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Open Access

Peer-reviewed

Research Article

Partial Agonism of Taurine at Gamma-Containing Native and Recombinant GABAA Receptors

  • Olaf Kletke,

    Affiliations Department of Cell Physiology of the Ruhr-University, Bochum, Germany, Department of Neurophysiology, Medical Faculty of Heinrich-Heine University, Düsseldorf, Germany

    ⨯
  • Guenter Gisselmann,

    Affiliation Department of Cell Physiology of the Ruhr-University, Bochum, Germany

    ⨯
  • Andrea May,

    Affiliation Department of Neurophysiology, Medical Faculty of Heinrich-Heine University, Düsseldorf, Germany

    ⨯
  • Hanns Hatt,

    Affiliation Department of Cell Physiology of the Ruhr-University, Bochum, Germany

    ⨯
  • Olga A. Sergeeva

    olga.sergeeva@uni-duesseldorf.de

    Affiliation Department of Neurophysiology, Medical Faculty of Heinrich-Heine University, Düsseldorf, Germany

    ⨯

Correction

24 Oct 2013: Kletke O, Gisselmann G, May A, Hatt H, Sergeeva OA (2013) Correction: Partial Agonism of Taurine at Gamma-Containing Native and Recombinant GABAA Receptors. PLOS ONE 8(10): 10.1371/annotation/fddd2ff3-c991-4c2f-8b84-a27eb20fba91. https://doi.org/10.1371/annotation/fddd2ff3-c991-4c2f-8b84-a27eb20fba91 View correction

Abstract

Taurine is a semi-essential sulfonic acid found at high concentrations in plasma and mammalian tissues which regulates osmolarity, ion channel activity and glucose homeostasis. The structural requirements of GABAA-receptors (GABAAR) gated by taurine are not yet known. We determined taurine potency and efficacy relative to GABA at different types of recombinant GABAAR occurring in central histaminergic neurons of the mouse hypothalamic tuberomamillary nucleus (TMN) which controls arousal. At binary α1/2β1/3 receptors taurine was as efficient as GABA, whereas incorporation of the γ1/2 subunit reduced taurine efficacy to 60–90% of GABA. The mutation γ2F77I, which abolishes zolpidem potentiation, significantly reduced taurine efficacy at recombinant and native receptors compared to the wild type controls. As taurine was a full- or super- agonist at recombinant αxβ1δ-GABAAR, we generated a chimeric γ2 subunit carrying the δ subunit motif around F77 (MTVFLH). At α1/2β1γ2(MTVFLH) receptors taurine became a super-agonist, similar to δ-containing ternary receptors, but remained a partial agonist at β3-containing receptors. In conclusion, using site-directed mutagenesis we found structural determinants of taurine’s partial agonism at γ-containing GABAA receptors. Our study sheds new light on the β1 subunit conferring the widest range of taurine-efficacies modifying GABAAR function under (patho)physiological conditions.

Citation: Kletke O, Gisselmann G, May A, Hatt H, A. Sergeeva O (2013) Partial Agonism of Taurine at Gamma-Containing Native and Recombinant GABAA Receptors. PLoS ONE 8(4): e61733. https://doi.org/10.1371/journal.pone.0061733

