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Differential modulation of human GABAC-ρ1 receptor by sulfur-containing compounds structurally related to taurine

BMC Neuroscience volume 19, Article number: 47 (2018) Cite this article

Abstract

Background

The amino acid taurine (2-Aminoethanesulfonic acid) modulates inhibitory neurotransmitter receptors. This study aimed to determine if the dual action of taurine on GABAC-ρ1R relates to its structure. To address this, we tested the ability of the structurally related compounds homotaurine, hypotaurine, and isethionic acid to modulate GABAC-ρ1R.

Results

In Xenopus laevis oocytes, hypotaurine and homotaurine partially activate heterologously expressed GABAC-ρ1R, showing an increment in its deactivation time with no changes in channel permeability, whereas isethionic acid showed no effect. Competitive assays suggest that hypotaurine and homotaurine compete for the GABA-binding site. In addition, their effects were blocked by the ion-channel blockers picrotixin and Methyl(1,2,5,6-tetrahydropyridine-4-yl) phosphinic acid. In contrast to taurine, co-application of GABA with hypotaurine or homotaurine revealed that the dual effect is present separately for each compound: hypotaurine modulates positively the GABA current, while homotaurine shows a negative modulation, both in a dose-dependent manner. Interestingly, homotaurine diminished hypotaurine-induced currents. Thus, these results strongly suggest a competitive interaction between GABA and homotaurine or hypotaurine for the same binding site. “In silico” modeling confirms these observations, but it also shows a second binding site for homotaurine, which could explain the negative effect of this compound on the current generated by GABA or hypotaurine, during co-application protocols.

Conclusions

The sulfur-containing compounds structurally related to taurine are partial agonists of GABAC-ρ1R that occupy the agonist binding site. The dual effect is unique to taurine, whereas in the case of hypotaurine and homotaurine it presents separately; hypotaurine increases and homotaurine decreases the GABA current.

Background

Incorporation of sulfur into amino acids, proteins, enzymes, vitamins, and other biomolecules makes sulfur essential for biological systems [1]. In mammals, methionine is an essential amino acid, whereas cysteine, homocysteine, and taurine are semi-essential amino acids because they can be synthetized from methionine, sulfur, and serine via trans-sulfuration [1, 2].

Taurine (2-ethanosulfonic acid) is a ubiquitous, non-protein β-amino acid that abounds in different mammalian tissues. In the central nervous system (CNS) and retina, taurine is the second most abundant amino acid after glutamic acid, with a concentration in different species that ranges from 10 to 90 mM [3]. Taurine plays a role in different cell functions, as well as in neuronal migration and CNS development. In general, extensive experimental evidence reported relates taurine with cell volume and osmolality regulation [4,5,6]. In addition, taurine is involved in other physiological processes of the CNS and retina, such as modulation of Ca2+ channels and neurotransmission [7,8,9,10,11]. Other physiological functions of taurine not described in retina are antioxidant defense, phase II detoxification reactions and a role as a neurotrophic factor. GABA (γ-aminobutyric acid) plays a key role in neurotransmission. For example, in the retina, GABA modulates transmission of information flowing from photoreceptors to the brain. GABA performs its inhibitory action via two types of receptors: (1) ionotropic, designated GABAA and GABAC (also known as GABAA-ρ); and, (2) metabotropic, named GABAB, all of them with different molecular and pharmacological properties [12,13,14,15]. GABAC receptor is particularly interesting because it is insensitive to barbiturates, benzodiazepines, bicuculline, and baclofen (modulators and inhibitors of GABAA and GABAB receptors, respectively) [16,17,18,19,20] but sensitive to picrotoxin and Methyl(1,2,5,6-tetrahydropyridine-4-yl) phosphinic acid (TPMPA) [21].

GABAA receptors are heteropentamers, constituted by α, β, γ, and δ subunits; the combinations of these subunits determine the pharmacological and physiological properties of the receptor, and the α1/β2/γ2 is the most common combination found in the CNS and retina [22, 23]. GABAC receptors are composed of the ρ1, ρ2, and ρ3 subunits, each of them capable of forming homomeric functional receptors when expressed heterologously. However, the native composition of these receptors in neurons is unknown. In the CNS, evidence of the expression of GABAC receptors has been demonstrated in cerebellar Purkinje neurons and in the amygdala [24,25,26]. Experiments “in vivo” using specific antagonists of GABAC receptors directly applied into the amygdala, suggest that GABAC-mediated activity participates in the modulation of fear and anxiety [26]. Although the role of GABAC receptors in the retina is still poorly understood, its function is tightly modulated by intracellular cascades triggered by neuroactive molecules and their receptors, and they are thought to play a major role in visual signaling [19, 20, 27, 28]. While GABAA receptors (α1/β2/γ2) are distributed in all neuronal types of the retinal circuit, GABAC conformed by ρ1 and ρ2 subunits are located in bipolar neurons, where they downregulate NMDA receptors and, consequently, modulate transient glutamate release in response to light. In addition, amacrine and ganglion cells express GABAC receptors [29,30,31].

