GlyRα₂β expressed in L cells were ~3 mM for both isoforms (n = 4 and 7, respectively). We did not compare L cell data with that obtained from HEK cells due to the uncertainty surrounding the fits. However, there was no significant difference in apparent taurine potency between the α₂and α₂β receptors expressed in HEK cells (P >> 0.05, t-test).

Taurine efficacy was obviously quite different between the two expression systems. In HEK cells, taurine efficacy was 48 ± 12% of glycine for GlyRα₂and 32 ± 4% of glycine for the GlyRα₂β isoform. Efficacy for these same receptors was reduced to approximately 6 ± 1% and 5 ± 0.7% of glycine when they were expressed in L-cells in these particular studies. The system difference was significant with two-way ANOVA (F = 17.4, P < 0.001) with no substantial effects of subunit composition or interactions between these variables.

Expression level and system-dependent pharmacology

The preceding results suggest that there may be a complex interaction between subunit composition and the expression system in which the receptor is produced. Specifically, the system-dependent agonist pharmacology could be related to differences in the relative expression levels between various systems. Expression level has clearly been demonstrated to influence agonist pharmacology for G protein-coupled receptors (e.g. [13]), where the levels of G-protein bound to receptor and thus the relative levels of high affinity receptor can vary from system to system. However, the influence of expression level on ligand-gated channel function has not been extensively explored (see Discussion). Unfortunately, it is problematic to compare expression levels between HEK and L-cells since the relative efficiency of transfection varied widely between these systems. Indeed, liposome-mediated transfection is remarkably efficient in HEK 293 cells (70–90% of cells based on GFP fluorescence) but only marginally effective in L-cells (10–20% of cells, not shown). To get around these differences in transfection efficiency, we examined the relative expression level of GlyRα₂protein using western analysis of total lysate derived from the same number of GFP⁺HEK 293 or L cells from transfected cultures (Fig. (Fig.2A).2A). For this experiment, cells were harvested under native conditions, GFP⁺cells were counted, and volumes of lysate corresponding to equivalent numbers of GFP⁺cells was loaded onto the gel. Western blots from two separate experiments demonstrate that transfected HEK 293 cells expressed 4- to 5-fold more GlyRα₂protein than transfected L-cells. The mean optical density from the two experiments was 83 ± 2 units for HEK cells and 17 ± 2 units for L-cells.

Figure 2

Relative expression levels does not influence taurine efficacy. (A) HEK and L-cells were co-transfected with the GlyRα₂subunit and GFP. Relative expression levels of α₂-protein were examined using western blot analysis of total lysate (more ...)

Maximal conductance is an independent measure of functional expression and was also larger for both α₂and α₂β receptors expressed in HEK cells compared to receptors expressed in L cells (Fig. (Fig.2B).2B). Across all experiments where maximal glycine concentrations were assayed, the conductance of α₂receptors expressed in L-cells was 65 ± 11 nS and was 114 ± 21 nS in HEK cells. Similarly, L-cells expressed α₂β receptors at 42 ± 9 nS while HEK cells expressed this isoform at 104 ± 17 nS. Two-way ANOVA using subunit and system as variables revealed a significant effect of system (F = 15.4, P < 0.001) but not subunit, nor was there a significant interaction between variables. Results from both westerns and functional experiments therefore indicate that relative expression levels of glycine receptor were different between HEK and L-cells.

