Abstract
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.
Similar content being viewed by others
Introduction
γ-Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the central nervous system (CNS), and its co-localization with other neurotransmitter pathways has been fully demonstrated. Interestingly, the co-release of GABA and dopamine (DA) has been reported in the striatum and retina1,2; however, the physiological consequences of this convergence are not fully understood. There are two main components that give rise to the ionotropic GABA responses. The first component is the most abundant GABA-A receptor widely distributed throughout the nervous system and effectively blocked by the alkaloid bicuculline. This receptor is formed by a combination of α, β and γ subunits and by other less abundant subunits (δ, θ, ε, π)3. The second component does not desensitize after prolonged exposure to the agonist, is insensitive to bicuculline4,5 and the (1,2,5,6-Tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) is a competitive antagonist6; is abundantly expressed in retina and has also been found in several areas of the brain; for example, the cerebellum7, hippocampus8 and striatum9. This GABA receptor is formed by ρ subunits (ρ1–ρ3) and is commonly known as GABA-C10,11. Today we know that the three GABAρ subunits are phylogenetically related to classic GABA-A subunits (α, β, γ) included in the Cys-loop family of neurotransmitter receptors, which form pentameric assemblies and gate a chloride channel upon activation. In addition, ρ1 subunits assemble with classic GABA-A subunits and form heteromeric complexes with different characteristics12,13,14.
One of the special characteristics of the human GABAρ1 receptor reported in a previous communication showed a negative modulation by monoamines such as DA, serotonin and tyramine15. The GABAρ1 subunit cloned from Sus scrofa 16 is also modulated by DA, thus, the effect does not seem to be species specific. More recently, it was found that GABA-A receptors from striatal neurons are efficiently gated by DA; however, the effect is dependent on the presence of the GABA-A β3 subunit17. Another example of cross-talk between the GABAergic system and other neurotransmitters involves the allosteric potentiation of ATP on GABA-A receptors18. This interaction is not exclusive to the GABAergic system, since similar effects have been described in the past between neurotransmitters such as the serotoninergic modulation of nicotinic receptors19,20.
The Cys-loop family of neurotransmitter receptors includes several ionotropic DA receptors, such as LGC-53, isolated from Caenorhabditis elegans 21, and other DA receptors known from invertebrates22,23. These findings suggest that during the evolution of the Cys-loop family of receptors, the mutations that increased sensitivity and specificity for an agonist24 did not necessarily fully eliminate the recognition for other ligands. The presence of a second ligand-binding site makes it possible to diversify the neurotransmitter signaling and would allow the receptor to be targeted not only by the second neurotransmitter but also by structurally similar molecules.
LGC-53 is activated by DA but not by serotonin (5HT), octopamine, tyramine or histamine, yet it is sensitive to modulators of mammalian metabotropic DA receptors such as haloperidol, risperidone and spiperone21. LGC-53 has several characteristics in common with GABA-A receptors17, and in particular with GABAρ1. For example, it is permeable to Cl− and forms functional homopentamers when expressed in heterologous systems. These characteristics make this receptor suitable for comparative functional and structural studies.
In this communication, we report the negative modulation of GABAρ1 by DA metabolites: 3-methoxytyramine (3-MT), 3, 4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and structural DA analogues 2-phenylethylamine (β-PEA) and 4-amino-1-butanol (4-AM-1-OH). In addition, we tested whether these compounds gate the inotropic DA receptor LGC-53. Our findings extend the list of compounds that act as modulators of GABAρ1 and suggest that these molecules may execute some effect in the GABAergic system in vivo, where they are commonly found (e.g., the brain25 and retina26).
Results
Oocytes injected with ρ1 generated non-desensitizing ionic currents upon perfusion of GABA, whereas those injected with LGC-53 generated fast, desensitizing ionic currents when DA was perfused. Currents showing run-down or run-up (>20% of change) during three consecutive applications were discarded. Non-injected oocytes and oocytes injected with human ρ1 did not generate evident currents when exposed to up to 1 mM DA.