Taurine exerts its inhibitory effect via activation of GABAA and glycine receptors, but has less affinity compared to the specific agonists of each receptor [10, 11]. However, the mechanisms by which taurine regulates ionotropic GABA receptors remain to be determined. A negative and positive modulation of the GABA-induced current in oocytes heterologously expressing human GABAC-ρ1 receptor, exposed at lower and higher taurine concentrations was reported [32]. These results suggest that taurine acts in a dual way and may compete with GABA for the same binding site. Our study aimed to determine if the dual effect of taurine observed on GABAC-ρ1R was associated with the molecular structure of taurine. To confirm this hypothesis, we used homotaurine (Homo), hypotaurine (Hypo), and isethionic acid (IA), all sulfur-containing compounds structurally related to taurine (SCC-tau), but with some differences in their chemical structure. For instance, unlike taurine, Homo has one additional carbon and the sulfonic group (SO3H) is in Cis position with respect to the amino (NH2) group; Hypo has a sulfinic group (SO2H) in Trans position with respect to the NH2 group, and IA has a hydroxyl group instead of NH2. Some of these compounds have been widely used to better understand the physiological functions of taurine and, likewise, to evaluate compounds that may be therapeutically applied in some diseases such as diabetes, alcoholism, ischemia, and others [33,34,35]. Although a variety of taurine analogues have been developed, their effects are still unknown in terms of structure–function in various systems [36].

Results

GABAC-ρ1 receptor (GABAC-ρ1R) heterologously expressed in oocytes generated typical non-desensitizing GABA currents (Fig. 1a) when SCC-tau was perfused onto the same oocyte; Homo- and Hypo- elicited a response in a concentration dependent manner. Both compounds gate the ion-channel at micromolar concentrations (Fig. 1b, c). In contrast, IA did not show any affect at the concentrations tested (Fig. 1d). Similar to the currents induced by GABA, Homo- and Hypo-induced well-maintained currents and did not desensitize, even after long exposure. Despite the lack of differences between the magnitude of the current elicited by Homo- and Hypo-, these SCC-tau show a clear difference in the EC50 of 70 ± 1.1 µM for Homo and 3 ± 1.5 mM for Hypo (Fig. 2a). The response induced by Homo and Hypo did not correlate with changes in the activation time of the currents (τact). Nevertheless, Homo showed an increase in deactivation time (τdeac). The τact and τdeac for GABA alone were 1 ± 0.2 min and 1.7 ± 0.5 min, respectively. In the presence of Hypo, τact was 1.5 ± 0.1 min and tdeact was 3.4 ± 1.1 min, whereas for Homo τact was 1.8 ± 0.2 min and τdeac was 6.7 ± 2.2 min (Fig. 2b). However, we did not observe any permeability changes of the channel activated, as suggested by their inversion potential, which was − 21 ± 1.0 mV for 3.5 µM GABA and − 25 ± 2.1 mV and − 26 ± 1.0 mV for 70 µM Homo and 3 mM Hypo, respectively (Fig. 2c). In all cases, the current–voltage (I–V) relationship was linear within the range explored (− 120 to + 40 mV), indicating that activation of GABAC-ρ1R by SCC-tau was voltage-independent and did not change the ion selectivity of the channel.

Fig. 1
figure1

Activation by GABA and SCC-tau of GABA-ρ1R heterologously expressed in oocytes. a Control: oocyte exposed to GABA at several concentrations (3–1000 µM); b–d Oocytes perfused with SCC-tau at the concentrations indicated. In each experiment the oocyte was first exposed to a GABA concentration equal to GABA’s EC50. The horizontal bars indicate the period of time when the compound was applicated. Oocytes were voltage-clamped at − 60 mV and inward currents are denoted as downward deflections. Chemical structure of GABA (a) and SCC-tau (b–d), are represent at right of each representative trace

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Fig. 2
figure2

GABA- and SCC-tau-induced currents in oocytes heterologously expressing GABAC-ρ1R. a Dose-response relationship showing the EC50 and Hill coefficient for each agonist. Data were normalized to the maximal current (I) obtained for each agonist. b Deactivation constant (τdeac) of currents activated by GABA, Hypo, and Homo. The difference in the deactivation constants between, GABA-, Hypo- or Homo-induced currents, was significant when P < 0.05. c Current–voltage relationship of GABA-, Homo-, and Hypo-induce currents at the indicated concentrations. Dose-response relationship (a) was constructed by measuring the maximum response evoked by each agonist concentration (see methods), for b and c, data are given as the mean ± S.E. from at least 8 oocytes (n = 8) from 3 frogs (N = 3)

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We also analyzed if the currents activated by Homo and Hypo were sensitive to TPMPA, a highly specific and selective antagonist of GABAC-ρ1R [21]. Figure 3 shows an IC50 of 1.2 ± 0.1 µM, 1.4 ± 0.1 µM or 2.0 ± 0.2 µM TPMPA when the receptor was activated by 3 µM GABA, 70 µM Homo or 3 mM Hypo, respectively (Fig. 3a). Hill coefficients were 1.3 ± 0.2 µM (GABA), 1.0 ± 0.1 µM (Homo), and 1.1 ± 0.8 µM (Hypo). It is well known that TPMPA competes with GABA for the same binding-site, so since the IC50 of TPMPA values are similar; it is probable that SCC-tau, Homo and Hypo, share the same binding site as GABA in the GABAC-ρ1R. As expected, picrotoxin, an allosteric antagonist of ionotropic GABA receptors [37, 38], irreversibly inhibits the currents activated by 3 µM GABA, 70 µM Homo or 3 mM Hypo in a dose-dependent manner, with an IC50 of 100 ± 0.1 µM, 105 ± 0.3 µM, and 100 ± 0.8 µM, respectively (Fig. 3b).