Editor: Pascale CHAVIS, INSERM U901, France

Received: November 1, 2012; Accepted: March 13, 2013; Published: April 30, 2013

Copyright: © 2013 Kletke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Supported by Deutsche Forschungsgemeinschaft SFB 575/TP C8 and a Heisenberg stipend to OAS (SE 1767). URL: http://www.dfg.de. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Taurine (2-aminoethane sulfonic acid) is very abundant in plasma and mammalian tissues including brain, where it regulates osmolarity, ion channel activity, neuronal growth and metabolism [1]–[4]. It remains controversial whether taurine can be called “neurotransmitter”: some but not other studies reported accumulation of taurine in the synaptic vesicles [5]; [6] and action potential-dependent release [7]. Taurine concentrations range from 3 to 9 mM in different species and brain regions and may reach 20 mM or higher in intracellular compartments [8]. Intracellular concentrations of taurine in the brain are about 400 times higher then extracellular [9] due to the high-affinity uptake system [10]. Taurine release from different CNS cells is observed under pathophysiological conditions such as hypoosmotic stress, ischemia or acute hyperammonemia, where its interaction with the receptors for inhibitory neurotransmitters GABA and glycine plays a neuroprotective role [8]. In many brain areas taurine in concentrations below 1 mM activates glycine but not GABAA receptors, except for the ventrobasal thalamus, where it activates α4β2δ GABAAR-type at physiological concentrations (10–100 µM) [11]. Mice deficient in taurine show impaired GABAergic inhibition in the striatum [12], indicating yet unrecognised role of taurine for the proper GABAergic signalling. If molecular structure of taurine binding site at different glycine receptor types are known [13]; [14], taurine binding site at GABAA receptor was not yet systematically analysed. Efficacy and potency of taurine is so far only known for a few subunit combinations. Taurine acts as a full agonist at α1β3 and a partial agonist at α1β3γ2 receptors [15]; at α4β2δ receptors taurine elicits even greater currents than GABA [11] and at α6β2δ GABAAR taurine is a partial agonist, with variable EC50s depending on the expression level [16]. The molecular mechanism that determines the efficacy of taurine at GABAARs is unknown. A comparative analysis of taurine gating of GABAAR containing different β subunits was not yet performed. GABAARs are heteropentameres composed of five subunits. A multitude of subunits can assemble to functional receptors (α1–6, β1–3, γ1–3, δ-, ε-, θ-, π-and ρ1–3) [17]. According to the current view GABAARs are composed of two α, two β and one γ (or δ) subunits aligned γ-β-α-β-α counter-clockwise when viewed from the synaptic cleft [18]. Mutational analysis studies demonstrated that the agonist binding site is located at the α/β interface and the benzodiazepine binding site at the α/γ interface [17]; [19]; [20]. The binding sites for the partial agonists at GABAAR are unclear [21]. The largest population of GABAARs in the rat brain has a subunit composition of α1β2γ2, whereas α2β3γ2 and α3βγ2/3 together constitute the next most prevalent subtypes [22]; [23]. Several subunit combinations such as α5β3γ2 [22] and α4/6β2/3δ [23]; [24] are found exclusively extrasynaptically, with the former type expressed in the hypothalamus. Histaminergic neurons from the tuberomamillary nucleus (TMN) of the hypothalamus were selected for the present study as functional and structural features of their GABAA receptors were previously characterised with the α1, α2, α5, β1, β3, γ1, γ2, ε, but not δ subunit- transcripts being regularly detected [25]–[28]. We compare now the taurine-sensitivity of native GABAAR versus selected GABAAR compositions recombinantly expressed in Xenopus oocytes. As we aimed to compare properties of recombinant GABAAR with the native receptors expressed in hypothalamic neurons we restricted the number of investigated subunits and receptor types to those present in TMN neurons [27]; [28]. We report that incorporation of the γ subunit reduces taurine efficacy. With the help of site-directed mutagenesis we describe structural determinants for the partial agonism of taurine at γ-containing GABAARs.

Materials and Methods

Electrophysiology in Native Neurons

Experiments were conducted according to the Animal Protection Law of the Federal Republic of Germany (Tierschutzgesetz BGBI.I,S.1206, revision 2006) and European Communities Council directive regarding care and use of animals for experimental procedures (86/609/EEC). Approval by the Ethics Committee for this kind of experiment is not necessary in accordance with the Animal Protection Law of the Federal Republic of Germany (§ 8 Abs.1 Tierschutzgesetz). All efforts were made to minimize the number of animals and their suffering. Brain tissue was removed from mice after decapitation by appropriately trained staff with approval of LANUV NRW (Landesamt für Umwelt, Natur und Verbraucherschutz Nordrhein Westfalen, Düsseldorf), permission number 058/91.

Five to eight week old male mice carrying a point mutation on GABAAR γ2 subunit (γ2F77I) further referred as KI (knock-in) mice and their wild type littermates were generated and genotyped as described previously [29]. Slice preparation, isolation of histaminergic neurons with the help of papain, whole-cell patch-clamp recordings in voltage clamp mode, fast drug application and single cell RT-PCR procedures was done as previously described [26]; [27]. Briefly, sterile patch electrodes were filled with the following solution: 140 mM KCl, 2 mM MgCl2, 0.5 mM CaCl2, 5 mM EGTA, and 10 mM HEPES/KOH (pH 7.2). After establishment of the whole-cell configuration (Vh = −50 mV), an acutely isolated cell was lifted into the major chute of the application system, where it was continuously perfused with the sterile control bath solution. The substances were applied through a glass capillary 0.08 mm in diameter. All solutions flowed continuously, gravity-driven, at the same speed and lateral movements of the capillaries exposed a cell either to control- or test-solutions. The kinetics of solution exchange at the open electrode tip were characterized by an exponential rise time constant of 7 ms, whereas the maximal GABA-evoked responses reached their maximum up to 2 times slower; thus peak responses represented the sum of activation, desensitization and delay of solution exchange around the large (15–25 µm) neurons (see Schubring et al [30]). For the comparison of apparent desensitization kinetics between zinc sensitive and zinc resistant neurons, only cells with a rise time constant below 10 ms were considered. Experiments were conducted and analyzed with commercially available software (TIDA for Windows, HEKA, Lambrecht, Germany). Fitting of concentration - response data points was done as previously described [25]; [26]. Post-hoc identification of recorded TMN neurons and GABAAR analysis was done with single cell RT-PCR according to the previously published protocols [27]; [28]. Real-time RT - PCR was used for the semiquantitative analysis of γ2 subunit expression in TMN (relative to the β -actin endogenous control according to the “2−ΔΔCt “(ΔFold) method as in [27]. After the final cycle the PCR were subjected to a heat dissociation protocol (PE Biosystems 5700 software). Each PCR product showed a single peak in the denaturation curve. Standard curves were obtained with the sequential dilution of one cDNA sample (from KI mouse #1). From these curves the linear regression coefficient (r = −0.99) and efficiency (E = 1.8) for the β –actin and γ2 subunit – cDNA amplification (r = −0.98, E = 1.9) were calculated, where E = 10[−1/slope]. Expression levels of the γ2-subunit in each sample are normalized to the sample with minimal expression (for this sample: ΔΔC = 0, 2−ΔΔCt = 1). The following primers were used: up: 5′-tat gtD aac agc att ggW ccW gt -3′ and lo: 5′-acc atc att cca aat tct cag cat-3′. The size of the PCR product (234 b.p.) was verified by electrophoresis in 2% agarose gel, whereas its identity with the known mouse γ2-subunit cDNA (M86572, Genbank) was confirmed by sequencing.