GABA currents were negatively and concentration-dependently modulated by DA metabolites like 3-MT (IC50 = 285 ± 30 µM, R2 = 0.986), DOPAC (IC50 = 2.25 ± 0.26 mM, R2 = 0.977) and HVA (IC50 = 1.97 ± 0.20 mM, R2 = 0.976). Representative recordings and concentration-response curves are shown in Fig. 1A–C (Eq. 1, 2 and 3). Other DA analogues modulated the GABA currents negatively (Fig. 1B–C) but were less potent; for example, 4-AM-1-OH (IC50 = 3.91 ± 0.54 mM, R2 = 0.978) and β-PEA (IC50 = 1.79 ± 0.10 mM, R2 = 0.957). This result shows that 3-MT inhibits the GABA currents with similar potency to DA and is more potent than other previously tested biogenic amines such as 5 hydroxy-tryptamine, tyramine and octopamine15. L-DOPA, adrenaline (AD) and noradrenaline (NAD) were evaluated at concentrations up to 20 mM and failed to elicit any effect on the responses generated by 3 μM GABA (data not shown). To determine that the inhibitory effect is not due to the oocyte expression system, we evaluated whether DA and 3-MT inhibit GABA responses of GABAρ1 expressed in HEK cells. As shown in Supplementary Fig. S1, the inhibitory effect was similar to that in oocytes.
Full inhibition of GABA currents was observed at high concentrations of 3-MT and DA (Fig. 2A), while the rest of the compounds did not fully block the GABA currents and consistently showed residual responses (Fig. 2B and C), suggesting a partial antagonism27. Data obtained from the concentration-response curves at the maximum inhibition was compared with the full inhibition (100%, Eq. 3) for each compound. The comparison revealed that DOPAC and HVA, which has an acidic moiety, was significantly different from the full inhibition (Fig. 2D). The summary (mean and SEM) of maximal effects of the analogues from concentration-response curves are indicated in the corresponding bar in Fig. 2D.
All these compounds are ionized at pH 7.4. In order to determine the effect of the ligand charge on the antagonist-receptor interaction, a current-voltage ramp protocol was performed from −120 to +70 mV in presence of the IC50 concentration for each compound (n = 6 oocytes, Supplementary Fig. 2). The results obtained did not show significant differences in their inhibitory effects on the ramp voltage, indicating that the formation of the receptor-compound complex is insensitive to the voltage and suggesting an independent mechanism of the pore-blockage inhibition in agreement with previous reports for DA15. Additionally, the reversal potential observed here for GABA currents was consistent with other reports28,29. This reversal potential did not change in presence of these compounds (−35.25 ± 1.04 mV, −35.69 ± 1.26 mV, −35.31 ± 0.90 mV, −37.05 ± 2.32 mV and −36.25 ± 1.03 mV, for 3-MT, HVA, β-PEA and 4-AM-1-OH, GABA alone, −35.07 ± 0.90 mV, Supplementary Fig. S2).
Concentration-response curves for the competition assays between 3-MT and GABA were performed in presence of three different concentrations of 3-MT (IC25, IC50 and IC75; Fig. 3A). These showed a shift to the right of the curves as the concentration of 3-MT increased (Fig. 3B), without significant change in the Hill number (1.38 ± 0.24 at IC25, 1.40 ± 0.27 at IC50 and 1.90 ± 0.36 at IC75); these values are statistically non-significant as compared to GABA alone (1.34 ± 0.37). The GABA EC50 (2.27 ± 0.40 µM) was higher as the concentration of 3-MT increased; IC25 = 2.68 ± 0.4; IC50 = 3.50 ± 0.74; and IC75 = 8.09 ± 0.65. β-PEA was also evaluated and the results are similar to those found for 3-MT and are shown in Supplementary Figs S3 and S4.
The results obtained in the competition assays led us to explore in a molecular docking model the possible ways in which the competitive antagonists bind to the GABA site. For these tests, the GABAρ1 structural model described by Limon et al.29 was used as a template to evaluate 3-MT and DOPAC. The GABA pocket is immersed within the inter-subunit interface. It is made up of 20 residues that are directly or indirectly involved in GABA binding30; two of these residues, located in one of each subunit (Y198 in the + subunit and S168 in the − subunit), are involved in the recognition of the amine group of GABA. The acidic group of GABA is bound to R10431 and T24432 (in the − and + subunit, respectively) (Fig. 4A). Figure 4 shows the GABA binding pocket occupied by GABA at distances no larger than 6 Å, where the amino moiety of GABA is at 2.18 and 3.17 Å from Y198 and S168, respectively; whereas the distances from the GABA acidic region to T244 and R104 are 3.35 and 4.27 Å, respectively (Fig. 4B). Similar observations were previously reported by Harrison and Lummis33. 3-MT fitted the GABA binding site and potentialy binds to Y198 (3.1 Å), S168 (3.2 Å), T244 (7.8 Å) and R104 (6.4 Å) (Fig. 4C). DOPAC also fitted inside the GABA binding site interacting with the same residues: Y198 (7.5 Å), S168 (7.2 Å), T244 (3.48 Å) and R104 (4.98 Å) (Fig. 4D).