To further explore the interaction between expression level and partial agonist efficacy, both current density and taurine efficacy were compared for α₂and α₂β glycine receptors in a number of different heterologous systems, as well as for native receptors expressed in rat lateral/basolateral amygdala. In addition to HEK and L-cells, the heterologous systems included mouse 3T3 fibroblasts and MDCK kidney cells. α₂β receptors expressed in mouse 3T3 fibroblasts had twice the current density (121 ± 34 pA/pF) of the mouse L-cells (59 ± 19 pA/pF) but had a similar taurine efficacy (13 ± 8% of glycine in 3T3 cells versus 8 ± 1% in L-cells). Similarly, α₂β receptors expressed in HEK293 cells had a current density similar to GlyRs expressed in 3T3 fibroblasts (115 ± 11 pA/pF) but had a taurine efficacy compared to glycine of 48 ± 3%. This efficacy was similar to glycine receptors expressed by acutely isolated adult rat basolateral amygdala neurons (46 ± 5% of glycine) although the current density in this native system was only 57 ± 14 pA/pF. Note that the channels expressed by these neurons are composed primarily of α₂+β subunits [4]. Canine kidney MDCK cells expressed the lowest α₂β current density (15 ± 5 pA/pF); yet the channels expressed by this system had the highest taurine efficacy of any cell tested (101+6%). For α₂GlyRs, the rank order of glycine receptor density was 3T3 (111 ± 21 pA/pF)> HEK cell (90 ± 11 pA/pF)> L-cell (50 ± 9 pA/pF); while the rank order of taurine efficacy for these same receptors was HEK (74 ± 9%)> 3T3 (23+7%)> L-cells (11 ± 2%). Across all subunit combinations and systems, there was no significant correlation (R²= 0.14, P >> 0.05) between taurine efficacy and glycine current density (Fig. 5C). Indeed, no correlation between expression level and taurine efficacy was evident within any given population of cells whether the receptors were expressed in native or heterologous systems. For example, the correlation coefficients for α₂β receptors between taurine efficacy and glycine current density in individual systems were 0.11, 0.13, 0.19, 0.05, and 0.31 for HEK, L-cells, 3T3 cells, MDCK cells, and amygdala neurons, respectively (P >> 0.05). Thus, while there is clearly a difference in expression level between both the systems as well as between individual cells in a given system, this particular characteristic cannot account for the apparent taurine efficacy.

The agonist-binding site is not affected by expression system

There are a variety of possible mechanisms to account for the disparities in partial agonist pharmacology between two expression systems. One way to address this is to examine competitive antagonist binding properties in HEK and L-cells. We therefore examined the potency of the glycine receptor competitive antagonist strychnine in both systems. Following a 30 second pretreatment with the antagonist [2], we co-applied strychnine and an EC₅₀concentration of glycine. The strychnine K_Bwas estimated for HEK and L-cells expressing either the GlyRα₂or GlyRα₂+β subunits using the Cheng-Prusoff relationship (see Methods). This relationship takes into account the divergent Hill-slope and potencies for glycine found in these two expression systems. Receptors composed of the GlyRα₂subunit (Fig. (Fig.4A)4A) had very similar K_Bvalues when expressed in either HEK (K_B= 49 ± 8 nM, n = 11) or L-cells (K_B= 38 ± 7 nM, n = 16; Fig. Fig.4C).4C). The same was true for cells expressing the GlyRα₂β subunits where strychnine apparent affinity was 32 ± 7 nM (n = 10) in HEK cells and 38 ± 8 nM (n = 10) in L-cells. Two-way ANOVA did not reveal any significant effect of either system or subunit composition. Since strychnine is a competitive antagonist and site-directed mutation studies suggests that strychnine and glycine interact with overlapping regions of the receptor [14-16], our results strongly suggest that functional strychnine affinity, and hence the general structure of the agonist binding pocket, was not substantially influence by expression system.

Figure 4

The association of glycine receptors with the tubulin-cytoskeleton may influence partial agonist efficacy. (A) Tubulin depolymerization with colchicine decreased both taurine and β-alanine efficacy of α₂β glycine receptors expressed (more ...)