Expression of Recombinant GABAA Receptors and Electrophysiology in Xenopus Oocytes

GABAAR subunit cDNAs were obtained as follows: rat α1 and β1 cDNAs were prepared using standard molecular biology procedures. Mouse γ2L, α2, and human β3 and δ cDNA were obtained from RZPD (Berlin, Germany). Chimeric γ2(δ 74–79) was generated using overlap extension PCR [31] with the following primer pairs for the exchanged area: fw-γ2(δ 74–79) ‘5-atg gaa tat aca atg acg gtg ttc ctg cac cag agc tgg cgg gac aga cgt ttg aaa ttt aac-3′ and rev- γ2(δ 74–79) ‘5-gtt aaa ttt caa acg tct gtc ccg cca gct ctg gtg cag gaa cac cgt cat tgt ata ttc cat-3′. All cDNAs were subcloned into pSGEM (courtesy of M. Hollmann, Bochum, Germany). Plasmids were linearized with PacI restriction endonuclease and corresponding cRNA was synthesized using the AmpliCap T7 high-yield message marker kit (Epicentre, Madison, WI), following the manufacturers protocol. 5 to 15 ng of the mixture of cRNAs with a ratio of 10∶1:10 for αβγ/δ was injected into every oocyte to prevent subpopulations of β homomultimeric GABAAR [32]. Two to six days after injection of cRNA, oocytes were screened for receptor expression by two-electrode voltage-clamp recording. Electrodes were made using a Kopf vertical micropipette puller and filled with 3 M potassium chloride, giving resistances of 0.1–0.5 MΩ. Eggs were placed in an oocyte chamber and superfused with Frog-Ringer’s solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 10 mM Hepes, pH 7.2). Current signals were recorded with a two-electrode voltage - clamp amplifier (TURBO TEC-03, npi, Tamm, Germany) and pCLAMP software (Axon Instruments, Union City, CA) or CellWorks (npi, Tamm, Germany) depending on the setup used. The membrane potential was clamped at −40 to −60 mV. All experiments were performed at room temperature. Complete concentration - response curves for GABA and taurine were recorded on the same oocyte. These agonists of GABAAR were dissolved in Frog-Ringer and applied in a volume of 200 µl into the entrance tube of the recording chamber, totally exchanging the bath solution within a second. Currents were analyzed using pCLAMP 10 software. Dataset was processed in Excel (Microsoft Corporation, Redmond, WA). Curve fitting by the 3 parameter Hill equation and statistics (t - test) was done using SigmaPlot V8.0 (Systat Software, San Jose, CA). Taurine efficacy was determined by the maximum of the taurine concentration - response curve calculated by the 3 parameter Hill equation in relation to the maximum current of the GABA concentration - response curve. Proper γ subunit integration into the GABAAR was analysed using zinc. Ternary αβγ GABAARs are insensitive to low micromolar concentrations of zinc, whereas binary αβ receptors are inhibited by those concentrations [33] (Figure S1). Delta (δ) - containing GABAAR have intermediate zinc sensitivities [34], therefore our criterion for δ subunit integration was the modulation of GABA - evoked currents by tracazolate (Figure S2). In accordance with Thompson et al. [35] we found that tracazolate potentiates ternary δ - containing GABAAR to a larger extent than the corresponding binary αxβx receptors.

Drugs and Statistical Analysis

Gabazine (SR 95531) and tracazolate were obtained from Tocris-Biotrend (Köln, Germany). All other chemicals were obtained from Sigma-Aldrich (Taufkirchen, Germany). Drugs were diluted and stored as recommended. Statistical analysis was performed with the non - parametrical Mann - Whitney U-test if not indicated otherwise. Significance level was set at p<0.05. Data are presented as mean ± standard error of the mean (SEM).

Results

Taurine Efficacy and Potency at GABAA Receptors Composed of α and β Subunits

In accordance with the study performed in HEK 293 cells by Dominguez-Perrot et al. [15] recombinant GABAAR composed of α1- and β3 -subunits in our study responded to GABA and to taurine with maximal currents of similar amplitude (Table 1). At receptors containing the β1-subunit taurine demonstrated super-agonism, eliciting maximal responses nearly two times larger than the maximal GABA responses (Table 1). When the α1-subunit was replaced by the α2-subunit GABA and taurine potencies were reduced at β1-containing receptors whereas taurine potency at β3-containing receptors was not affected. Taurine efficacy was independent of the α-subunit type and was determined by the β-subunit type.