We tested whether DA and its analogues modulated GABAρ1 while maintaining the receptor activated with GABA 3 µM; co-application of DA analogues during GABA perfusion are shown in the representative traces (Fig. 5A,B). All compounds maintained the negative modulation on activated GABAρ1. Molecules with amine moieties showed a tendency to decrease their potency (3-MT and β-PEA), but not in a statistically significant manner since the decay was only 2-fold. However, DA showed the most significant decrease, around 10-fold, whereas the potency of 4-AM-1-OH increased by about 25% and was not statistically significant (Fig. 5C). Molecules with an acidic moiety did not show changes in their potency (Fig. 5B, Eq. 3). Efficacy to inhibit GABA responses under the co-application protocol was considerably lower for DA, 3-MT, β-PEA and 4AM–1-OH, which decreased to 61.94 ± 6.66, 51.65 ± 6.93, 63.62 ± 11.44 and 65.50 ± 4.77%, respectively (all changes are significantly different; Fig. 5A,D).
These results suggest a different binding site or inhibitory mechanism that depends on the presence of an amine or acid moiety in the antagonist. To determine whether the binding sites or action mechanism are the same for the open and closed state of the receptor we constructed isobolograms34 in three experimental conditions: GABA/analogue1, GABA/analogue2, and GABA/analogue1/analogue2. The theoretical value was obtained from the addition of the individual effects of each analogue in the same oocyte and compared to the effect found in the experimental condition of the mix of both analogues in the same cell. The isobolograms were derived from assays of comparing competency between: (1) GABA and 3-MT and (2) GABA, 3-MT and β-PEA. Figure 6(A,C and E) shows representative traces of the recordings from this series of assays. The first part revealed that the inhibition by the combination 150 µM 3-MT and 1.5 mM DOPAC is more potent than the individual effects of each molecule when the receptor is activated or not (Fig. 6A and B).
In the second part we assessed the interactions between 3-MT/GABA, β-PEA/GABA and 3-MT/β-PEA/GABA. Representative recordings from oocytes expressing GABAρ1 and exposed to 3 µM GABA either alone or with 150 µM 3-MT and/or 1.5 mM β-PEA are shown in Fig. 6C and E.
In contrast to the effect of DOPAC, the addition of β-PEA did not inhibit or potentiate the effect of 3-MT on GABA responses, either when the molecules were perfused all together with GABA or when the receptor was first activated by 3 µM GABA. Figure 6D and F summarizes the results described above.
Finally, to determine whether the DA analogues activate an ionotropic DA receptor, and thus infer if the agonist-binding site fits these molecules, we expressed LGC-53 in frog oocytes. LGC-53 gates a chloride channel that inactivates promptly and exhibits little or no desensitization after consecutive applications of DA21. Neither GABA nor 3-MT gated the channel or showed an evident modulatory effect even at high concentrations (up to 1 mM). DA EC50 (4.15 ± 1.1 µM) was similar to the previously reported21, whereas NAD activated the receptor with an EC50 = 139 ± 35 µM and was more potent than other biogenic amines such as β-PEA (1.63 ± 0.15 mM), Tyr (2.22 ± 0.16 mM) and AD (3.8 ± 0.8 mM, Fig. 7B). None of these amines acted like a full agonist27 (Fig. 7A and C). Figure 7A shows sample recordings of oocytes expressing LGC-53 and exposed to several DA analogues. Figure 7B plots the corresponding concentration-response relations, and Fig. 7C compares the maximal effect for each DA analogue.