Fig. 3
figure3

Pharmacological modulation of GABA- and SCC-tau-induced currents by TPMPA (a) and picrotoxin (b). Currents were normalized to the maximum amplitude elicited by GABA or SCC-tau in absence of inhibitors. Data points are the mean ± S.E. from at least 7 oocytes (n = 7) from 3 frogs (N = 3)

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Figure 4 shows that during GABA-induced activation of the receptor, Homo reduced the currents when co-applied at different concentrations (3 µM to 30 mM). The effect of Homo on the GABA-induced current changes depended on GABA concentrations. Figure 4a shows that at a concentration of 1.5 µM and 3 µM of GABA, Homo decreased GABA-activated currents. However, at a higher concentration of GABA (6 µM), the effect of Homo diminished; the IC50 for Homo were 40 ± 1.0 µM, 90 ± 1.5 µM, and 410 ± 2.9 µM at 1.5 µM, 3 µM and 6 µM GABA, respectively (Fig. 4b). In all cases, the GABA-induced currents without SCC-tau, increased in a dose-concentration fashion.

Fig. 4
figure4

Effect of Homo on GABA-induced currents in oocytes heterologously expressing GABAC-ρ1R. a Representative traces of currents induced by 1.5, 3, and 6 µM GABA and co-applied with Homo at the indicated concentrations. b Homo dose-response relation of currents elicited by 1.5, 3, and 6 µM GABA. The currents were normalized to the maximum amplitude elicited by the agonist in absence of modulators. Data points are the mean ± S.E. from at least 9 oocytes (n = 9) from 4 frogs (N = 4)

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We also analyzed whether the stimulatory effect of Hypo changed by the extracellular GABA concentration and we found that unlike Homo, Hypo potentiated GABA currents. As observed in Fig. 5a, at 1.5 µM GABA, Hypo induced a current increase, that was even greater than the current activated by GABA alone. At 3 µM GABA, the effect of Hypo was still present almost with the same characteristics as 1.5 µM GABA; however, at 6 µM GABA, the increase induced by Hypo on GABA currents diminished. GABA-induced current increased in a dose-concentration fashion. The EC50 values obtained for Hypo were 87 ± 1.5 µM, 170 ± 1.7 µM, and 480 ± 2.1 µM for 1.5 µM, 3 µM, and 6 µM GABA, respectively (Fig. 5b). These results suggest a competitive action of SCC-tau with GABA for the same binding site (Additional file 1: Figure S1). In the case of IA, we did not observe any effect (data not shown).

Fig. 5
figure5

Effect of Hypo on GABA-induced currents in oocytes heterologously expressing GABAC-ρ1R. a Representative traces of currents induced by 1.5, 3, and 6 µM GABA and co-applied with Homo at the indicated concentrations. b Hypo dose-response relation of currents elicited by 1.5, 3, and 6 µM GABA. The currents were normalized to the maximum amplitude elicited by the agonist in absence of a modulator. Data points are the mean ± S.E. from at least 9 oocytes (n = 9) from 4 frogs (N = 4)

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Molecular modeling

To prove theoretical studies on the interactions of the different ligands with the human GABAC-ρ1receptor, we performed homology modeling. We selected as target the structure of the GABAC-ρ1 R-beta3 homopentamer, since it has coverage of around 70% and an identity of 48%. The original sequence of this gene (GenBank: AAA52509.1) has an initial segment of 44 amino acids with a disordered/unstructured identity. We constructed this segment using an “ab inition” modeling, which shows good structural correlation. With this segment and the crystallographic structure, the GABAA-ρ1R model was constructed and validated (Additional file 2: Figure S2). This model presents an initial segment corresponding to the transmembrane section, which may have an effective recognition function. Subsequently, using this constructed model, we performed docking studies with different ligands (GABA, Homo, Hypo), IA, and one GABAA receptor antagonist (picrotoxin). For docking probes, we included taurine to compare the binding sites with SCC-tau and GABA. Figure 6 shows the results obtained after performing 1000 runs for each compound, observing three different binding sites. In the first one we located (BS1) GABA, taurine, Homo, Hypo, and IA; the second binding site (BS2) apparently is exclusive for Homo, additional to the site it shares with GABA and SCC-tau, and the third one (BS3) corresponds to picrotoxin, located inside the receptor. The binding parameters (Ki) obtained by AutoDock4 are in the mM range, except for picrotoxin that is of a lesser order of magnitude. The order of affinity predicted is as follows: picrotoxin (0.012 mM) < IA (3.25 mM) < Homo (3.56 mM) < GABA (8.48 mM) < Hypo (10.50 mM) < taurine (10.86 mM). There are different binding sites at the GABAC-ρ1R that function as modulators of the activity of this receptor. Therefore, we complemented the experimental results with theoretical studies, observing some correlation, such as: (1) the structural relationship between Homo and Hypo to activate GABAC-ρ1R, demonstrates that Homo is a more potent agonist than Hypo; also, it has a lower Ki than Hypo; (2) competition experiments between TPMPA and GABA by GABAC-ρ1R indicate that Homo and Hypo (even taurine), interact at the same site (BS1), supporting the data obtained by docking; (3) Homo has a second binding site (BS2), that is not used by GABA or SCC-tau; and (4) picrotoxin, an allosteric antagonist of ionotropic GABA receptors, its binding was predicted in the anion channel of the receptor (BS3), a unique site not used by the SCC-tau compounds with a strong affinity according to literature reports for this blocker [37, 38].

Fig. 6
figure6

Structural models of GABAA side view (a) and top view (b) of the receptor and results of docking with different ligands, represented by colored spheres GABA (red circle), Tau-SCC (black circle), Hypo (orange circle), IA (violet circle), Homo (green circle), and Picro (brown circle). The structures were drawn using the PyMOL and LIGPLOT v.4.5.3 programs

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Discussion

The use of SCC-tau, that possesses only some of the properties of taurine, has increased our understanding about the role of this β-amino acid in the cell physiology. Previous reports show that SCC-tau modulates the inhibitory action triggered by GABA [39,40,41]. However, the structural characteristics that determine these effects are unclear. In this work, we report that even though Homo, Hypo, and IA share similar core structures, Homo was a potent partial agonist of GABAC-ρ1R compared to Hypo. This observation could indicate that the presence of an additional carbon and the SO3H group in Cis position with respect to the NH2 group in Homo´s structure allows it to form a moiety that enhances the affinity and binding of Homo for GABAC-ρ1R. In contrast, it is probable that the absence of an NH2 group and the short carbon skeleton are responsible for the non-response effect with IA.