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Table 1. GABA- and taurine- gating of different GABAA receptor types recombinantly expressed in Xenopus oocytes.

https://doi.org/10.1371/journal.pone.0061733.t001

Presence of γ2-subunit Reduces Taurine Efficacy

When co-assembled with the α- and β-subunits the γ2L subunit negatively affected taurine efficacy in all receptor types. At α2β3γ2L taurine efficacy (0.87) showed the slightest but significant deviation in efficacy reduction compared to the binary α2β3 receptors (Table 1, Fig. 1). After integration of the γ2L or γ1 subunit into β1-containing receptors, the efficacy of taurine was reduced to about 1/3 of the binary αxβ1 receptor and taurine could be called a “partial agonist”. Previous studies have shown that the putative assembly signals, the residues determining selective co-assemblies of α-β or α-γ2, in GABAARs [36] as well as in nicotinic acetylcholine receptors [37], are adjacent to, or identical to the residues that actually form the ligand-binding site. Thus, the α1 residues 56–67, in particular glutamine 67 (α1Q67), are important for the assembly with the β3 subunit and are involved in the formation of the low affinity GABA-binding site [36]; [38]. The γ2A assembly signal (MEYTIDIFFAQTW) [36] which interacts with the α subunit includes phenylalanine at position 77 (F77). This residue plays an important role for the zolpidem modulation of γ2-containing GABAAR [39].

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Figure 1. Comparison of taurine- and GABA-evoked maximal currents recorded from binary αxβx (A) or ternary αxβxγ2L (B) GABAA receptors.

Note that gating by taurine of γ2 subunit containing GABAA receptors is significantly less efficacious compared to the corresponding binary receptors. Representative current traces (comparison of taurine (600 mM) - and GABA (0.1–1 mM) -evoked maximal currents at different receptor subtypes) are shown at the left. Scale markers represent 0.1 µA vertically and 20 s horizontally for all figures with oocyte recordings. Right: averaged concentration - response curves. Concentration of agonist (filled symbols for GABA -, open symbols for taurine - responses) is plotted versus normalized response amplitudes. Each individual measurement was first normalized to the observed maximal GABA - current amplitude and subsequently averaged. Number of investigated oocytes, Hill coefficients (nHill) and concentrations evoking a half - maximal response (EC50) are presented in Table 1.

https://doi.org/10.1371/journal.pone.0061733.g001

Role of Zolpidem Binding Site for the GABAAR Gating by Taurine

The mutation of phenylalanine to isoleucine at position 77 of GABAAR γ2 subunit (γ2F77I) leads to the loss of zolpidem-modulation of GABA-responses in recombinant and native receptors [29]; [39]. This is the only residue, which is different between the γ2 and the γ1 subunit within the putative assembly signal γ2A (see above). As taurine was significantly less efficient at α2β3γ1- than at α2β3γ2- receptors (Table 1) we generated a mutated γ2F77I subunit using overlap extension PCR techniques [31]. Proper incorporation of γ2F77I into GABAAR was verified by zinc-resistance (Figure S1). In all investigated receptor types taurine efficacy was significantly reduced at mutated compared to the corresponding wild type receptors: p<0.05 for α1β1γ7F77I; p<0.01 for α2β1γ2F77I; p<0.001 for α2β3γ2F77I (Table 1, Fig. 2). As previous studies examining macroscopic (whole-cell currents) and microscopic (single-channel currents) kinetics of recombinant GABAAR with a mutation within the GABA-binding site came to the conclusion that the reduction in agonist potency (e.g. a 70-fold increase in EC50 for GABA after mutation β2-R207C) may be accompanied by the apparent reduction (by half) of the relative efficacy of a partial agonist (e.g. piperidine-4-sulfonic acid, P4S) under the slow, but not under the fast solution exchange conditions [40]. In order to control for this possibility we performed a correlation analysis between the relative potency of taurine (EC50 taurine/EC50 GABA) versus relative efficacy of taurine for all individual measurements from γ7F77I –containing and corresponding WT receptors. There was no significant correlation (Pearson coefficient: −0.07, p = 0.75). Taurine was 1965±219 times (n = 15) and 1692±154 times (n = 11) less potent than GABA in KI and WT receptors, respectively (p = 0.8), but its relative efficacy was significantly lower in KI receptors (45.6±3.7% vs 74.6±3.7%, respectively, p = 0.0002).

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Figure 2. Mutation γ2F77I reduces taurine efficacy at recombinant GABAA receptors.

(A) Representative current traces show responses to the maximal GABA and taurine concentrations at different receptor types. For two representative receptor types (marked with symbols) concentration-response plots for GABA (filled symbols) and taurine (open symbols) are shown in (B) and (C). Data obtained in corresponding wild type receptors (γ2 instead of γ2F77I) are plotted in red.

https://doi.org/10.1371/journal.pone.0061733.g002

GABAA Receptors in Zinc-resistant Neurons from Mutant γ2F77I Mice show Reduced Taurine Gating