Discussion
The results shown here indicate that there may be some interactions between DA and DA metabolites with GABAρ1. In frog oocytes, the effect of DA metabolites on GABAρ1 was concentration-dependent and voltage-independent. These observations extend those described in earlier papers15,16 and other evidence that showed that GABA-A receptors are gated by DA in the striatum and other expression systems17. Adding to these observations, it was previously demonstrated that DA indirectly modulates GABAρ receptors in isolated cone horizontal cells of catfish35 and at the bipolar cell terminals of tiger salamander retina36. These findings provided strong evidence for DA modulation of GABA receptor function in the nervous system; however it remains to be explored whether the direct and indirect DA activation/modulation of ionotropic GABA receptors depends on the different subunit combinations that form the receptor in the retina and striatum. Given the present evidence this should be regarded only as a tentative explanation. On the other hand, the role of GABAρ in neural inhibition is well known in retina13 and other areas of the nervous system, in which GABA and DA are co-realeased1 and where DA can reach concentrations of up to 1.6 mM in synaptic cleft37. Our observations were consistent in multiple experiments in different frog donors and RNA preparations, however the question remains whether the metabolites of DA modulate GABAρ1 in vivo.
Compared to the other DA metabolites evaluated here, 3-MT turned out to be the most potent modulator of GABAρ1, with a similar potency to DA, suggesting that the methyl moiety in 3-MT does not interfere in ligand binding and may even be important for selectivity of the molecule on ρ1, as described by Krall38 with imidazolic core antagonists. Therefore, it is possible that the substitution of the catechol core by a benzodioxol could maintain the effect of DA on the ρ1 receptor as well as the selectivity of 3-MT.
The compounds that showed a modulatory effect on GABAρ1 can be chemically grouped into those that include an amine (3-MT, β-PEA and 4-AM-1OH) or an acidic group (DOPAC and HVA). Since GABA contains both moieties, which interact with the binding site in the receptor, it is feasible for the three compounds to elicit some effect on the receptor. Although none of the tested molecules gate the ion channel of GABAρ1, these three molecules bind to the receptor when it is already in the open conformation; thus, their effect is explained by the structural differences that fluctuate during the cycle of activation-inactivation of GABAρ1. Clear differences in the modulation were observed when DA and DA metabolites were applied after exposing GABAρ1 to GABA. In this protocol, 3-MT showed the largest difference in efficacy as compared to the rest of the compounds.
On the other hand, the structure of HVA and DOPAC includes an acidic moiety and they did not show differences in the efficacy or potency. This is particularly interesting since two other GABAρ1 antagonists with an acidic moiety, (±) −4-ACPAM and SR-95813, show the same effect39. DA and 3-MT showed differences in their effect when applied while the receptor was activated or not. This effect is similar to that observed for ginkgolides at GABAρ1. It has been suggested that ginkgolides have two binding sites which are selectively exposed depending on the conformational state of the receptor, thus conferring different affinities for the open and closed states of the channel40.
The competition assays indicated that 3-MT, β-PEA and DOPAC share the same binding site with GABA; however, the isobolograms and docking suggested two different mechanisms for inhibition as explained below:
-
(1)
Steric impediment. The 3-MT (antagonist, amine) anchored to the GABA binding site prevents the binding of GABA and the activation of GABAρ1. The residues (Y198, S168) described by Sedelnikova30 are responsible for the recognition of the amine moiety of GABA. The S168A mutation in GABAρ1 generates functional receptors but 300 less sensitive to the agonist31, suggesting that this site is involved in the stabilization of the agonist. Docking modeling shows that 3-MT potentially interacts with the S168 and Y198 residues (at about 5 Å), whereas distances to R104 and T244 residues (involved in channel opening) are considerably larger (more than 7 Å). However, when the agonist-receptor complex is established, 3-MT has a low efficacy to displace the GABA from its binding site.
-
(2)
Reduced efficacy by weak interactions. The effect of DOPAC is dependent on the presence of GABA; however, DOPAC does not fully inhibit the GABA currents generated on GABAρ1. This may be explained if the receptor reaches a reduced state of conductivity. Previous reports showed that residues T224 and R104 are critical for interacting with the acidic moiety of GABA and channel gating. Mutations in R104 induce a reconfiguration of the GABA binding site unable to gate the channel32, whereas mutation T224A reduces the efficacy of GABA33. In our docking model, DOPAC is less than 5 Å from T224 and R104, but the larger distance to Y198 and S168 (more than 7 Å) would make the hydrogen bonds unstable. This structural conformation would be associated to a reduced efficacy and/or a weak interaction with the GABA binding site, thus explaining the remaining current at high concentrations of DA analogues.