Cytoskeletal components influence glycine receptor pharmacology

A third possible mechanism for reduced efficacy in L-cells compared to HEK cells or neurons could be related to intracellular factors that influence channel gating [17]. This hypothesis was examined by disrupting the cytoskeletal protein tubulin, which has been shown to be important for glycine receptor localization [18]. Direct application of 100 μM colchicine did not elicit any membrane currents. Furthermore, acute application of 100 μM colchicine and an EC₅₀concentration of glycine (300 μM) did not significantly affect glycine-gated currents themselves. Glycine currents were 17.3 ± 2.3 pA/pF while glycine+colchicine currents were 16.7 ± 2.2 pA/pF (p > 0.5, paired two-tail t-test, n = 7).

The relative efficacy of β-alanine and taurine was examined in HEK cells expressing α₂β subunits following 30 min incubation with 100 μM colchicine at 37°C, enough time to allow irreversible tubulin disruption [19]. As an additional control, γ-lumicolchicine, an inactive analog of colchicine [20], was also used to treat α₂β-expressing HEK cells (Fig (Fig4A).4A). These brief treatments had no obvious effect on the survival of untransfected cells. There was a trend for colchicine treatment to reduce the overall current density at 300 μM glycine, 56.7 ± 9.1 pA/pF in control cells (n = 8), 41.2 ± 2.3 pA/pF in colchicine-treated cells (n = 10), and 50.1 ± 9.9 pA/pF (n = 6) in γ-lumicolchicine-treated cells; however, this was not significant (p > 0.05, ANOVA) and was probably not related to any direct action of colchicine given that the glycine current density was also slightly reduced in α₂β-expressing cells exposed to γ-lumicolchicine compared to controls. However, the efficacy of both taurine (p < 0.01, One-way ANOVA) and β-alanine (p < 0.05, ANOVA) were significantly decreased by colchicine but not γ-lumicolchicine treatment. Taurine efficacy was 33 ± 6% of glycine in controls, 13 ± 3% following colchicine, and 28 ± 3% following γ-lumicolchicine. Similarly, β-alanine efficacy was 70 ± 7% of glycine in controls, 49 ± 6% following colchicine, and 72 ± 7% following γ-lumicolchicine. Similar treatment of α₂-expressing HEK cells with colchicine (Fig. (Fig.4B)4B) did not reveal any significant effect on glycine current density (54 ± 14 pA/pF in controls, 60 ± 15 pA/pF in treated), on taurine efficacy (34 ± 16% in controls vs. 38 ± 10% in treated), or on β-alanine efficacy (71 ± 12% in controls vs. 86 ± 8% in treated). Cholchicine treatment also significantly reduced β-alanine efficacy in L-cells expressing GlyRα₂β (23 ± 2% in controls vs. 12 ± 3% in treated, P < 0.05, t-test) but not in GlyRα₂-expressing L-cells (Fig. (Fig.4C).4C). We did not attempt to examine taurine in L-cells treated with colchicine given the exceptionally low efficacy of receptors expressed in this cell line.

Because the glycine receptor- and tubulin-binding protein gephyrin provides an obvious link between the receptor and the tubulin cytoskeleton, we used western analysis of HEK and L-cell lysates with a gephyrin monoclonal antibody specific for the C-terminus. These experiments revealed that gephyrin-like immunoreactivity was expressed in both expression systems (Fig. (Fig.4D).4D). Notably, a ca. 100 kD band dominated the HEK cell gephyrin immunoreactivity, while multiple bands of varying intensity could be seen in lysate from L-cells. When taken with our colchicine data, differences in glycine receptor pharmacology between α₂β receptors expressed in HEK and L-cells may be partially due to distinct, system-dependent interactions with distinct isoforms of the cytoskeletal protein gephyrin.

Discussion

We have expressed several the 'embryonic' (α₂homomeric) and 'forebrain' (α₂β heteromeric) isoforms in two distinct expression systems to understand the influence of endogenous and exogenous factors on receptor partial agonist pharmacology. Although the pharmacology of the 'embryonic' GlyRα₂isoform and the 'adult spinal' isoform (GlyRα₁β) have been explored more frequently in the literature, the pharmacology of GlyRα₂β receptors has remained largely unexplored. Despite this, there is strong evidence that the adult 'forebrain' isoforms, specifically in the rat basolateral amygdala, is indeed α₂β [4]. The current study indicates a general trend for decreased apparent affinity and reduced relative efficacy of agonists when receptors consist of the α₂β subunits compared to their homomeric α₂counter parts.

