Baur R et al. (2013), Do N-arachidonyl-glycine (NA-glycine) and 2-ara...

XB-ART-50215

PeerJ 2013 Sep 10;1:e149. doi: 10.7717/peerj.149.

Show Gene links Show Anatomy links

Do N-arachidonyl-glycine (NA-glycine) and 2-arachidonoyl glycerol (2-AG) share mode of action and the binding site on the β2 subunit of GABAA receptors?

Baur R , Gertsch J , Sigel E .

???displayArticle.abstract???
NA-glycine is an endogenous lipid molecule with analgesic properties, which is structurally similar to the endocannabinoids 2-AG and anandamide but does not interact with cannabinoid receptors. NA-glycine has been suggested to act at the G-protein coupled receptors GPR18 and GPR92. Recently, we have described that NA-glycine can also modulate recombinant α1β2γ2 GABAA receptors. Here we characterize in more detail this modulation and investigate the relationship of its binding site with that of the endocannabinoid 2-AG.

???displayArticle.pubmedLink??? 24058880
???displayArticle.pmcLink??? PMC3775635
???displayArticle.link??? PeerJ

Species referenced: Xenopus
Genes referenced: gabarap gpr18 tbx2

???attribute.lit??? ???displayArticles.show???

	Figure 1. Chemical structure of NA-glycine and 2-AG.
	Figure 2. Concentration-dependent potentiation of currents mediated by recombinant α1β2γ2GABAA receptors.(A) Receptors were expressed in Xenopus oocytes and currents were measured by using electrophysiological techniques at a GABA concentration eliciting 0.5–1.0% of the maximal current amplitude (EC0.5–1.0). GABA was applied twice (single bars) and subsequently in combination with increasing concentrations of NA-glycine. The numbers above the double bars indicate the concentration of NA-glycine in µM. NA-glycine was pre-applied for 30 s. Original current traces are shown. (B) shows the averaged concentration-dependent potentiation of currents elicited by GABA by NA-glycine. Four experiments as shown under (A) were averaged. Data are shown as mean ± SD (n = 4). Such an averaged curve has been shown before based on 3 experiments and missing the point at 10 µM NA-glycine (Baur et al., 2013).
	Figure 3. Influence of the GABA concentration.Current potentiation by 3 µM NA-glycine (closed circles) or 2-AG (closed squares) was determined at different concentrations of GABA. Potentiation decreased with increasing concentrations of GABA. The GABA concentration response curve was fitted with a mean EC50 of 35 µM and a mean Hill coefficient of 1.5 (not shown).
	Figure 4. Mode of action of NA-glycine.Simplified model (A) The model assumes two agonist binding sites 1 and 2 with different affinities. 2-AG affects the closed/open transition of the two singly ligated states. The receptor R can first bind GABA (A) either to the site 1 (AR) or the site 2 (RA). The receptor occupied by two agonist molecules ARA can isomerize to the open state ARA, the receptors occupied by a single agonist molecule can isomerize to the open states AR and RA*. Binding is described with K as dissociation constants and gating with L as closed state/open state equilibrium. (C) Theoretical GABA concentration response curves in the absence and presence of NA-glycine. The following parameters were assumed: 0.24 for L, 10 and 2.2 for L1 in the absence and presence of NA-glycine, respectively, 11 and 2.4 for L2 in the absence (line) and presence (dashed line) of NA-glycine, respectively, 30 µM for K1, 90 µM for K2. (C) Dependence of the potentiation by NA-glycine on the concentration of GABA obtained by the ratio of the computed current in the presence of NA-glycine divided by the current in its absence.
	Figure 5. Concentration inhibition curve of DEA.Increasing concentrations of DEA were co-applied with 1 µM 2-AG (open circles), 3 µM 2-AG (open squares), 15 µM 2-AG (open diamonds), 0.5 µM NA-glycine (filled circles), or 3 µM NA-glycine (filled squares). Data are shown as mean ± SEM (n = 4).
	Figure 6. Effect of subunit combination and co-application with 2-AG.(A) Current potentiation by the combined application of 3 µM NA-glycine and 3 µM 2-AG is compared with the individual application of the two substances. (B) Current potentiation by 3 µM NA-glycine in α1β2γ2 receptors and α1β1γ2 receptors. Potentiation is strongly dependent on the presence of the β2 subunit.
	Figure 7. Concentration-dependent potentiation of currents mediated by concatenated GABAA receptors.Concatenated α1-β1-α1/γ2-β1, α1-β1-α1/γ2-β2, α1-β2-α1/γ2-β1 or α1-β2-α1/γ2-β2 receptors were expressed in Xenopus oocytes and currents were measured at a GABA concentration eliciting 0.5–1.0% of the maximal current amplitude (EC0.5−−1.0). Current potentiation by increasing concentrations of NA-glycine was determined. Four such experiments were averaged. Data are shown as mean ± SD (n = 4).
	Figure 8. Effect of point mutations that reduced potentiation by 2-AG on the potentiation of NA-glycine.(A) Potentiation by 3 µM NA-glycine is compared between wild type receptors and receptors containing the point mutation S429C in the β2 subunit. This mutation results at the beginning of the drug application in an about 50% reduction of potentiation and after 1 min drug application potentiation is abolished. (B) Wild type receptors are compared with mutant receptors. Current potentiation is indicated at the beginning of the drug application (filled bars) and after 1 min drug exposure (open bars).
	Figure 9. Time course of the potentiation by NA-glycine.An oocyte expressing α1β2γ2 receptors was sequentially exposed to medium alone, to 1 µM GABA, to the same concentration of GABA in combination with 5 µM NA-glycine, to 1 µM GABA alone and the to medium. This experiment was repeated two more times with similar results.

References [+] :

Barbara, T-type calcium channel inhibition underlies the analgesic effects of the endogenous lipoamino acids. 2009, Pubmed