On the other hand, it was unexpected to find the modulation of GABAρ1 by 4-AM-1-OH. This can be explained given the similarity with the monoamines and the GABA, in addition the comparison of the alcohol with the agonist suggests the importance of the keto group for the process of opening the channel
Concerning the ionotropic LGC-53 receptor, it is highly selective to DA and does not generate ionic currents when exposed to high concentrations of 5-HT, octopamine and histamine21,41. Moreover, we found that it is not modulated by GABA (data not show). We also found that NAD activated LGC-53 with an EC50 of 137 µM; thus, this molecule may well gate the ion channel of LGC-53 in vivo, whereas AD, Tyr and β-PEA were found to be partial agonists and activated the receptor at high concentrations. Certain interesting possibilities arise in connection with the potential of NAD to activate LGC-53. It might be expected that this biogenic amine interacts with the dopaminergic regulation in C. elegans, despite the fact that a noradrenergic system has not been identified in this nematode, it has been observed that NAD induced an oscillatory chloride current in frog oocytes injected with mRNA isolated from C. elegans 42.
In this work, we show the effect of DA metabolites on GABAρ1 and LGC-53 receptors expressed in X. laevis oocytes (Table 1). In GABAρ1, 3-MT showed potency and efficacy similar to that of DA, and in terms of potency, the effects of 3-MT and β-PEA do not depend on the state of receptor activation. This contrasts with the effect of DA, which exhibits lower affinity when the receptor channel is open, and with the higher affinity of 4-AM-OH when the GABAρ1 channel is open. In LGC-53, NAD was found to be a full agonist, whereas AD, Tyr and β-PEA are partial agonists. GABA neither activated LGC-53 nor modulated the DA responses. All this evidence suggests: (1) that the human GABAρ1 receptor is inhibited by DA and some DA metabolites, which may be important in areas of the brain where the GABAeric and dopaminergic systems converge; and (2) that the nematode ionotropic receptor LGC-53 is activated by DA analogues but not activated by GABA.
Material and Methods
Expression of GABAρ1 and LGC-53 receptors in X. laevis oocytes
All frogs were handled in accordance with the guidelines of the National Institute 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. Frogs were anesthetized, and follicles were removed manually and treated with 0.3 µg/µl collagenase type I in Ca2+-free Ringer’s at room temperature for 30 min. Oocytes were maintained at 16 °C in Barth’s medium: 88 mM NaCl, 1 mM KCl, 0.33 mM Ca2(NO)3, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM NaHCO3, 5 mM HEPES, pH 7.4, and 0.1 mg/mL gentamycin sulfate. The next day, 50 nL (1 µg /µL) of human GABAρ1 mRNA (cDNA encoding in pcDNA3 plasmid43 or LGC53 mRNA21 (for in vitro transcription, we used the mMessage mMachine kit) were injected into each oocyte, and the electrophysiological recordings were obtained 1–5 days after injection.
Voltage clamp recordings
Membrane currents produced by the agonists were recorded using the two-microelectrode voltage-clamp technique42,43,44 with an AXOCLAMP-2B amplifier (Axon Instruments) and DIGIDATA 1440 A (Molecular Devices) and pClamp 10.5 software (Molecular Devices). Oocytes were placed in a recording chamber of 0.5 mL and impaled with two glass microelectrodes filled with 3 M KCl, with resistances in the range of 0.5 to 2.0 MΩ, and the membrane potential was held at −60 mV. All recordings were done at room temperature. Oocytes were continually perfused (20 mL/min) with Ringer’s solution: 115 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, and 5 mM HEPES, pH 7.4. All compounds were purchased from Sigma-Aldrich, GABA, A5835-25G; DA, PHR1090-1G; 3-MT, 4251-100MG; DOPAC, 850217-1 G; HVA, H1252-1G; 4-AM-1-OH, 178330; β-PEA, P6513-25G; AD, E4250-500MG; NAD, A7257-500MG; Tyr, T-7255 and L-DOPA, D-9628-5G. Aliquots of 1 M GABA were stored at −30 °C. DA and analogues were prepared in Ringer’s solution right before application. The pH of all solutions was adjusted to 7.4. All compounds were applied in bath solution. To determine the concnetration-response relation, the agonist (GABA 3 µM) was applied before and after the mix of GABA 3 µM/analogue to evaluate the ability of DA and analogues to modulate GABAρ1 while the receptor was activated. The analogue was applied during the current plateau upon activation with 3 µM GABA. To activate the LGC-53 receptor and determine its modulation by DA analogues, DA was applied before and after the analogue.