Acutely isolated mouse TMN neurons responded to GABA with EC50s around 15 µM. There was no difference in GABA-sensitivity between γ2F77I mice and their WT littermates. All data presented in the manuscript are obtained from neurons expressing histidine decarboxylase (cell identification with single-cell RT-PCR). In contrast to the rat [25], where GABAARs are “zinc-resistant” in all TMN neurons, about 30% of mouse TMN neurons are zinc-sensitive. No difference in the occurrence of zinc- sensitivity was found between γ2F77I (knock-in, KI) mice and their WT littermates (27.7% and 27% of cells, respectively). GABA EC15 responses were inhibited by 30 µM of ZnCl2 in zinc-sensitive cells to 28.2±5.3% of control and by 10 µM to 52.1±5.9% of control (pooled data from 3 WT and 5 KI neurons, where a complete analysis of GABAAR expression with single-cell RT-PCR was successfully done, Fig. 3). In zinc-resistant cells, where 10 µM of zinc did not affect GABA-responses, inhibition by 30 µM zinc amounted to 74.1±2.2% of control (significantly different from “zinc-sensitive” cells; p<0.01). The apparent macroscopic desensitization of current responses to saturating GABA concentration (plateau/peak ratio at the end of a 2s-application period) amounted to 73.3±3.3% (n = 5) vs 67.6±3.7% (n = 10), in zinc-sensitive and zinc-resistant cells respectively (the difference is not significant). In 25% of the zinc-sensitive cells mRNAs encoding for γ subunits were not detected, whereas in the same cells α- and β-subunit transcripts were present. Two different γ subunits were never found coexpressed in zinc-sensitive neurons, whereas 48% of zinc-resistant cells coexpressed γ1 and γ2 subunits (p<0.05, Fisher’s test). All zinc-resistant cells (n = 21) expressed either γ1 (57%) or γ2 (90.5%) subunit or both. The detection frequency of any of the GABAAR subunits did not differ between 11 WT and 18 KI neurons (% of positive cells: WT vs KI): α1 in 18% vs 28%, α2 in 100% vs 94%, α5 in 18% vs 17%, β1 in 18% vs 44%, β2 in 9% vs 17%, β3 in 91% vs 78%, γ1 in 36% vs 50% and γ2 in 82% vs 83%. None of the cells expressed a detectable amount of γ3 subunit transcripts. Semiquantitative real-time PCR analysis of γ2 subunit expression revealed no difference in mRNA levels between TMN of γ2F77I KI mice (n = 5) and their WT littermates (n = 5): 1.5±0.1 vs 1.5±0.2 (p = 0.83). In WT mice taurine was more effective (p<0.05) in “zinc-sensitive” cells compared to “zinc-resistant” ones (Fig. 3B and C). In “zinc resistant cells” taurine was significantly more efficient in WT (72±2.4% of maximal GABA-currents, n = 10) compared to KI mice (53±2%, n = 14; Fig. 4A and B). Neither taurine potency (13±1 mM vs. 19.4±1.3 mM) nor slope functions of dose-response curves (nHill 1.7±2 vs. 1.74±0.14) differed between WT and KI neurons. The GlyR-mediated component of taurine-responses was subtracted from each response-amplitude (remaining component after co-application of taurine and gabazine 20 µM, Fig. 4C).

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Figure 3. Zinc-sensitive TMN neurons show similar efficacies for GABA and taurine.

(A) Zinc-inhibition of GABA-evoked currents in two representative neurons. Note that these neurons respond differently to ZnCl2 10 µM. Block of the GABA-response by this concentration served as a criterion for the selection of “zinc-sensitive” neurons. (B) Photographs of two neurons and gels illustrating single-cell RT-PCR analysis of γ-subunit (γ1–γ3) expression. Note the lack of a detectable amount of γ-subunit transcripts in zinc-sensitive cell (#2). (C) Superimposed responses to different concentrations of taurine in comparison to the maximal GABA response recorded in one zinc-sensitive neuron. (D) Averaged concentration - response plots for the two neuronal groups. Significant difference between individual data points is indicated: * = p<0.05. The maximal taurine-evoked currents represented 100±5% (filled squares, EC50 = 12.6±0.6 mM, n = 5) vs 74±2% (open squares, EC50 = 14.9±0.9 mM, n = 6) of maximal GABA-evoked currents.

https://doi.org/10.1371/journal.pone.0061733.g003

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Figure 4. Gating of native GABAA receptors by taurine is impaired by the mutation γ2F77I.

(A) Whole-cell voltage-clamp recordings (Vh = −50 mV) from adult WT or KI mouse TMN neurons isolated from hypothalamic slices. Taurine evokes maximal responses (at 50 and 100 mM) which are comparable in amplitude to the maximal GABA (0.5 mM)-evoked currents in wild-type (WT) mouse but represents only half of the GABA-response in the knock-in (KI) γ2F77I mouse. (B) Averaged concentration - response curves obtained from 10 WT and 14 KI neurons. Significant difference between individual data points is indicated: * p<0.05; *** p<0.005. (C) GABAAR- versus GlyR-involvement in taurine-responses was tested by the co-application of taurine with gabazine (gz, GABAAR antagonist). Amplitude of the remaining response was subtracted in each neuron from the control taurine response, to construct the concentration - response curves in (B).

https://doi.org/10.1371/journal.pone.0061733.g004

Super-agonistic Properties of Taurine at αxβ1δ-receptors can be Transferred to the αxβ1γ2L Receptors by Introducing into the γ2L Subunit the δ-motif: MTVFLH

