Selective antagonism of rat inhibitory glycine receptor subunits

Abstract

Retinal ganglion cells exhibit fast and slow inhibitory synaptic glycine currents that can be selectively inhibited by strychnine and 5,7-dichlorokynurenic acid (DCKA), respectively. In this study we examined whether strychnine and DCKA selectivity correlated with the subunit composition of the glycine receptor. Homomeric alpha1, alpha2 or alpha2* glycine subunits were in vitro expressed in human embryonic kidney cells (HEK 293). In cells expressing the alpha1 subunit, responses to 200 microm glycine were blocked by 1 microm strychnine but not by 500 microm DCKA. In cells expressing the alpha2 subunit, both 1 microm strychnine and 500 microm DCKA were effective antagonists of 200 microm glycine. In cells expressing alpha2* subunits, which are much less glycine-sensitive, 10 mm glycine was inhibited by 500 microm DCKA but not by 1 microm strychnine. A single amino acid mutation in the alpha1 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 alpha1, alpha2 and alpha2* 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 alpha subunits.

Figure 1. Glycine responses in HEK 293 cells expressing homo-oligomers of α1, α2, or α2* subunits

Upper panel, typical currents from receptors formed from each subunit are shown at several glycine concentrations. The scale bar is 500 pA for α1 and α2 and 50 pA for α2*. The time scale is 10 s. Cells were clamped, using the KCl electrode filling solution, at –20 mV for α1 and α2, and at –60 mV for α2*. This provided a chloride driving force of ∼20 mV and ∼60 mV, respectively. Recordings are from cells transfected using FuGENE-6. The EC₅₀ at the α1 subunit was 60.6 ± 3.1 μm(n= 5), at the α2 receptor it was 62.2 ± 0.9 μm(n= 8), and at the α2* it was about 3.1 ± 0.2 mm(n= 8). In these examples drugs were applied by a rapid puff but wash out was by bath exchange, which slowed recovery especially at high glycine concentrations. Lower panel, dose–response curves for receptors formed from each subunit, using the calcium phosphate transfection technique which produced smaller currents and higher EC₅₀ values than when using FuGENE-6. The vertical lines show that 200 μm glycine produced approximately 80% of the peak response in α1 and α2 subunit expression, while 10 mm glycine produced a similar relative response in cells expressing α2* subunits. Curves were fitted to the data using the Hill equation as described in Methods.

Figure 2. Strychnine sensitivity of GlyR α1, α2, and α2*

In HEK 293 cells transfected with one of the subunits, glycine was applied alone (a) or after pretreatment and in the presence of 1 μm strychnine (b). The glycine concentration was 200 μm in cells expressing α1 or α2 subunits and was 10 mm in cells expressing α2* subunits. The bar at the top of each panel indicates the timing of glycine application. Cells were voltage clamped to 0 mV using the potassium gluconate filling solution, providing a chloride driving force of ∼50 mV.

Figure 3. DCKA sensitivity of GlyR α1, α2 and α2*

HEK 293 cells were transfected with cDNA of the different alpha subunits. In each case, the cells were tested by applying glycine alone (curve a in each panel), or in the presence of 500 μm DCKA (curve b). The glycine concentration was 200 μm in cells expressing α1 or α2 subunits and was 10 mm in cells expressing α2* subunits. Cells were voltage clamped to 0 mV using the potassium gluconate filling solution, providing a chloride driving force of ∼50 mV.

Figure 4. DCKA inhibition dose–response curves

Glycine was applied alone or in the presence of various concentrations of DCKA. The amplitude of the glycine currents (normalized to the glycine current in the absence of DCKA) are plotted against DCKA concentration. The glycine concentration was 200 μm in HEK cells expressing α1 or α2 subunits and was 10 mm in cells expressing α2* subunits. Inhibition curves were fitted as described in Methods.

Figure 5. Alignment of the extracellular, N-terminal domains of GlyR α1, α2 and α2*

The amino acid sequence of α1 is shown and compared to α2 and α2*. The differences in amino acid sequence are marked and the amino acid sites subject to site-directed mutagenesis are enclosed in boxes.

Figure 6. DCKA sensitivity of GlyR α1 and α2 mutations

Glycine was applied in the presence of various concentrations of DCKA. The response to glycine at each DCKA concentration was normalized to the response to glycine alone. Left panel, glycine (200 μm) was applied to HEK 293 cells expressing homo-oligomers of the wild-type α1 (▪) subunit or two mutations of this subunit (A175P (▴) and R196G (▾)). Mutation R196G in GlyR α1 resulted in DCKA sensitivity with an IC₅₀= 448 ± 180 μm. Right panel, comparison the response to 200 μm glycine in the presence of various DCKA concentrations to HEK 293 cells expressing wild-type GlyR α2 (▪) or α2* (•) homo-oligomers or the P182A mutant (▴) of the α2* subunit. The glycine responses of α2 homo-oligomer were inhibited by DCKA with an IC₅₀= 188 ± 13 μm(n= 6), while α2* had an IC₅₀= 243 ± 61 μm(n= 10). On the other hand, α2* mutation P182A reduced DCKA sensitivity to an IC₅₀= 724 ± 53 μm(n= 6). Inhibition curves were derived as described in Methods.

Figure 7. DCKA is a competitive antagonist at α2 and α1(R196G) glycine receptors

The top two panels show glycine dose–response curves in control Ringer solution and in the presence of 250 μm or 500 μm DCKA for α2 and α1(R196G) homo-oligomers. Cells were clamped at –20 mV, providing a chloride driving force of ∼20 mV. Data were fitted as described in Methods. The lower panel is a Schild plot (log scale in both axes) of DCKA antagonism at the α2 and α1(R196G) GlyRs