Oocytes that heterologously express the GABAC-ρ1R can generate currents when exposed to GABA, Homo, or Hypo. When SCC-tau and GABA were co-applied, we observed a concentration-dependent decrease (Homo) or increase (Hypo) of GABA induced-currents. Interestingly, in both cases, we did not observe a dual action of Homo and/or Hypo in the modulation of GABA-induced currents, as in the case of taurine [32]. In this sense, unlike the human glycine receptor (Gly-α1R), where taurine and other β-amino acids act like agonists or antagonists [42], in GABAA-ρ1R only taurine follows this pattern. One interpretation for this lack of dual effect of Homo and Hypo is that probably its action it is not associated to a change in the molecular structure of the compounds [42], and the positive and negative modulation on the GABA-induced current could be a result of the occupation of the agonist binding sites in the binding pocket by each of these compounds. A second possibility is and effect of either negative or positive cooperativity between GABA-Homo or GABA-Hypo.

In the case of taurine, it was previously reported that it generates a dual effect on GABA-induced currents, suggesting that this β-amino acid (at low concentrations) can compete with GABA for the same binding site [32]. This hypothesis is reinforced with our molecular model, where Hypo, Homo, IA, taurine and GABA share the same binding site. The fact that taurine inhibits GABA response at high concentrations [32], just like Homo in this work, could be due to an interaction of taurine and Homo with a second binding site.

These data suggest a competitive behavior of SCC-tau and, thus, reflects an antagonistic binding within a common ligand-binding pocket. The Hill coefficient obtained in both the negative and positive modulations generated by Homo and Hypo, respectively, indicates a contribution of GABA displacement despite its affinity to the binding site. This structural phenomenon would allow the interaction of SCC-tau in different binding conformations, inducing agonist or antagonist effects.

GABA induced-currents were modulated when SCC-tau and GABA were co-applied, while Homo reduced, Hypo increased the currents. This effect is explained with the data obtained from the docking, where Homo presents a second site with a greater binding probability, different from the binding site it shares with GABA; these two sites are located at different places of the receptor. Therefore, considering that GABA shares the same binding site as Hypo and Homo, this would confirm the fact that they compete for the same binding site as reflected in the experimental protocol. However, in the case of Homo, it does not share the second binding site with GABA, so there is no competition between them. Therefore, it is likely that this second Homo binding site may act as a negative regulatory site of the GABAC-ρ1R, when GABA activates the receptor.

Homo acts like an antagonist of GABA- and Hypo-induced currents; however, is a partial agonist at high concentrations without GABA or Hypo. This could be explained by negative cooperativity between Homo and GABA or Hypo, and this interaction is possibly due to a dependence of the second Homo binding site on the action of the agonist, that is available only when the first site (BS1) is previously occupied by GABA or Hypo. Therefore, it is likely that the affinity of Homo depends on the previous binding of a first agonist, which changes the conformational structure of the receptor.

Conclusions

Interest in receptors for γ-aminobutyric acid, the major inhibitory transmitter in the central nervous system, has been development over the last four decades. Given their widespread distribution, lower abundance and relative simplicity compared to GABAA and GABAB receptors, GABAC receptors are attractive drug targets. GABAC receptors pharmacology is different from GABAA and GABAB receptors, therefore, the development or characterization of novel compounds for GABAC receptor is imperative. This will allow to determine with certainty the function of this receptor in the central nervous system. In this sense, small molecules such as SCC-tau are compounds with great potential for this purpose. Here, we observed that SCC-tau are partial agonists of the GABAC-ρ1R that occupy the agonist binding site. The dual effect observed with SCC-tau was present in a separate way: while Hypo increases, Homo decreases the GABA-currents. These observations suggest that taurine could induce the dualistic effect by a change in the molecular structure in response to taurine concentration. However, the fact that Hypo and Homo act differently on GABA-currents could potentially be used to design pharmacological tools for the modulation of GABAergic receptors.

Methods

Expression of homomeric human GABAC-ρ1 receptors in Xenopus laevis oocytes

All the animals were handled in accordance with guidelines of the National Institutes of Health Guide for Care and Use of Laboratory Animals and with the approval of the Institutional Animal Care and Use Committee of the National University of Mexico. We used Ethyl 3-aminobenzoate methanesulfonate (MS-222) at 0.17% for 30 min, to anesthetize the Xenopus laevis frogs. The oocytes were manually removed and enzymatically defolliculated at room temperature using collagenase type I (0.3 μg/μl) for 45 min. Later, oocytes were kept in Barth’s medium (in mM): 88, NaCl; 1, KCl; 0.33, Ca2 (NO)3; 0.41, CaCl2; 0.82, MgSO4; 2.4, NaHCO3; 5, HEPES; pH 7.4 and 0.1 mg/ml gentamicin sulfate. After 24 h, 0.5 μg/μl of human GABAC-ρ1R mRNA was injected in vegetal hemisphere of the oocyte, and electrophysiological recordings were obtained 2–3 days after injection.