Data analysis
All data were analyzed using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego CA) and expressed as mean ± S.E.M. Each data point was obtained from to 6 cells from at least 3 frogs. The effects of dopamine analogues were evaluated by comparing the response obtained by the application of 3 μM GABA, and the inhibition was determined as follows:
where, Ihn [Ana]: % of inhibition by the analogue at concentration i when co-applied with 3 μM GABA; I[Ana]i: current obtained by the co-application of the analogue at concentration i and 3 μM GABA; I[GABA 3μM] pre: current obtained by 3 μM GABA before the co-application; I[GABA 3μM]post: current obtained by 3 μM GABA after the co-application. Concentration-response relations were plotted according to equation:
where X is the logarithm of analogue concentrations, IC50 is the antagonist concentration ([Ana]) causing half-maximal inhibition of GABA, and nH is the Hill coefficient. When complete inhibition was not obtained, the following equation was applied:
where Imin represents the residual GABA current remaining with a maximal concentration of analogue and Imax current obtained by 3 μM GABA. IC50 is the concentration of antagonist that gives a response halfway between Bottom and Top.
Molecular Docking
We used the homology model described by Lima et al.29, the ligands were optimized with Avogadro45, and the docking was done with Autodock vina 4.246 for visualization of the solutions and measurement of distances used USFC Chimera47.
Significant differences between groups were determined by a t-test for unpaired two-tailed groups. Significant differences were P: ns >0.05 >* >0005 >** >0.0008 >*** <0.0001.
References
Hirasawa, H., Betensky, R. A. & Raviola, E. Corelease of dopamine and GABA by a retinal dopaminergic neuron. J Neurosci. 32, 13281–13291 (2012).
Tritsch, N. X., Ding, J. B. & Sabatini, B. L. Dopaminergic neurons inhibit striatal output through non-canonical release of GABA. Nature. 490, 262–266 (2012).
Olsen, R. W. & Sieghart, W. International Union of Pharmacology. LXX. Subtypes of γ-aminobutyric acid (A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update. Pharmacol. Rev. 60, 243–60 (2008).
Polenzani, L., Woodward, R. M. & Miledi, R. Expression of mammalian γ-amino butyric acid receptors with distinct pharmacology in Xenopus oocytes. Proc. Natl. Acad. Sci. USA 88, 4318–4322 (1991).
Cutting, G. R. et al. Cloning of the γ-aminobutyric acid (GABA) ρ1 cDNA: a GABA receptor subunit highly expressed in the retina. Proc. Natl. Acad. Sci. USA 88, 2673–2677 (1991).
Ragozzino, D. et al. Design and in vitro pharmacology for a selective -aminobutyric acid C receptor antagonist. Mol. Pharmacol. 50, 1024–30 (1996).
Mejia, C. et al. Expression of subunits GABAρ During rat cerebellum development. Neurosci. Lett. 432, 1–6 (2008).
Rosas-Arellano, A. et al. TheGABA (A) receptors in hippocampal ρ spontaneous activity and their distribution in hippocampus, amygdala and visual cortex. Neurosci Lett. 500, 20–25 (2011).
Rosas-Arellano, A., Machuca-Parra, A. I., Reyes-Haro, D., Miledi, R. & Martinez-Torres, A. Expression of GABAρ receptors in the neostriatum: Localization in aspiny, medium spiny neurons and GFAP-positive cells. J. Neurochem. 122, 900–910 (2012).
Martínez-Delgado, G., Estrada-Mondragón, A., Miledi, R. & Martínez-Torres, A. An Update on GABAρ Receptors. Curr Neuropharmacol. 8, 422–33 (2010).
Ng, C. et al. Medicinal chemistry of ρ GABAC receptors. Future Med. Chem. 3, 197–209 (2011).
Qian, H. & Ripps, H. Response kinetics and pharmacological properties of heteromeric receptors formed by coassembly of GABA rho- and gamma 2-subunits. Proc Biol Sci. 266, 2419–2425 (1999).
Harvey, V. L., Duguid, I. C., Krasel, C. & Stephens, G. J. Evidence that GABA rho subunits contribute to functional ionotropic GABA receptors in mouse cerebellar Purkinje cells. J. Physiol. 577, 127–139 (2006).