This and previous studies show that δ-containing receptors are more potently and efficiently gated by taurine than γ-containing receptors. The molecular determinants for high sensitivity to taurine are unknown. We exchanged the γ2 motif around phenylalanine 77 which we found to be responsible for the reduced efficacy of taurine with the corresponding motif of the δ subunit (Fig. 5). The resulting chimeric receptors αxβ1γ2(δ74–79) displayed superagonistic properties of taurine, which did not differ significantly from the αxβ1δ receptors (Table 1, Fig. 6). Interestingly, co-assembly of the chimeric γ2 subunit with αx and β3 subunits did not render taurine agonism superior to GABA (Table 1). Chimeric α2β3γ2(δ74–79) receptors were insensitive to zolpidem, like α2β3γ2F77I or α2β1γ1 receptors (Figure S3). When GABA (at EC10) was co-applied with 1 µM zolpidem, the resulting currents represented 97±7% of control (n = 5). At wild type α2β3γ2 receptors the same concentration of zolpidem increased control GABA-response to 430±70% of control (n = 5). Thus, zolpidem insensitivity could be transferred from δ to γ2 through the motif MTVFLH.

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Figure 5. Sequence alignment of GABAA receptor subunits between amino acids 58 and 92 (γ2 mouse numbering).

Underlined is a putative assembly signal conserved in different GABAA receptor subunits (36)). Note no difference between all three β- subunits in the putative assembly signal: MDYTLTMYFQQ_W with the exception for the position 81 (different residues are indicated in different colour). Interestingly, these coloured β subunit-specific residues were shown previously to affect stabilization of a homomeric assembly (45). Fat letters show δ: MTVFLH and γ2: IDIFFA motifs which were exchanged in the chimeric γ2(δ74–79) subunit. Orange field indicates location of γ2F77 site involved in zolpidem binding as well as homologous or same residues at other GABAAR subunits.

https://doi.org/10.1371/journal.pone.0061733.g005

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Figure 6. Chimeric αxβ1γ2(δ74–79) receptors show superagonistic properties of taurine.

(A and B) Representative current traces (comparison of taurine (600 mM) - and GABA (0.3–3 mM) - evoked maximal currents) are shown for different receptor types. (A) Concentration - response curves for the β1 -containing receptors. Concentrations of agonist are plotted versus current amplitudes normalized on maximal GABA response (filled symbols for GABA -, open symbols for taurine - responses). Red curves are given for comparison with γ2 (WT) - containing receptors. Note the dramatic increase in taurine efficacy over GABA in chimeric β1 - containing GABAAR, which renders them similarity with the α2β1δ receptors.

https://doi.org/10.1371/journal.pone.0061733.g006

Discussion

We demonstrate that taurine gating depends on the type of β subunit and is negatively affected by the γ subunit of the GABAAR. The mutation γ2F77I which makes the GABAAR zolpidem-insensitive reduces the efficacy of taurine-gating in recombinant and native GABAA receptors. Substitution of the γ2 subunit motif around phenylalanine 77 (mouse γ2 subunit numbering) with the corresponding δ subunit motif (MTVFLH) results in a receptor with superagonistic properties of taurine in β1- but not in β3- containing receptors.

Our results obtained on recombinant GABAAR expressed in Xenopus oocytes are in line with a previous report on decreased efficacy and potency of taurine at ternary α1β3γ2 receptors compared to binary α1β3 receptors expressed in HEK293 cells [15]. This decrease in efficacy was accompanied by decreased taurine potency at α1β3γ2 and α2β3γ2 receptors (Table 1; present study and [15]). Interestingly, β1-coassembly with the γ2 subunit resulted in a reduction of taurine efficacy and potency in α2- but an increased potency in α1-containing pentamers in our study. We are aware of the technical limitations in our measurements of maximal efficacies and potencies of GABAAR agonists due to the slow speed of the solution exchange around an oocyte. Apparent efficacies and potencies were calculated from the peak responses which represent the sum of different processes such as fast kinetics of receptor activation and desensitization and the slow concentration ramp. Theoretical predictions formulated in a study by Wagner et al [40] are the following: i) the true maximal efficacy or open probability is underestimated in experiments on oocytes as desensitization during the agonist concentration ramp blunts the peak amplitude of the response; ii) the degree of this blunting depends on ligand affinity, such that high affinity ligands reach higher effective concentrations sooner during the agonist concentration ramp than do low affinity ligands. Although absolute efficacy and potency values can only be obtained from experiments recording single channel activity, our results from Xenopus oocytes are in line with those from HEK293 cells [15] and native neurons (present study) where a much faster solution exchange around smaller cells was achieved. Combining patch-clamp recordings from hypothalamic neurons with single-cell RT-PCR we observed the same structure-function relation for taurine gating of native GABAAR as seen in Xenopus oocytes. Thirty percent of mouse histaminergic neurons expressed GABAARs with high zinc sensitivity indicating the prevalence of binary (αxβx) receptors over ternary (αxβxγx) in these cells. The functionality of such receptors was demonstrated by Gunther et al. [41] in γ2 - subunit knockout mice. Taurine efficacy was comparable to GABA in zinc-sensitive cells, whereas taurine was less efficient than GABA in zinc-resistant cells, in keeping with the findings on recombinant γ-containing receptors expressed in Xenopus oocytes (Table 1), where taurine efficacy varied between 60–70% (α1β3γ2, α2β1γ1, α2β1γ2) and 80–90% (α1β1γ2, α1β2γ2, α2β3γ2) of maximal GABA-responses. Note that in TMN neurons, which variably express 9 subunits of GABAAR [27], all aforementioned GABAAR types are likely occurring. The potency of taurine was not different between zinc-sensitive and zinc-resistant neurons, indicating that α1- and α2-containing GABAAR-populations, which show different changes in taurine potency upon co-assembly with the γ subunit (see above), may both contribute to the TMN pharmacology.