Voltage-clamp recordings

Membrane currents elicited by GABA and SCC-tau were recorded using the two-microelectrode voltage-clamp technique. Oocytes were placed in a chamber, with a volume of 500 μl, and impaled with two microelectrodes previously filled with 3 M KCl (0.5–2.5 MΩ) and clamped at − 60 mV. To determine the equilibrium membrane potential of the agonist action, I–V relationships were constructed by stepping the oocyte’s membrane potential from − 60 to − 120 mV for 1 s and then from − 60 to + 40 mV (in 20 mV steps) in the absence or presence of GABA or SCC-tau (Homo, Hypo, IA). All recordings were done at 20–23 °C in a chamber continuously perfused at 5–10 ml/min of Ringer solution (in mM): 115, NaCl; 2, KCl; 1.8, CaCl2; 5, HEPES, pH 7.4. All drugs were purchased from SIGMA-ALDRICH (San Louis Missouri, USA). The stock solution of GABA (1 M), Homo (0.5 mM), Hypo (0.5 mM) or IA (0.5 mM) was kept frozen until thawed for the experiments. The pH of all solutions was adjusted to 7.4.

Data analysis

Results are reported as mean ± S.E. of the values obtained from several cells, each value being the average of measurements in different cells. Data from each experiment were collected from at least seven oocytes. Agonist concentration–response curves were constructed by measuring the maximum response evoked by each agonist concentration. The half-maximal concentration (EC50) and Hill coefficient (nH) of GABA, Hypo, and Homo were estimated for each curve by fitting the data to the logistic type equation (Origin 6.0, Northampton, MA): \({\text{A}} = {\text{A}}_{ \text{max} }/(1 + 10^{{{{ [ {\text{logEC}}_{50}}}^{{{-}[{\text{agonist] nH}}}}}})\). The half-inhibitory concentration (IC50) of Homo was estimated by fitting the following equation: \({\text{A}} = {\text{ A}}_{ \text{max} } /(1 + 10^{{[[{\text{agonist}}] - {\text{logEC}}_{ 5 0} }} )\). Differences among groups were statistically analyzed by ANOVA and Tukey–Kramer post-test, and were considered significant when P < 0.05. Control responses to GABA were obtained before and after each drug application to account for possible shifts in the amplitude of the control current. To determine the time constants for the activation (τact) and deactivation (τdeac) of GABA-, Homo- and Hypo-currents responses, a decay function of the form: I(τ) = exp(− t/τ d) + C, where I is the current and t is time, was fit to the experimental data (Origin 6.0 software; Northampton, MA). Differences between groups were statistically analyzed by ANOVA and a Tukey–Kramer post-test, and were considered significant at the level P < 0.05.

Homology modeling of Type A gamma-aminobutyric acid (GABAA) receptors (Homo sapiens)

This homology modeling started with the retrieval of the amino acid sequence of GABAA (GenBank: AAA52509.1). The mature protein, predicted from this cDNA sequence in 458 amino acids, displays between 30 and 38% amino acid similarity to the previously identified GABAA subunits [29]. Using the program Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi) [43], and the database of the Protein Data Bank, the structural model was built based on the structure of a human gamma-aminobutyric acid receptor, the Gaba(a) r-beta3 homopentamer (4COF.pdb) [44], which revealed a coverage of 70% and the highest sequence identity (48%) with the target. A multiple alignment was performed using the constraint-based multiple alignment tool (COBALT; https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) server, where an initial segment of 44 amino acids with no structural identity appears. An ab initio model of this fragment was made using the Rosetta 3.4 program [45]; 1000 models of this segment were made. Subsequently, using MODELLER 9.17 r10881 with the multiple models protocol, 500 GABAA models were constructed, using the most stable model of the peptide and the 4COF.pdb file as a template. Later, a simple structural refinement of full atom was performed using “relax” application of Rosetta. The final model was validated using the Verify-3D (structure evaluation software) and Whatcheck (protein verification tools software) computer programs [46, 47].

Docking protocol

Docking was made with the previously constructed three-dimensional structure (Type A gamma-aminobutyric acid receptors—GABAA), and the ligands were built with HyperChem 8 software. The ligands were minimized employ Gaussian 09, revision A.02 (Gaussian Inc., Wallingford, CT) at DTF B3LYP/3-21G level of theory. AutoDockTools 1.5.4 (http://mgltools.scripps.edu/), it was used to prepare the structures before carrying out the docking. The preparation of the structures consisted in adding all hydrogen atoms as well as the Kollman united-atom partial charges to the receptor, while Gasteiger–Marsili charges and rotatable groups were assigned automatically to the ligands. AutoGrid4 was occupying to generate the electrostatic grid maps for each atom type. The initial grid box size was 60 Å × 60 Å × 60 Å in the x, y, and z dimensions. Docking was produced out with AutoDock4 version 4.2 (http://autodock.scripps.edu/) [48, 49] using default parameters; for the Lamarckian genetic algorithm with local search, number of individuals in population (150), maximum number of energy evaluations (2.5 million), maximum number of generations (27,000), rate of gene mutation (0.02), rate of crossover (0.8), and 1000 runs for docking. Finally, the docking was analyzed with AutoDockTools using cluster analysis and PyMOL software [50].

Abbreviations

GABA:

γ-aminobutyric acid

Taurine:

2-Aminoethanesulfonic acid

Homotaurine:

3-Aminopropane-1-sulfonic acid

Hypotaurine:

2-Aminoethanesulfinic acid

Isethionic acid:

2-hydroxyethanesulfonic acid

TPMPA:

Methyl(1,2,5,6-tetrahydropyridine-4-yl) phosphinic acid

MS-222:

Ethyl 3-aminobenzoate methanesulfonate

NMDA:

N-methyl-d-aspartate

HEPES:

4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid

SCC-tau:

sulfur-containing compounds structurally related to taurine

References

  1. 1.

