Alaniz-Palacios A and Martínez-Torres A (2017), Antagonistic effect of dopamine structural anal...

XB-ART-54355

Sci Rep 2017 Dec 12;71:17385. doi: 10.1038/s41598-017-17530-8.

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Antagonistic effect of dopamine structural analogues on human GABAρ1 receptor.

Alaniz-Palacios A , Martínez-Torres A .

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GABAergic and dopaminergic pathways are co-localized in several areas of the central nervous system and recently several reports have shown co-release of both neurotransmitters. The GABA-A receptor (β and ρ1 subunits) is modulated by dopamine (DA) and, interestingly, GABAρ1 can be modulated by several biogenic amines. Here we explored the effects of the metabolites of the dopaminergic pathway and other structural analogues of DA on GABAρ1 and the DA gated ion channel (LGC-53) from Caenorhabditis elegans expressed in Xenopus laevis oocytes. Our findings show an antagonistic effect of the metabolite 3-Methoxytyramine (3-MT, IC50 = 285 ± 30 µM) with similar potency compared to DA on induced GABA currents; however, it was inactive on LGC-53. The structural DA analogues and metabolites, 3, 4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 2-phenylethylamine (β-PEA) and 4-amino-1-butanol (4-AM-1-OH), antagonized GABAρ1 currents, whereas β-PEA acted as partial agonists on LGC-53, indicating that the putative binding sites of both receptors may share structural characteristics. These results suggest that the DA metabolites 3-MT, DOPAC and HVA modulate GABAρ1 and possibly affect the activity of the receptors that include this subunit in vivo.

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Species referenced: Xenopus laevis
Genes referenced: adm

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	Figure 1. Effect of DA analogues on GABA currents. (A) Sample currents of co-applications of 3 µM GABA and DA metabolites. (B) Sample currents of co-applications of 3 µM GABA and a DA analogues. (C) Concentration-response relations. IC50 for each compound is shown in inset. Data were normalized to the response to 3 µM GABA. 3-MT shows a similar potency to DA (previously reported by Ochoa de la Paz, 2012). Each point was evaluated in 6 oocytes from 3 frogs.
	Figure 2. Maximal effect of DA analogues on GABA currents. Only amines for (A) Sample currents of co-applications of 3 µM GABA and DA or 3-MT; amine moiety with current remnant for (B) Sample currents of co-applications of 3 µM GABA and β-PEA or 4-AM-1-OH. Acid moiety. (C) Sample currents of co-applications of 3 µM GABA and/or HVA. (D) Maximal effect of the analogues. Mean and SEM are indicated inside the corresponding bar. Only HVA and DOPAC did not fully inhibit GABA-induced currents. Data are from to 6 cells from 3 frogs.
	Figure 3. Competition assays. (A) Sample currents generate by co-application of GABA (100 nM to 1 mM) and 3-MT (140, 280 and 510 µM), and (B) concentration−response relation. Data were normalized to the response to 1 mM GABA. (C) Effect of 3-MT concentration of GABA EC50. Significant difference (P value) versus GABA alone, 0.0076, ***<0.0001; &significant difference (P value) versus GABA/3-MT [510] µM, &&&& <0.0001; #significant difference (P value) versus GABA/3-MT [280] µM, #0.0496. Data are from to 6 oocytes from 2 frogs.
	Figure 4. Docking model for GABA and antagonist. (A) Structural model of GABAρ1. For clarity, only two of the five subunits are shown. Inset: Enlargement of the agonist pocket formed by 20 residues (wire and stick). Residues from + subunit are in orange and residues from - subunit are in light orange. The key residues Y198, T244, R104 and S268 are represented in stick. (B) GABA docked into the key residues and the calculated distances 1 = 2.18, 2 = 3.17, 3 = 3.55 and 4 = 4.27 Å. (C) 3-MT docked into the key residues and the calculated distances 1 = 3.06, 2 = 4.18, 3 = 7.78 and 4 = 8.20 Å. The last two distances are too long for establishing a hydrogen bond. (D) DOPAC docked into the key residues and the calculated distances 1 = 7.50, 2 = 7.24, 3 = 3.48 and 4 = 4.98 Å. The first two distances are too long for establishing a hydrogen bond. The result suggests a different way for 3-MT and DOPAC anchorage.
	Figure 5. Modulation of GABA responses by DA analogues on activated GABAρ1. (A) Sample currents generated by applying the maximal concentrations of DA analogues with an amine moiety on activated receptors (insets) and their corresponding concentration-response curve (continuous line), and concentration-response curve of structural analogous during co-application of GABA (dotted line, from Fig. 1C). Data were normalized to the response to 3 µM GABA. (B) Sample currents generated by applying the maximal concentrations of DA analogues with acidic moiety on activated receptors (insets) and their corresponding concentration-response curve (continuous line), and concentration-response curve for analogues during co-application of GABA (dotted line, from Fig. 1C). (C) IC50 for each analogue when co-applied with GABA perfusion or previously activated receptor. Only DA showed a significant difference between protocols (). (D) Comparison of efficacies between the two protocols (filled bars: same time application, empty bars: activated GABA channels). Molecules that include an amine moiety show significant differences (). Data in B, D and F are from to 6 oocytes from 3 frogs.
	Figure 6. Construction of isobolograms to explore inhibition mechanism. Sample currents of co-applications of 3 µM GABA plus an analogue [at the IC25 for each compound and the mix 3 µM GABA/Analogue1[IC25]/Analogue2[IC25] in (A,C and E,A) 3-MT/GABA and /GABA and 3-MT//GABA. (B) Normalized responses (bars 1, 2, 4 and 5), experimental responses to the application of the GABA//3-MT mix. Calculated values (bars 3 and 6). *Significant difference versus theoretical value (P = 0.019, 0.046, co-application and prior to perfusion of GABA). (C) GABA/3-MT and GABA/β-PEA and mix GABA/3-MT/β-PEA. (D) Response normalized to the sum of the individual effects (bars 1, 2, 4), observed responses to the application of the GABA/β-PEA/3-MT mix, the response was normalized to the theoretical value (calculated values in bars 3 and 6, left-right). (E) 3-MT/GABA and mix GABA/3-MT/β-PEA. (F) Response was normalized to the sum of the individual effects (3-MT for co-application and β-PEA for pre-application of GABA, bars 1 and 2, respectively) on the experimental response to the application of the GABA/3-MT/β-PEA mix. Bar 3, response normalized to the calculated values (data in B, D and F are shown with mean ± S.E.M. Data are from 6 oocytes).
	Figure 7. Effect of DA and analogues on new receptor (LGC-53 from C. elegans). (A) Sample currents generated by DA (300 µM, maximal effect) and other biogenic amines (maximal concentration tested, maximal effect). (B) Concentration-response curves of DA and other biogenic amines. (C) Maximal effect of the biogenic amine versus maximal response to DA. Data in B and C correspond to the mean ± S.E.M. from 5 oocytes.

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