Pétriz, A., Reyes-Haro, D., González-González, M. A., Miledi, R. & Martínez-Torres, A. GABAρ subunits confer a bicuculline-insensitive component to GFAP+ cells of cerebellum. Proc Natl Acad Sci USA 111, 17522–7 (2014).
Ochoa-de la Paz, L. D., Estrada-Mondragon, A., Limón, A., Miledi, R. & Martinez-Torres, A. Dopamine and serotonin Modulate human GABAρ1 receptors Expressed in Xenopus laevis oocytes. ACS Chem Neurosci. 3, 96–104 (2012).
Reyes-Ruiz, J. M., Limón, A. & Miledi, R. Cloning and characterization of the ionotropic GABA receptor subunit ρ1 from pig (Sus scrofa). Neurosci Lett. 558, 78–81 (2014).
Hoerbelt, P., Lindsley, T. A. & Fleck, W. M. Dopamine Directly Modulates Receptors GABA. J. Neurosci. 35, 3525–3536 (2015).
Liu, J. & Wang, Y. T. Allosteric modulation of GABAA receptors by extracellular ATP. Mol. Brain. 7, 6 (2014).
Woodward, R. W., Panicker, M. M. & Miledi, R. Actions of dopamine and dopaminergic drugs on cloned serotonin receptors expressed in Xenopus oocytes. Proc Natl Acad Sci USA 89, 4708–4712 (1992).
Garcia-Colunga, J. & Miledi, R. Effects of serotonergic agents on neuronal nicotinic acetylcholine receptors. Proc. Natl. Acad. Sci. USA 92, 2919–2923 (1995).
Ringstad, N., Abe, N. & Horvitz, H. R. Ligand-gated chloride channels are receptors for biogenic amines in C. elegans. Science. 325, 96–100 (2009).
Beech, R. N., Callanan, M. K., Rao, V. T., Dawe, G. B. & Forrester, S. G. Characterization of cys-loop receptor genes involved in inhibitory amine neurotransmission in parasitic and free living nematodes. Parasitol Int. 62, 599–605 (2013).
Rao, V. T. et al. Characterization of a novel tyramine-gated chloride channel from Haemonchus contortus. Mol Biochem Parasitol. 173, 64–68 (2010).
Beg, A. A. & Jorgensen, E. M. EXP-1 is an excitatory GABA-gated cation channel. Nat Neurosci. 6, 1145–1152 (2003).
Wilk, S. & Stanley, M. Dopamine metabolites in human brain. Psychopharmacology. 57, 77–81 (1978).
Di Paolo, T., Harnois, C. & Daigle, M. Assay of dopamine and its metabolites in human and rat retina. Neurosci. Lett. 74, 250–254 (1987).
Ariëns, E. J. Intrinsic activity: partial agonists and partial antagonists. J. Cardiovasc Pharmacol. 5, 8–15 (1983).
Pan, Z. H., Zhang, D., Zhang, X. & Lipton, S. A. Agonist-induced closure of constitutively open γ-aminobutyric acid channels with mutated M2 domains. Proc. Natl. Acad. Sci. USA 94, 6490–6495 (1997).
Limón, A., Estrada-Mondragón, A., Reyes-Ruiz, J. M. & Miledi, R. Dipicrylamine Modulates GABAρ1 Receptors through Interactions with Residues in the TM4 and Cys-loop Domains. Mol Pharmacol. 89, 446–456 (2016).
Sedelnikova, A. et al. Mapping the ρ1 GABAC receptor agonist binding pocket. J. Biol. Chem. 280, 1535–1542 (2005).
Melis, C., Lummis, S. C. R. & Molteni, C. Molecular dynamics simulations of GABA binding to the GABA(C) receptor: the role ofArg(104). J. Biophys. 95, 4115–4123 (2008).
Naffaa, M. M. et al. Investigating the Role of Loop C Hydrophilic Residue ‘T244’ in the Binding Site of ρ1 GABAC Receptors via Site Mutation and Partial Agonism. PLoS ONE. 11, e0156618, https://doi.org/10.1371/journal.pone.0156618. (2016).
Harrison, N. J. & Lummis, S. C. Locating the carboxylate group of GABA in the homomeric rho GABAA receptor ligand-binding pocket. J. Biol Chem. 281, 24455–24461 (2006).