    Komarnisky LA, Christopherson RJ, Basu TK. Sulfur: its clinical and toxicologic aspects. Nutrition. 2003;19(1):54–61.

    Article  PubMed  CAS  Google Scholar 

  2. 2.

    Atmaca G. Antioxidant effects of sulfur-containing amino acids. Yonsei Med J. 2004;45(5):776–88.

    Article  PubMed  CAS  Google Scholar 

  3. 3.

    Sturman JA. Taurine in development. Physiol Rev. 1993;73(1):119–47.

    Article  PubMed  CAS  Google Scholar 

  4. 4.

    Pasantes-Morales H, Franco R, Torres-Marquez ME, Hernandez-Fonseca K, Ortega A. Amino acid osmolytes in regulatory volume decrease and isovolumetric regulation in brain cells: contribution and mechanisms. Cell Physiol Biochem. 2000;10(5–6):361–70.

    Article  PubMed  CAS  Google Scholar 

  5. 5.

    Pasantes-Morales H. Volume regulation in brain cells: cellular and molecular mechanisms. Metab Brain Dis. 1996;11(3):187–204.

    Article  PubMed  CAS  Google Scholar 

  6. 6.

    Pasantes-Morales H, Schousboe A. Release of taurine from astrocytes during potassium-evoked swelling. Glia. 1989;2(1):45–50.

    Article  PubMed  CAS  Google Scholar 

  7. 7.

    Del Olmo ND, Galarreta M, Bustamante J, Martin del Rio R, Solis JM. Taurine-induced synaptic potentiation: dependence on extra- and intracellular calcium sources. Adv Exp Med Biol. 2000;483:283–92.

    Article  PubMed  Google Scholar 

  8. 8.

    Lombardini JB. Taurine: retinal function. Brain Res Brain Res Rev. 1991;16(2):151–69.

    Article  PubMed  CAS  Google Scholar 

  9. 9.

    Pasantes-Morales H, Gamboa A. Effect of taurine on 45Ca2+ accumulation in rat brain synaptosomes. J Neurochem. 1980;34(1):244–6.

    Article  PubMed  CAS  Google Scholar 

  10. 10.

    Bureau MH, Olsen RW. Taurine acts on a subclass of GABAA receptors in mammalian brain in vitro. Eur J Pharmacol. 1991;207(1):9–16.

    Article  PubMed  CAS  Google Scholar 

  11. 11.

    Horikoshi T, Asanuma A, Yanagisawa K, Goto S. Taurine modulates glycine response in Xenopus oocytes injected with messenger RNA from mouse brain. Brain Res. 1988;464(3):243–6.

    PubMed  CAS  Google Scholar 

  12. 12.

    Polenzani L, Woodward RM, Miledi R. Expression of mammalian gamma-aminobutyric acid receptors with distinct pharmacology in Xenopus oocytes. Proc Natl Acad Sci U S A. 1991;88(10):4318–22.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. 13.

    Woodward RM, Polenzani L, Miledi R. Effects of steroids on gamma-aminobutyric acid receptors expressed in Xenopus oocytes by poly(A)+ RNA from mammalian brain and retina. Mol Pharmacol. 1992;41(1):89–103.

    PubMed  CAS  Google Scholar 

  14. 14.

    Woodward RM, Polenzani L, Miledi R. Effects of hexachlorocyclohexanes on gamma-aminobutyric acid receptors expressed in Xenopus oocytes by RNA from mammalian brain and retina. Mol Pharmacol. 1992;41(6):1107–15.

    PubMed  CAS  Google Scholar 

  15. 15.

    Zhu WJ, Vicini S. Neurosteroid prolongs GABAA channel deactivation by altering kinetics of desensitized states. J Neurosci. 1997;17(11):4022–31.

    Article  PubMed  CAS  Google Scholar 

  16. 16.

    Woodward RM, Polenzani L, Miledi R. Characterization of bicuculline/baclofen-insensitive gamma-aminobutyric acid receptors expressed in Xenopus oocytes. I. Effects of Cl- channel inhibitors. Mol Pharmacol. 1992;42(1):165–73.

    PubMed  CAS  Google Scholar 

  17. 17.

    Feigenspan A, Bormann J. Differential pharmacology of GABAA and GABAC receptors on rat retinal bipolar cells. Eur J Pharmacol. 1994;288(1):97–104.

    Article  PubMed  CAS  Google Scholar 

  18. 18.

    Lukasiewicz PD, Maple BR, Werblin FS. A novel GABA receptor on bipolar cell terminals in the tiger salamander retina. J Neurosci. 1994;143(Pt 1):1202–12.

    Article  Google Scholar 

  19. 19.

    Lukasiewicz PD, Wong RO. GABAC receptors on ferret retinal bipolar cells: a diversity of subtypes in mammals? Vis Neurosci. 1997;14(5):989–94.

    Article  PubMed  CAS  Google Scholar 

  20. 20.

    Pan ZH, Lipton SA. Multiple GABA receptor subtypes mediate inhibition of calcium influx at rat retinal bipolar cell terminals. J Neurosci. 1995;15(4):2668–79.

    Article  PubMed  CAS  Google Scholar 

  21. 21.

    Ragozzino D, Woodward RM, Murata Y, Eusebi F, Overman LE, Miledi R. Design and in vitro pharmacology of a selective gamma-aminobutyric acidC receptor antagonist. Mol Pharmacol. 1996;50(4):1024–30.

    PubMed  CAS  Google Scholar 

  22. 22.