We were particularly surprised to find that receptors expressed in different expression systems possessed markedly different partial agonist efficacies. The remainder of our study focused on identifying extrinsic factors that influence difference in ligand-gated receptor pharmacology in distinct expression systems. While differences in efficiency of cDNA expression/transfection between systems could explain such differences, the expression levels of GlyRα₂β receptors measured by current density was not correlated with taurine efficacy across several different cell types or within any given system. Importantly, the efficacy of β-alanine and taurine in HEK cells agree with previous findings where cells expressing GlyRα₂show almost full efficacy for taurine and β-alanine [21]. Similarly, distinct ligand binding characteristics of receptors expressed in different expression systems seemed to be another possible mechanism governing agonist efficacy or potency. For the glycine receptor, the binding site for the competitive antagonist strychnine is believed to be adjacent to the agonist-binding site, sterically hindering agonist binding. A gross alteration in the agonist binding pocket, particularly one that hindered agonist binding, would most likely affect strychnine binding as well. To examine this, strychnine K_Bwas calculated for GlyRα₂and GlyRα₂β isoforms expressed in both HEK and L-cells. In order to decrease the error in estimating K_B, a derivation of the Cheng-Prusoff equation was used that takes in account variations in the slopes of the inhibition curves [22]. Differences in K_Bwere negligible between expression systems, indicating the strychnine binding site, and presumably the agonist binding site, was altogether similar in these different systems. Differences in pharmacology between systems therefore cannot be explained by substantial alterations in the agonist/competitive antagonist binding pocket.

Receptor gating is another mechanism by which receptor function may be altered. Cytoskeletal elements have been shown to play a crucial role in neurotransmitter receptor clustering [17] and may have a role in receptor function as well. For example, cytoskeletal stabilization has been shown to reduce Ca⁺⁺-dependent inactivation of Ca⁺⁺channels in snail ganglia [23]; and, actin has been shown to modulate several different types of membrane ion channel [24-26]. Cytoskeletal depolymerization has also been found to inhibit the function of GABA_Areceptors, which share significant sequence homology and functional characteristics with strychnine-sensitive glycine receptors [27]. And the tubulin-gephyrin-glycine receptor interaction is critical for establishing functional glycinergic synapses [28]. The current study suggests that cytoskeletal elements may play a functional role in α₂β glycine receptor pharmacology as well. β-containing glycine receptors are intimately associated with the tubulin-associated protein gephyrin [29] via the gephyrin binding site that lies within the intracellular domain of this subunit [30]. In our studies, the efficacy of both taurine and β-alanine were reduced in cells expressing GlyRα₂β subunits. This despite the finding that gephyrin-like immunoreactivity in L-cells was apparently distinct from that in HEK cells, suggesting that distinct cytoskeletal components in these systems may have profound influence over GlyR α₂β pharmacology. This is further supported by suggestions that gephyrin may exist in multiple, tissue-specific isoforms with potentially distinct functional roles [31-33]. It should be noted however that colchicine treatment of α₂β-expressing HEK cells did not suppress partial agonist efficacy to a level that approached that found in L-cells or 3T3-fibroblasts. Given that colchicine had no perceptible effect of α₂-homomeric channels expressed in HEK cells, our results suggest that additional system-dependent factors may have a more pronounced influence on the partial agonist pharmacology of strychnine-sensitive glycine receptors.