Hans-Georg Breitinger. “Drug Synergy“ Mechanisms and Methods of Analysis, Toxicity and Drug Testing, Prof. Bill Acree (Ed.), ISBN: 978-953-51-0004-1, InTech, https://doi.org/10.5772/30922. Available from: http://www.intechopen.com/books/toxicity-and-drug-testing/drug-synergy-mechanisms-and-methods-of-analysis.
Dong, C. J. & Werblin, F. S. Dopamine modulation of GABAc receptor function in an isolated retinal neuron. J. Neurophysiol. 71, 1258–1260 (1994).
Wellis, D. P. & Werblin, F. S. Dopamine modulates GABAc receptors mediating inhibition of calcium entry into and transmitter release from bipolar cell terminals in tiger salamander retina. J. Neurosci. 15, 4748–4761 (1995).
Garris, P. A., Ciolkowski, E. L., Pastore, P. & Wightman, R. M. Efflux of dopamine from the synaptic cleft in the nucleus accumbens of the rat brain. J Neurosci. 14, 6084–6093 (1994).
Krall, J. et al. Discovery of a-Substituted Imidazole-4-acetic Acid Analogues as a Novel Class of ρ1 γ-Aminobutyric Acid Type A Receptor Antagonists with Effect on Retinal Vascular Tone. Chem Med Chem. 11, 2299–2310 (2016).
Yamamoto, I. et al. Structurally diverse GABA antagonists interact differently with open and closed conformational states of the receptor ρ1. ACS Chem. Neurosci. 3, (293–301 (2012).
Huang, S. H. et al. Mixed antagonistic effects of the ginkgolides at rho1 GABAC recombinant human receptors. Neuropharmacology. 63, 1127–1139 (2012).
Kress, G. J. et al. Fast phasic release properties of dopamine studied with a channel biosensor. J. Neurosci. 34, 11792–802 (2014).
Martínez-Torres, A. & Miledi, R. Expression of Caenorhabditis elegans neurotransmitter receptors and ion channels in Xenopus oocytes. Proc. Natl. Acad. Sci. USA 103, 5120–5124 (2006).
Martinez-Torres, A. & Miledi, R. Expression of γ-aminobutyric acid ρ1 and ρ1Δ450 as gene fusions with the green fluorescent protein. Proc. Natl. Acad. Sci. USA 98, 1947–1951 (2001).
Miledi, R., Parker, I. & Sumikawa, K. Synthesis of chick brain GABA receptors by frog oocytes. Proc. Roy. Soc. Lond. Biol. Sci. 216, 509–515 (1982).
Hanwell, M. D. et al. Avogadro: An advanced semantic chemical editor, visualization, and analysis platform. J. Cheminf. 4, 17, https://doi.org/10.1186/1758-2946-4-17 (2012).
Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput Chem. 31, 455–461 (2010).
Pettersen, E. F. et al. UCSF Chimera—A visualization system for exploratory research and analysis. Journal of Computational Chemistry. 25, 1605–1612, https://doi.org/10.1002/jcc.20084 (2004).
Acknowledgements
The authors are in debt with Dr. R. Miledi who supported this project from the start. We thank A.E. Espino-Saldaña, M. Ramírez-Romero, A. limón, L. Robles-Martínez. and R. Arellano. for providing technical assistance. A. Alaniz-Palacios is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and received fellowship 342519 from CONACYT. The authors thank Dr. Dorothy Pless and Jessica Gonzalez Norris for editing the manuscript, Dr. A Hernández-Cortes for his comments and Dr. Niels Ringstad from NYU Skirball Institute for donating LGC-53 expression plasmid. This work was supported by grants from CONACYT (220224) and PAPIIT-DGAPA (IN206616) to AMT.
Author information
Authors and Affiliations
Contributions
A.M.T. and A.A.P. designed the project. A.M.T. and A.A.P. designed the experiments. A.A.P. performed the experiments and analyzed data. A.M.T. and A.A.P. interpreted the results and wrote the paper.
Corresponding author
Ethics declarations
Competing Interests
The authors declare that they have no competing interests.
Additional information
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Alaniz-Palacios, A., Martínez-Torres, A. Antagonistic effect of dopamine structural analogues on human GABAρ1 receptor. Sci Rep 7, 17385 (2017). https://doi.org/10.1038/s41598-017-17530-8
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-017-17530-8
- Springer Nature Limited
This article is cited by
-
A reassessment of the safety profile of monoamine oxidase inhibitors: elucidating tired old tyramine myths
Journal of Neural Transmission (2018)