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. 1A). The apparent EC₅₀ of 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 EC₅₀ of 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 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.

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. 1B), the average β-alanine EC₅₀ values calculated from individual cells were 770 μM (n = 6) and 570 μM (n = 7) for α₂ and 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. EC₅₀ values for α₂ and α₂/β receptors were 2.0 mM (n = 8) and 2.9 mM (n = 7), respectively. Two-way ANOVA on LogEC₅₀ values 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 α₂ and 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 α₂ and 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.

Similar analysis of taurine pharmacology in HEK and L-cells revealed more dramatic effects of system and subunit on this partial agonist (Fig. 1C). In HEK cells, the apparent EC₅₀ for 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 GlyRα₂ and 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 α₂ and 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 GlyRα₂ and 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.

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 GlyRα₂ protein using western analysis of total lysate derived from the same number of GFP⁺ HEK 293 or L cells from transfected cultures (Fig. 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 GlyRα₂ protein 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.

Maximal conductance is an independent measure of functional expression and was also larger for both α₂ and α₂β receptors expressed in HEK cells compared to receptors expressed in L cells (Fig. 2B). Across all experiments where maximal glycine concentrations were assayed, the conductance of α₂ receptors 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 α₂ and 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 α₂ GlyRs, 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

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 EC₅₀ concentration of glycine. The strychnine K_B was estimated for HEK and L-cells expressing either the GlyRα₂ or 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 GlyRα₂ subunit (Fig. 4A) had very similar K_B values when expressed in either HEK (K_B = 49 ± 8 nM, n = 11) or L-cells (K_B = 38 ± 7 nM, n = 16; Fig. 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 141516, 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.

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 EC₅₀ concentration 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 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. 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. 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. 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.

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 α₂ and 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 α₂ homomeric channels.

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, CaCl₂ 1, Mg-ATP 4, pH 7.2 with methane sulfonic acid; adjusted to 290–295 mmol kg^-1 with 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, CaCl₂ 2.5 MgCl₂ 1.0, pH 7.4, 305–320 mmol kg^-1 Data 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_B from functional IC₅₀ and EC₅₀ data, the Cheng-Prusoff equation was used:

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

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.

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).