Conclusions

It is of particular interest that the β subunit appears to play a functional role in the pharmacology of α₂-containing receptors regardless of expression system. For example, the beta subunit decreased the apparent efficacy of the partial agonists. Since the β-subunit itself does not appreciably interact with the competitive antagonist strychnine [34], these results are at least consistent with some allosteric interaction between the β-subunit and the agonist binding site present the α subunit. This may indicate that α₂β glycine receptors in the forebrain may be distinguishable from other receptor isoforms given the appropriate pharmacologic agent. Although cytoskeletal components potentially play some role for these 'forebrain' receptors, there appear to be other 'extrinsic' factors governing expression system-dependent effects on agonist pharmacology. Since it is conceivable that such factors may be differentially distributed between different forebrain regions, the large apparent differences between glycine receptor pharmacology reported by various studies may not necessarily depend upon differential expression of glycine receptor subunits per se. At the very least, our findings suggest that great care should be taken when utilizing different expression systems to develop screens for novel pharmacophores acting on this receptor.

Methods

Cell culture and transfection

HEK 293 (TSA 201; gift from Michael J. Davis, Dept. Med. Physiol., Texas A&M Univ. Health Science Center, College Station, USA), mouse L-cells (NCTC-929; American Type Culture Collection), NIH/3T3 fibroblasts (ATCC), and MDCK cells (gift from Alan Parrish, Dept. Med. Pharmacol. & Toxicol., Texas A&M Univ. Health Sci. Center) were grown in Dulbecco's modified Eagle's medium (DMEM, SIGMA) with 10% fetal bovine serum (HyClone Laboratories, Logan, UT, USA) and 1X penicillin/streptomycin (Life Technologies) on Thermanox cover slips in 35 mm culture dishes. Cells were transfected during log-phase growth (30–50% confluent) using the Superfect reagent (Qiagen, Valencia, CA, USA), according to manufacturers instructions. Rat glycine receptor α₂and β subunits were cloned previously [4]. Briefly, a total of 2–3 μg of plasmid constructs was added to 100 μL serum-free media along with 10 μL Superfect reagent, vortexed for several seconds, and incubated for 10 minutes to allow DNA/liposome formation. Standard media (600 μL) was added and this mixture applied the cells and incubated for 2–3 hours at 37°C. The cells were subsequently washed twice with phosphate-buffered saline, fresh media was applied and cells were then incubated for 24–48 hours prior to recording. Cells were co-transfected with green fluorescent protein (pEGFP-C1, Clontech, Pal Alto, CA, USA) to identify transfected cells before recording. Mass ratios of 1:1:5 were used in the GFP:α₂:β transfections. An equal mass of the cloning vector pCI (Promega Corp) replaced the β subunit when examining α₂homomeric channels.

Electrophysiology

Whole cell recordings were performed at room temperature using standard patch-clamp techniques and the axopatch-1D amplifier (Axon Instruments, Inc., Foster City CA, USA) in the voltage clamp mode. Gigaohm seals were formed using patch pipettes made from borosilicate glass (World Precision Instruments, Sarasota FL, USA). For most experiments, the internal solution contained (in mM): CsCl 100, EGTA 11, HEPES 10, CaCl₂1, Mg-ATP 4, pH 7.2 with methane sulfonic acid; adjusted to 290–295 mmol kg^-1with sucrose. Whole cell capacitance and series resistance was manually compensated after opening the cell. Cells were continuously bath perfused with a HEPES-buffered saline (in mM): NaCl 150, Glucose 10, HEPES 10, KCl 2.5, CaCl₂2.5 MgCl₂1.0, pH 7.4, 305–320 mmol kg^-1Data will be analyzed off-line using pClamp software (Axon). Numerical analysis was performed using commercially available software. Independent student's t-test and two-way ANOVA were used for comparisons where appropriate; and statistical significance was based on p < 0.05. Concentration-response curves were generated from fits of data to a standard logistic equation as previously described (McCool & Botting, 2000). To derive K_Bfrom functional IC₅₀and EC₅₀data, the Cheng-Prusoff equation was used:

An external file that holds a picture, illustration, etc.
Object name is 1471-2210-4-16-i1.gif

where S = hillslope of agonist curve, A = concentration of agonist, EC₅₀= half-maximal agonist concentration, and IC₅₀= half-maximal antagonist concentration.