Receptors lacking a benzodiazepine (BZ) -binding site, such as α1/2β1, and α1/2β1δ, are better gated by taurine than by GABA (Table 1, Fig. 1). Our observation that taurine gating of β3-containing receptors with the same stoichiometry was weaker compared to β1-containing receptors could be explained by the presence of a low - affinity binding site for BZ at β2/3 but not at β1 receptors [42]. The mutation γ2F77I which abolished zolpidem - potentiation did not rescue taurine gating. In contrast, taurine efficacy significantly dropped in this mutation, resembling now the taurine efficacy at the equivalent γ1 - containing receptors, which are poorly potentiated by a variety of BZ - site ligands [39] and naturally carry isoleucine at the position 77. We conclude that steric intersubunit - interactions (see below), rather than the BZ - binding site per se, play a decisive role for taurine or GABA gating as well as for the modulatory action of BZ.

In line with the data obtained on recombinant receptors containing the mutant γ2F77I subunit, taurine efficacy was reduced in zinc - resistant native neurons from KI (γ2F77I) mice from 72% to 54% of maximal GABA efficacy. This efficacy drop corresponds very well to the values obtained from recombinant α2β1γ2 receptors (70% WT vs. 50% in α2β1γ2F77I ) and supports our previous conclusion that the α2β1γ2 receptor type plays a dominant role for the pharmacology of TMN neurons [27]; [28]. Thus the mutation γ2F77I modified taurine - efficacy at the γ2 - containing GABAARs. The (patho)physiological conditions for the gating of these receptors by taurine are unknown. The normal extracellular concentration of taurine is >20 times below their activation threshold. The expression of the α4 and δ subunits increases in the hippocampus of γ2F77I KI mice, indicating that δ - containing receptors might be up -regulated as compensatory response for the impaired taurine efficacy at γ2 - containing receptors. This may be a reason for the lack of behavioural abnormalities in these mice compared to WT littermates [29]; [34]. Jia et al. [11] reported that, at extrasynaptic receptors of the α4β2δ-type, taurine shows agonistic properties superior to GABA and controls the excitability of mouse ventrobasal thalamic neurons. Jia et al. found a big difference in taurine sensitivity between recombinant α4β2δ receptors (threshold concentration 300 µM, EC50 = 7.5 mM) and native extrasynaptic receptors of possibly the same subunit composition (threshold concentration 10 µM, potency is not determined). In our study taurine showed higher potency at neuronal receptors (EC50 = 13–19 mM) compared to the corresponding recombinant α2β1γ2-receptors (EC50 = 120 mM) with threshold concentrations just above 1 mM. This disparity may result from the absence of GABAAR - associated proteins or yet unknown intracellular modulators in recombinant systems [11]. The exceptionally high potency and efficacy of taurine at α4β2δ receptors reported by Jia et al [11] together with the partial agonism of taurine at α6β2δ receptors [16] support our observation that the type of α subunit influences taurine binding or the transduction to receptor gating. Neither α1βxδ nor α2βxδ receptors in our study showed higher sensitivities to GABA when compared to the corresponding αxβx receptors. This is in line with previous studies [43]; [44] where incorporation of the δ subunit was verified by concatenation. We applied tracazolate [35] at the end of each experiment to confirm the presence of a δ subunit in functional receptors. All data presented here are obtained from oocytes with different modulation of ternary versus binary receptors in parallel experiments. We cannot rule out the possibility of a sub-population of αxβx receptors along with αxβxδ, which we tried to prevent by injection of 10∶1:10 cRNA ratios. Furthermore, significantly different parameters derived from the agonist concentration - response relationship (Table 1) indicates a prevalence of αxβxδ receptor types. We compared taurine agonism between the restricted number of GABAAR types expressed in histaminergic neurons and used δ- containing receptors (which are not expressed in TMN) only for the comparison with chimeric receptors, composed of the γ2 subunit with a δ-motif (MTVFLH). Structural determinants for super- and partial- agonism of taurine at δ-containing receptors await further characterisation.

The differences between β1 and β3 subunits seen in our study may rely on a number of subunit - specific residues involved in the stabilisation of receptor assembly. Bracamontes and Steinbach [45] described a number of β3 - specific residues allowing the formation of functional homomultimeric receptors. Some of them are located near or within the assembly signal, e.g. tyrosine at the position 81 (see Fig. 5, present study). Others are at remote places and unlikely involved in gating or agonist binding; they may play a role in the stabilization of different receptor conformations. Steric intersubunit interactions in heteromeric β1-containing receptors may support the stable transition from the closed to the open state after taurine binding at the αxβ1δ, αxβ1γ2(δ74–79) and αxβ1 receptor, with taurine acting as a superagonist. In contrast, at β3 - containing receptors taurine acts as a partial agonist compared to the analogous receptor types.