    McKernan RM, Whiting PJ. Which GABAA-receptor subtypes really occur in the brain? Trends Neurosci. 1996;19(4):139–43.

    Article  PubMed  CAS  Google Scholar 

  23. 23.

    Wassle H, Koulen P, Brandstatter JH, Fletcher EL, Becker CM. Glycine and GABA receptors in the mammalian retina. Vis Res. 1998;38(10):1411–30.

    Article  PubMed  CAS  Google Scholar 

  24. 24.

    Harvey VL, Duguid IC, Krasel C, Stephens G. Evidence tha GABAρ subunits contribute to functional ionotropic GABA receptors in mouse Purkinje cells. J Physiol. 2006;577(Pt 1):127–39.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. 25.

    Mejía C, García-Alcocer G, Berumen LC, Rosas-Arellano A, Miledi R, Martínez-Torres A. Expression of GABArho subunits during rat cerebellum development. NeurosciLett. 2008;432(1):1–6.

    Google Scholar 

  26. 26.

    Flores-Gracia C, Nuche-Bricaire A, Crespo-Ramírez M, Miledi R, Fuxe K, Pérez de la Mora M. GABA(A) ρ receptor mechanisms in the rat amygdala and its role in the modulation of fear and anxiety. Psychopharmacology. 2010;212(4):475–84.

    Article  PubMed  CAS  Google Scholar 

  27. 27.

    Koulen P, Brandstatter JH, Enz R, Bormann J, Wassle H. Synaptic clustering of GABA(C) receptor rho-subunits in the rat retina. Eur J Neurosci. 1998;10(1):115–27.

    Article  PubMed  CAS  Google Scholar 

  28. 28.

    Qian H, Dowling JE. GABA responses on retinal bipolar cells. Biol Bull. 1993;185(2):312.

    Article  PubMed  CAS  Google Scholar 

  29. 29.

    Cutting GR, Lu L, O’Hara BF, Kasch LM, Montrose-Rafizadeh C, Donovan DM, Shimada S, Antonarakis SE, Guggino WB, Uhl GR, et al. Cloning of the gamma-aminobutyric acid (GABA) rho 1 cDNA: a GABA receptor subunit highly expressed in the retina. Proc Natl Acad Sci U S A. 1991;88(7):2673–7.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. 30.

    Ogurusu T, Shingai R. Cloning of a putative gamma-aminobutyric acid (GABA) receptor subunit rho 3 cDNA. Biochim Biophys Acta. 1996;1305(1–2):15–8.

    Article  PubMed  Google Scholar 

  31. 31.

    Olsen RW, Bureau M, Khrestchatisky M, MacLennan AJ, Chiang MY, Tobin AJ, Xu W, Jackson M, Sternini C, Brecha N. Isolation of pharmacologically distinct GABA-benzodiazepine receptors by protein chemistry and molecular cloning. Adv Biochem Psychopharmacol. 1990;46:35–49.

    PubMed  CAS  Google Scholar 

  32. 32.

    Ochoa-de la Paz LD, Martinez-Davila IA, Miledi R, Martinez-Torres A. Modulation of human GABArho1 receptors by taurine. Neurosci Res. 2008;61(3):302–8.

    Article  PubMed  CAS  Google Scholar 

  33. 33.

    Wilde MI, Wagstaff AJ. Acamprosate. A review of its pharmacology and clinical potential in the management of alcohol dependence after detoxification. Drugs. 1997;53(6):1038–53.

    Article  PubMed  CAS  Google Scholar 

  34. 34.

    McCarty MF. The reported clinical utility of taurine in ischemic disorders may reflect a down-regulation of neutrophil activation and adhesion. Med Hypotheses. 1999;53(4):290–9.

    Article  PubMed  CAS  Google Scholar 

  35. 35.

    Kendler BS. Recent nutritional approaches to the prevention and therapy of cardiovascular disease. Prog Cardiovasc Nurs. 1997;12(3):3–23.

    PubMed  CAS  Google Scholar 

  36. 36.

    Della Corte L, Crichton RR, Duburs G, Nolan K, Tipton KF, Tirzitis G, Ward RJ. The use of taurine analogues to investigate taurine functions and their potential therapeutic applications. Amino Acids. 2002;23(4):367–79.

    Article  PubMed  CAS  Google Scholar 

  37. 37.

    Zhang D, Pan ZH, Zhang X, Brideau AD, Lipton SA. Cloning of a gamma-aminobutyric acid type C receptor subunit in rat retina with a methionine residue critical for picrotoxinin channel block. Proc Natl Acad Sci U S A. 1995;92(25):11756–60.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. 38.

    Wang TL, Hackam AS, Guggino WB, Cutting GR. A single amino acid in gamma-aminobutyric acid rho 1 receptors affects competitive and noncompetitive components of picrotoxin inhibition. Proc Natl Acad Sci U S A. 1995;92(25):11751–5.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. 39.

    Fariello RG, Golden GT. Homotaurine: a GABA agonist with anticonvulsant effects. Brain Res Bull. 1980;5:691–9.

    Article  CAS  Google Scholar 

  40. 40.

    Frosini M, Sesti C, Dragoni S, Valoti M, Palmi M, Dixon HBF, Machetti F, Sgaragli G. Interactions of taurine and structurally releated analogues with the GABAergic system and taurine binding sites of rabbit brain. Br J Pharmacol. 2003;138(6):1163–71.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. 41.

    El Idrissi A, Trenkner E. Taurine as modulator of excitatory and inhibitory neurotransmission. Neurochem Res. 2004;29(1):189–97.

    Article  PubMed  Google Scholar 

  42. 42.