Drugs

Stocks of glycine, taurine, β-alanine, strychnine (Tocris) and colchicine (SIGMA) were prepared fresh each day. Agonists and antagonists were applied for 4–10 sec from an array of eight HPLC-grade capillary tubes (150 μm i.d.; Hewlett Packard Analytical Direct) placed within 100 μm of the cell of interest.

Western analysis

Cells were cultured, transfected as stated above except in 10 cm Petri dishes, and harvested by scrapping with 500 μL of lysis buffer (10 mM Tris pH 7.5, 1% SDS). Proteins were quantified using the Bradford assay and loaded onto 8–10% SDS-polyacrylamide gels, separated, and transferred to a nitrocellulose membrane (Hybond C, Amersham). The membrane was blocked overnight TBS (200 mM NaCl, 10 mM Tris pH 7.5) containing 0.2% Tween-20 and 10% low-fat dry milk. Blots were then incubated in TBS/0.1% Tween-20 containing primary GlyR antibody (monoclonal GlyR4a antibody, 1:200, Alexis Biochemicals) and monoclonal anti-gephyrin antibody (1:2000, Transduction Laboratories) for two hours at room temperature. After several washes, the HRP-coupled rabbit anti-mouse secondary antibody (SIGMA) was added for one hour (1:2000). The detection was performed by the ECL method (Amersham).

List of abbreviations

GlyR – glycine receptor; BLA – lateral/basolateral amygdala; HEK – human embryonic kidney cells; MDCK – Manin-Darby canine kidney cells

Author's contributions

JF carried out the electrophysiological recordings and participated in the western analysis. BM conceived of the study, participated in its design and coordination, performed the western analysis and some electrophysiology experiments, and drafted the manuscript. All authors read and approved the final manuscript.

Figure 3

Glycine receptors expressed in different expression systems have similar 'functional' strychnine binding. Cells were pre-treated for 30 seconds with strychnine alone then exposed to a strychnine admixture with an EC₅₀concentration of glycine. (A) Strychnine-mediated (more ...)

Acknowledgements

This work was supported in part by a Research Starter Grant from the Pharmaceutical Research & Manufacturers of America Foundation, Inc., and by the Texas A&M University Center for Environmental and Rural Health. We thank Dr. Jerry Trzeciakowski for his helpful discussions on the strychnine K_Banalysis.