Our study reveals the importance of the γ2 motif around phenylalanine 77 for the reduction of taurine efficacy at γ - containing receptors and shows that all three subunit types (α,β,γ) in the GABAA receptor can influence taurine agonism. Recent studies showed that the partiality of ligand agonism is pre - determined by the earliest step of agonist binding [46]; [47]. According to models suggested by these studies, partial agonist-binding generates an unstable conformational change, leading to receptor-flipping between closed and opened states [46]. Thus, the difference between taurine - and GABA - gating of GABAAR shown in this study indicates either a different but overlapping location of their binding sites or different transduction mechanisms at different receptor types.

By revealing structural demands for high efficacy GABAAR gating by taurine our study has broad physiological implications. Low taurine plasma level correlates with prediabetic and diabetic states and taurine supplementation is able to rescue insufficient insulin secretion by pancreatic islets [48]. Our data predict that a glucose-dependent up-regulation of the GABAAR γ2-subunit in pancreatic islets can reduce taurine action [49] and increase the risk of diabetes. (Patho)physiological correlates of GABAAR expression in pancreas await to be determined. Taurine deficiency in the brain results in GABAergic disinhibition, which models pathophysiological conditions of hepatic encephalopathy [12]. Thus the disclosure of structural demands for high efficacy taurine gating of GABAAR provides the basis for future studies analysing the role of GABAAR in diabetes mellitus and hepatic encephalopathy.

Conclusions

Our study provides new insight into molecular determinants of taurine gating at γ - subunit containing receptors. The mutation of phenylalanine to isoleucine at position 77 in the γ2 subunit decreases, whereas introduction of the δ subunit-motif (MTVFLH) increases the efficacy of GABAAR gating by taurine. We show, that β1 (but not β3)-containing receptors display a wide range of taurine efficacies: from superagonism at αxβ1 or αxβ1δ receptors to partial agonism at γ-containing receptors. These findings shed light on the modification of GABAAR under (patho)physiological conditions accompanying the loss of endogeneous taurine, such as diabetes mellitus or hepatic encephalopathy.

Supporting Information

Figure S1.

Zinc sensitivity of recombinant GABAA receptors. (A) Binary (αxβx, in black) GABAA receptors are inhibited by 1 µM zinc, whereas ternary αxβxγ2 (dark grey) receptors are insensitive to zinc. Delta-containing αxβxδ receptors (in white) do not differ in zinc-sensitivity from the corresponding αxβx receptors. Note that the zinc sensitivity is increased for αβγ2(δ74–79) (light grey) receptors compared to the αxβxγ2F77I receptors. Values represent mean ± SEM, p values are indicated by asterisk. * <0.05, ** <0.01, n.s. = not significant. (B) Representative current traces from oocyte recordings. Application is marked by horizontal bars. Scale markers represent 0.1 µA vertically and 20 s horizontally.

https://doi.org/10.1371/journal.pone.0061733.s001

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Figure S2.

Tracazolate (10 µM)-potentiation of GABA-evoked currents is different between ternary δ-containing and corresponding binary αxβx GABAAR types if GABA at ∼EC10 (for the β1-) and at ∼EC99 (for the β3-containing receptors) is used. (A) When GABA concentration around EC10 is used, tracazolate-potentiation of binary αxβ1 (but not αxβ3) receptors is significantly smaller compared to the ternary δ-containing receptors. (B) When the same experiments were done at saturating GABA concentrations (∼EC99) ternary αxβ3δ-GABAA receptors were potentiated to a larger extent than the corresponding binary receptors. Note no difference between β1-containing ternary and binary receptors in experiments with this GABA concentration. p values are indicated by asterisk. * <0.05, ** <0.01, *** <0.001, n.s. = not significant.

https://doi.org/10.1371/journal.pone.0061733.s002

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Figure S3.

Zolpidem potentiation of different GABAA receptor types. (A) Zolpidem modulation of chimeric α2β3γ2(δ 74–79) GABAARs. Introduction of the δ 74–79 motif MTVFLH into the γ2 subunit resulted in loss of potentiation by zolpidem, compared to the WT shown in (B). (B) Comparison of zolpidem-potentiation between α2β3γ2, α2β1γ2, α2β1γ1 and α2β3γ2F77I receptors. Note much larger bi-phasic potentiation by zolpidem at β3-containing receptors (in contrast to the β1-containing receptors) in accordance with involvement of the low - affinity binding site for BZ at β3 but not at β1 receptors (44). This site is most likely responsible for the potentiation of GABA – responses at “zolpidem-resistant” (γ2F77I-containing) receptors by 100 µM zolpidem. Data represent mean ± SEM of at least 4 individual oocytes.

https://doi.org/10.1371/journal.pone.0061733.s003

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Acknowledgments

We wish to thank W. Wisden (Imperial College London, UK) for the generous donation of γ2F77I mice.

Author Contributions

Conceived and designed the experiments: OAS OK GG HH. Performed the experiments: OK OAS GG AM. Analyzed the data: OK OAS GG AM. Contributed reagents/materials/analysis tools: OAS HH. Wrote the paper: OAS OK GG HH AM.

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