Targets for ethanol action and antagonism in loop 2 of the extracellular domain of glycine receptors

Abstract

The present studies used increased atmospheric pressure in place of a traditional pharmacological antagonist to probe the molecular sites and mechanisms of ethanol action in glycine receptors (GlyRs). Based on previous studies, we tested the hypothesis that physical-chemical properties at position 52 in extracellular domain Loop 2 of alpha1GlyRs, or the homologous alpha2GlyR position 59, determine sensitivity to ethanol and pressure antagonism of ethanol. Pressure antagonized ethanol in alpha1GlyRs that contain a non-polar residue at position 52, but did not antagonize ethanol in receptors with a polar residue at this position. Ethanol sensitivity in receptors with polar substitutions at position 52 was significantly lower than GlyRs with non-polar residues at this position. The alpha2T59A mutation switched sensitivity to ethanol and pressure antagonism in the WTalpha2GlyR, thereby making it alpha1-like. Collectively, these findings indicate that (i) polarity at position 52 plays a key role in determining sensitivity to ethanol and pressure antagonism of ethanol; (ii) the extracellular domain in alpha1- and alpha2GlyRs is a target for ethanol action and antagonism and (iii) there is structural-functional homology across subunits in Loop 2 of GlyRs with respect to their roles in determining sensitivity to ethanol and pressure antagonism of ethanol. These findings should help in the development of pharmacological agents that antagonize ethanol.

Figure 1. Concentration-response curves for glycine (10–3,000 μM) activated chloride currents in Xenopus oocytes expressing WT and mutant α1GlyR subunits

Glycine induced chloride currents were normalized to the maximal current activated by a saturating concentration of glycine (3 mM) when tested under 1 ATA air conditions. The curves represent non-linear regression analysis of the glycine concentration responses in the α1 mutant GlyRs compared to WT α1GlyRs. Details of EC₅₀, Hill Slope and Maximal current amplitude (I_max) are provided in Table 1. Glycine was applied for 30 sec. Washout time was 5–15 min after application of glycine. Each data point represents the mean ± SEM from 4 different oocytes.

Figure 2. Polarity of the residue at position 52 in α1GlyRs determines sensitivity to ethanol

Figure shows ethanol % potentiation of glycine induced chloride current at 100mM ethanol. White bars represent non-polar substitutions at position 52 in α1 GlyRs and grey bars represent polar substitutions. Ethanol responses for the polar mutants α1A52S, α1A52T, α1A52Y and α1A52W GlyRs were significantly reduced compared to WT α1GlyRs. There were no significant differences among the polar mutants with respect to ethanol sensitivity at 100mM. Significance is *p<0.05 vs. WT α1GlyRs.

Figure 3. The polarity and hydrophobicity of substitutions at position 52 are significantly correlated with sensitivity of α1GlyRs to ethanol

The physical-chemical properties of the residues at position 52 (A) Polarity, (B) Hydrophobicity, (C) Molecular Volume and (D) Molecular Weight respectively, were correlated against sensitivity to 100mM ethanol.

Figure 4

(A). Pressure significantly antagonizes ethanol potentiation in α1 GlyRs: The figure depicts WT α1GlyRs tested at both 1ATA air control (white bars) and 12 ATA heliox (black bars). In agreement with previous findings, pressure antagonized ethanol potentiation from 50–200mM but not at 25mM; (B) Pressure Does not Antagonize Ethanol potentiation in mutant α1A52S GlyRs: The figure depicts results from 25–100 mM ethanol in the mutant (A52S) α1GlyRs form the same batches of oocytes shown in (A), tested at both 1 ATA air control (white bars) and 12 ATA heliox (black bars). Data is presented as mean ± SEM of 4–6 oocytes. Data was subjected to two-way repeated measures ANOVA followed by Bonferroni post-hoc analyses. Statistical significance is *p < 0.05 or ***p<0.001.

Figure 5. Pressure antagonizes ethanol in α1GlyRs that have a non-polar residue at position 52

The figure depicts mutant α1GlyRs tested at both 1ATA air control (white bars) and 12 ATA heliox (black bars). Exposure to 12 ATA heliox did not significantly affect ethanol potentiation at 100mM in (A) α1A52C (intermediate, intermediate-polar) (B) α1A52T (intermediate, polar), (C) α1A52W (bulky, polar) or (D) α1A52Y (bulky, polar) GlyRs. Pressure did significantly antagonize ethanol potentiation of (E) α1A52F (bulky, non-polar), (F) α1A52I (intermediate, non-polar) and (G) α1A52V (small, non-polar) GlyRs. Data were analyzed using unpaired t-tests to determine the effect of atmospheric condition on ethanol potentiation of glycine-activated chloride currents. Statistical significance is **p<0.01. Data are presented as mean ± SEM of 4–6 oocytes.

Figure 6. The polarity of the residue at position 52 in α1GlyRs determines the receptors sensitivity to pressure antagonism of ethanol

The physical-chemical properties of the residues at position 52 (A) Polarity, (B) Hydrophobicity, (C) Molecular Volume and (D) Molecular Weight respectively, were correlated against change in ethanol potentiation upon exposure to 12ATA (ΔP).

Figure 7

(A) Concentration-response curves for glycine (10–3,000 μM) activated Cl- currents in Xenopus oocytes expressing WT and mutant α2 glycine receptor subunits: Glycine induced chloride currents were normalized to the maximal current activated by a saturating concentration of glycine (3mM) when tested under 1 ATA air conditions. The curves represent non-linear regression analysis of the glycine concentration responses in the α2 WT and mutant GlyRs compared to WT α1GlyRs. Details of EC₅₀, Hill Slope and Maximal current amplitude (I_max) are provided in Table 1. Glycine was applied for 30 sec. Washout time was 5–15 min after application of glycine. Each data point represents the mean ± SEM from 4 different oocytes; (B) Replacing the polar threonine at position 59 in WT α1GlyRs with the non-polar alanine changes the α2GlyRs to be α1GlyR-like in response to ethanol and pressure antagonism of ethanol: The figure depicts mutant α2T59A GlyRs tested at both 1ATA air control (white bars) and 12 ATA heliox (black bars). As predicted, pressure antagonized ethanol potentiation from 50–200mM but not at 25mM. The ethanol responses in the mutant were similar to WT α1GlyRs. Data were subjected to two-way repeated measures analysis of variance (ANOVA) followed by Bonferroni post-hoc analyses. Statistical significance is *p < 0.05 or ***p<0.001. Data are presented as mean ± SEM of 4–5 oocytes.