References

Davis M. The role of the amygdala in conditioned and unconditioned fear and anxiety. In: Aggleton JP, editor. The Amygdala: A Functional Analysis. New York, Oxford Press; 2000. pp. 213–288.
McCool BA, Botting SK. Characterization of strychnine-sensitive glycine receptors in acutely isolated adult rat basolateral amygdala neurons. Brain Res. 2000;859:341–351. doi: 10.1016/S0006-8993(00)02026-6. [PubMed] [Cross Ref]
Danober L, Pape HC. Strychnine-sensitive glycine responses in neurons of the lateral amygdala: an electrophysiological and immunocytochemical characterization. Neuroscience. 1998;85:427–441. doi: 10.1016/S0306-4522(97)00648-9. [PubMed] [Cross Ref]
McCool BA, Farroni JS. Subunit composition of strychnine-sensitive glycine receptors expressed by adult rat basolateral amygdala neurons. Eur J Neurosci. 2001;14:1082–1090. doi: 10.1046/j.0953-816x.2001.01730.x. [PMC free article] [PubMed] [Cross Ref]
Sergeeva OA, Haas HL. Expression and function of glycine receptors in striatal cholinergic interneurons from rat and mouse. Neuroscience. 2001;104:1043–1055. doi: 10.1016/S0306-4522(01)00130-0. [PubMed] [Cross Ref]
Killcross S, Robbins TW, Everitt BJ. Different types of fear-conditioned behaviour mediated by separate nuclei within amygdala. Nature. 1997;388:377–380. doi: 10.1038/41097. [PubMed] [Cross Ref]
Schmieden V, Kuhse J, Betz H. Agonist pharmacology of neonatal and adult glycine receptor alpha subunits: identification of amino acid residues involved in taurine activation. Embo J. 1992;11:2025–2032. [PubMed]
Moorhouse AJ, Jacques P, Barry PH, Schofield PR. The startle disease mutation Q266H, in the second transmembrane domain of the human glycine receptor, impairs channel gating. Mol Pharmacol. 1999;55:386–395. [PubMed]
Hussy N, Deleuze C, Pantaloni A, Desarmenien MG, Moos F. Agonist action of taurine on glycine receptors in rat supraoptic magnocellular neurones: possible role in osmoregulation. J Physiol. 1997;502 ( Pt 3):609–621. [PubMed]
Wang DS, Xu TL, Pang ZP, Li JS, Akaike N. Taurine-activated chloride currents in the rat sacral dorsal commissural neurons. Brain Res. 1998;792:41–47. doi: 10.1016/S0006-8993(98)00119-X. [PubMed] [Cross Ref]
Farroni JS, McCool BA. The Amygdala in Brain Function: Basic & Clinical Approaches. Galveston, Texas, NY Acad Sci; 2002. Expression system-induced changes in adult rat glycine receptor partial agonist pharmacology; p. P22.
Farroni JS, McCool BA. Alterations in the pharmacology of adult rat glycine receptor isoforms in diverse heterologous expression systems. Soc Neurosci Abst. 2001. p. 38.1.
Vilardaga JP, Ciccarelli E, Dubeaux C, De Neef P, Bollen A, Robberecht P. Properties and regulation of the coupling to adenylate cyclase of secretin receptors stably transfected in Chinese hamster ovary cells. Mol Pharmacol. 1994;45:1022–1028. [PubMed]
Schmieden V, Kuhse J, Betz H. A novel domain of the inhibitory glycine receptor determining antagonist efficacies: further evidence for partial agonism resulting from self-inhibition. Mol Pharmacol. 1999;56:464–472. [PubMed]
Vandenberg RJ, Handford CA, Schofield PR. Distinct agonist- and antagonist-binding sites on the glycine receptor. Neuron. 1992;9:491–496. doi: 10.1016/0896-6273(92)90186-H. [PubMed] [Cross Ref]
Kuhse J, Schmieden V, Betz H. A single amino acid exchange alters the pharmacology of neonatal rat glycine receptor subunit. Neuron. 1990;5:867–873. [PubMed]
Whatley VJ, Brozowski SJ, Hadingham KL, Whiting PJ, Harris RA. Microtubule depolymerization inhibits ethanol-induced enhancement of GABAA responses in stably transfected cells. J Neurochem. 1996;66:1318–1321. [PubMed]
Kirsch J, Betz H. The postsynaptic localization of the glycine receptor-associated protein gephyrin is regulated by the cytoskeleton. J Neurosci. 1995;15:4148–4156. [PubMed]