    Schmieden V, Betz H. Pharmacology of the inhibitory glycine receptor: agonist and antagonist actions of amino acids and piperidine carboxylic acid compounds. Mol Pharmacol. 1995;48(5):919–27.

    PubMed  CAS  Google Scholar 

  43. 43.

    Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25(17):3389–402.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. 44.

    Miller PS, Aricescu AR. Crystal structure of a human GABAA receptor. Nature. 2014;512(7514):270–5.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. 45.

    Bradley P, Misura KM, Baker D. Toward high-resolution de novo structure prediction for small proteins. Science. 2005;309(5742):1868–71.

    Article  PubMed  CAS  Google Scholar 

  46. 46.

    Luthy R, Bowie JU, Eisenberg D. Assessment of protein models with three-dimensional profiles. Nature. 1992;356(6364):83–5.

    Article  PubMed  CAS  Google Scholar 

  47. 47.

    Hooft RW, Vriend G, Sander C, Abola EE. Errors in protein structures. Nature. 1996;381(6580):272.

    Article  PubMed  CAS  Google Scholar 

  48. 48.

    Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson AJ. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem. 1998;19(14):1639–62.

    Article  CAS  Google Scholar 

  49. 49.

    Huey R, Morris GM, Olson AJ, Goodsell DS. A semiempirical free energy force field with charge-based desolvation. J Comput Chem. 2007;28(6):1145–52.

    Article  PubMed  CAS  Google Scholar 

  50. 50.

    DeLano WL. Use of PYMOL as a communications tool for molecular science. Abstr Pap Am Chem Soc. 2004;228:U313–4.

    Google Scholar 

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Authors’ contributions

LDOP conceived the project and coordinate the experiments, and did the electrophysiology and pharmacology assays in oocytes; RF did microinjections of mRNA into the oocytes; MG-A did the “in silico” model and docking probes; RZ-A, HP-M, and RG-S. did the purification and extraction of the DNA, and synthesized the mRNA; EZ, HQ-M, and RG-C did the statistics probes. All authors read and approved the final manuscript.

Acknowledgements

Authors want to thank Dr. Rogelio Arellano for electrophysiology equipment and Dr. Ataulfo Martinez-Torres for plasmids carrying the human GABAC-ρ1 receptor, and the facilities granted by Departamento de Bioquímica, Facultad de Medicina, UNAM and Departamento de Investigación APEC-UNAM, Asociación para Evitar la Ceguera en México I.A.P., Hospital Dr. Luis Sánchez Bulnes. The authors are very grateful to Mrs. Maria Josefina Bolado Garza from the División de Investigación, Facultad de Medicina, UNAM for the review and edition of the English version of this manuscript.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The data set during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Consent for publication

Not applicable.

Ethics approval and consent to participate

All the animals were handled in accordance with guidelines of the National Institutes of Health Guide for Care and Use of Laboratory Animals and with the approval of the Institutional Animal Care and Use Committee of the National University of Mexico.

Funding

This work was supported by grants from PAPIIT-UNAM (IA208116 and IA205918), LANCAD-UNAM-DGTIC-313 and Departamento de Investigación APEC-UNAM, Asociación para Evitar la Ceguera en México I.A.P. Hospital “Dr. Luis Sánchez Bulnes”.

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Affiliations

  1. Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Avenida Universidad 3000, Copilco Universidad, Cd. Universitaria, 04510, México City, México

    Lenin David Ochoa-de la Paz, Martin González-Andrade & Edgar Zenteno

  2. Departamento de Investigación APEC, Asociación para Evitar la Ceguera en México I.A.P. Hospital Dr. Luis Sánchez Bulnes, Fernández Leal 60, Col. La Concepción Coyoacán, 04020, Mexico City, Mexico

    Lenin David Ochoa-de la Paz, Rubén Zamora-Alvarado, Hugo Quiroz-Mercado, Roberto Gonzales-Salinas & Rosario Gulias-Cañizo

  3. División de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Coyoacán, Ciudad Universitaria, 04510, Mexico City, Mexico

    Herminia Pasantes-Morales

  4. Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE, 68583, USA

    Rodrigo Franco

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  1. Lenin David Ochoa-de la Paz
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  2. Martin González-Andrade
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Corresponding author

Correspondence to Lenin David Ochoa-de la Paz.

Additional files

Additional file 1: Figure S1.

Effect of Homo on Hypo-induced currents in oocytes heterologously expressing GABAC-ρ1R. (A) Representative traces of currents induced by 30 µM, 3 mM and 30 mM Hypo and co-applied with Homo at the indicated concentrations. (B) Homo dose-response relation of currents elicited by 30 µM, 3 mM and 30 mM Hypo. The currents were normalized to the maximum amplitude elicited by the agonist in absence of modulators. Data points are the means ± S.E. from at least 9 oocytes (n = 9) from 4 frogs (N = 4).

Additional file 2: Figure S2.

Three-dimensional models of GABAA and template. In slate blue cartoon (GABAA), splitpea green cartoon (template) (Protein Data Bank [PDB] code: 4COF). The structures were drawn using the PyMOL program.

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Ochoa-de la Paz, L.D., González-Andrade, M., Pasantes-Morales, H. et al. Differential modulation of human GABAC-ρ1 receptor by sulfur-containing compounds structurally related to taurine. BMC Neurosci 19, 47 (2018). https://doi.org/10.1186/s12868-018-0448-6

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Keywords

  • Receptor modulation
  • GABA receptor
  • Xenopus oocytes
  • Sulfur-containing compounds
  • Taurine
  • Homotaurine
  • Hypotaurine
  • Isethionic acid

BMC Neuroscience

ISSN: 1471-2202

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