Wilson L, Meza I. The mechanism of action of colchicine. Colchicine binding properties of sea urchin sperm tail outer doublet tubulin. J Cell Biol. 1973;58:709–719. doi: 10.1083/jcb.58.3.709. [PMC free article] [PubMed] [Cross Ref]
Banks P, Till R. A correlation between the effects of anti-mitotic drugs on microtubule assembly in vitro and the inhibition of axonal transport in noradrenergic neurones. J Physiol. 1975;252:283–294. [PubMed]
Rajendra S, Lynch JW, Pierce KD, French CR, Barry PH, Schofield PR. Mutation of an arginine residue in the human glycine receptor transforms beta-alanine and taurine from agonists into competitive antagonists. Neuron. 1995;14:169–175. doi: 10.1016/0896-6273(95)90251-1. [PubMed] [Cross Ref]
Leff P, Dougall IG. Further concerns over Cheng-Prusoff analysis. Trends Pharmacol Sci. 1993;14:110–112. doi: 10.1016/0165-6147(93)90080-4. [PubMed] [Cross Ref]
Johnson BD, Byerly L. Ca2+ channel Ca(2+)-dependent inactivation in a mammalian central neuron involves the cytoskeleton. Pflugers Arch. 1994;429:14–21. [PubMed]
Wang WH, Cassola A, Giebisch G. Involvement of actin cytoskeleton in modulation of apical K channel activity in rat collecting duct. Am J Physiol. 1994;267:F592–8. [PubMed]
Prat AG, Cunningham CC, Jackson G. R., Jr., Borkan SC, Wang Y, Ausiello DA, Cantiello HF. Actin filament organization is required for proper cAMP-dependent activation of CFTR. Am J Physiol. 1999;277:C1160–9. [PubMed]
Krupp JJ, Vissel B, Heinemann SF, Westbrook GL. Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific. Mol Pharmacol. 1996;50:1680–1688. [PubMed]
Grenningloh G, Rienitz A, Schmitt B, Methfessel C, Zensen M, Beyreuther K, Gundelfinger ED, Betz H. The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors. Nature. 1987;328:215–220. doi: 10.1038/328215a0. [PubMed] [Cross Ref]
van Zundert B, Alvarez FJ, Tapia JC, Yeh HH, Diaz E, Aguayo LG. Developmental-dependent action of microtubule depolymerization on the function and structure of synaptic glycine receptor clusters in spinal neurons. J Neurophysiol. 2004;91:1036–1049. doi: 10.1152/jn.00364.2003. [PubMed] [Cross Ref]
Betz H, Kuhse J, Fischer M, Schmieden V, Laube B, Kuryatov A, Langosch D, Meyer G, Bormann J, Rundstrom N., et al. Structure, diversity and synaptic localization of inhibitory glycine receptors. J Physiol Paris. 1994;88:243–248. doi: 10.1016/0928-4257(94)90087-6. [PubMed] [Cross Ref]
Meyer G, Kirsch J, Betz H, Langosch D. Identification of a gephyrin binding motif on the glycine receptor beta subunit. Neuron. 1995;15:563–572. doi: 10.1016/0896-6273(95)90145-0. [PubMed] [Cross Ref]
Ramming M, Kins S, Werner N, Hermann A, Betz H, Kirsch J. Diversity and phylogeny of gephyrin: tissue-specific splice variants, gene structure, and sequence similarities to molybdenum cofactor-synthesizing and cytoskeleton-associated proteins. Proc Natl Acad Sci U S A. 2000;97:10266–10271. doi: 10.1073/pnas.97.18.10266. [PubMed] [Cross Ref]
Kawasaki BT, Hoffman KB, Yamamoto RS, Bahr BA. Variants of the receptor/channel clustering molecule gephyrin in brain: distinct distribution patterns, developmental profiles, and proteolytic cleavage by calpain. J Neurosci Res. 1997;49:381–388. doi: 10.1002/(SICI)1097-4547(19970801)49:3<381::AID-JNR13>3.0.CO;2-2. [PubMed] [Cross Ref]
Hermann A, Kneussel M, Betz H. Identification of multiple gephyrin variants in different organs of the adult rat. Biochem Biophys Res Commun. 2001;282:67–70. doi: 10.1006/bbrc.2001.4553. [PubMed] [Cross Ref]
Grenningloh G, Pribilla I, Prior P, Multhaup G, Beyreuther K, Taleb O, Betz H. Cloning and expression of the 58 kd beta subunit of the inhibitory glycine receptor. Neuron. 1990;4:963–970. doi: 10.1016/0896-6273(90)90149-A. [PubMed] [Cross Ref]

Articles from BMC Pharmacology are provided here courtesy of
BioMed Central