@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Medicine, Department of"@en, "Non UBC"@en, "Medicine, Faculty of"@en ; edm:dataProvider "DSpace"@en ; ns0:identifierCitation "Molecular Brain. 2014 Jan 24;7(1):6"@en ; dcterms:contributor "Djavad Mowafaghian Centre for Brain Health"@en ; ns0:rightsCopyright "Liu and Wang; licensee BioMed Central Ltd."@en ; dcterms:creator "Liu, Jun"@en, "Wang, Yu T"@en ; dcterms:issued "2015-10-24T02:56:42"@en, "2014-01-24"@en ; dcterms:description """Background: The γ-aminobutyric acid type A receptor (GABAAR) is the primary receptor mediating fast synaptic inhibition in the brain and plays a critical role in modulation of neuronal excitability and neural networks. Previous studies have demonstrated that ATP and its nucleotide analogs may regulate the function of GABAARs via Ca²⁺-dependent intracellular mechanisms, which require activation of purinergic 2 (P2) receptors or cross-talk between two receptors. Results Here, we report a potentiation of GABAARs by extracellular ATP via a previously un-recognized allosteric mechanism. Using cultured hippocampal neurons as well as HEK293 cells transiently expressing GABAARs, we demonstrate that extracellular ATP potentiates GABAAR mediated currents in a dose-dependent manner with an EC50 of 2.1 ± 0.2 mM. The potentiation was mediated by a postsynaptic mechanism that was not dependent on activation of either ecto-protein kinase or P2 receptors. Single channel recordings from cell-free excised membrane patches under outside-out mode or isolated membrane patches under cell-attached mode suggest that the ATP modulation of GABA currents is achieved through a direct action of ATP on the channels themselves and manifested by increasing the single channel open probability without alteration of its conductance. Moreover, this ATP potentiation of GABAAR could be reconstituted in HEK293 cells that transiently expressed recombinant rat GABAARs. Conclusions Our data strongly suggest that extracellular ATP allosterically potentiates GABAAR-gated chloride channels. This novel mode of ATP-mediated modulation of GABAARs may play an important role in regulating neuronal excitability and thereby in fine-tuning the excitation-inhibition balance under conditions where a high level of extracellular ATP is ensured."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/55001?expand=metadata"@en ; skos:note "RESEARCH Open AccessAllosteric modulation of GABAA receptors byextracellular ATPJun Liu1,3 and Yu Tian Wang1,2*AbstractBackground: The γ-aminobutyric acid type A receptor (GABAAR) is the primary receptor mediating fast synapticinhibition in the brain and plays a critical role in modulation of neuronal excitability and neural networks. Previousstudies have demonstrated that ATP and its nucleotide analogs may regulate the function of GABAARs viaCa2+-dependent intracellular mechanisms, which require activation of purinergic 2 (P2) receptors or cross-talkbetween two receptors.Results: Here, we report a potentiation of GABAARs by extracellular ATP via a previously un-recognized allostericmechanism. Using cultured hippocampal neurons as well as HEK293 cells transiently expressing GABAARs, wedemonstrate that extracellular ATP potentiates GABAAR mediated currents in a dose-dependent manner with anEC50 of 2.1 ± 0.2 mM. The potentiation was mediated by a postsynaptic mechanism that was not dependent onactivation of either ecto-protein kinase or P2 receptors. Single channel recordings from cell-free excised membranepatches under outside-out mode or isolated membrane patches under cell-attached mode suggest that the ATPmodulation of GABA currents is achieved through a direct action of ATP on the channels themselves and manifestedby increasing the single channel open probability without alteration of its conductance. Moreover, this ATPpotentiation of GABAAR could be reconstituted in HEK293 cells that transiently expressed recombinant rat GABAARs.Conclusions: Our data strongly suggest that extracellular ATP allosterically potentiates GABAAR-gated chloridechannels. This novel mode of ATP-mediated modulation of GABAARs may play an important role in regulating neuronalexcitability and thereby in fine-tuning the excitation-inhibition balance under conditions where a high level ofextracellular ATP is ensured.Keywords: GABAA receptor, ATP, Allosteric potentiation, Neuronal culturesBackgroundThe γ-aminobutyric acid type A receptor (GABAAR) is aligand-gated chloride ion channel, activation of whichresults in membrane hyperpolarization and hence inhib-ition of the neuronal excitability in the adult mammalianbrain. Dysfunction of GABAARs is associated with thepathogenesis of a number of neurological diseases andneuropsychiatric disorders such as epilepsy, Alzheimerdisease, and anxiety [1-6]. GABAARs are also targets ofmany clinically-relevant drugs including benzodiazepine,barbiturates and general anesthetics [7]. Moreover,endogenously produced substances such as neuro-steroids [8] and zinc [9,10] modulate GABAARs viadirect interaction with the putative binding sites onthe receptor subunit. Therefore, allosteric modula-tion is an important mode in regulating GABAARfunctions and hence maintaining homeostasis forneuronal excitability.In the CNS, adenosine 5′-triphosphate (ATP) not onlyacts as a major intracellular energy source and phosphatedonor, but also functions extracellularly as a neurotrans-mitter via activation of purinergic 2 (P2) receptors.Previous studies demonstrate that extracellular ATPcan modulate GABAAR function by activation of P2 re-ceptors [11-13]. In addition, a physical cross-talk betweenGABAARs and P2 receptors which influencess inhibitionof GABAAR-mediated currents has also recently been* Correspondence: ytwang@brain.ubc.ca1Brain Research Centre and Department of Medicine, Vancouver CoastalHealth Research Institute, University of British Columbia, Vancouver, BC V6T2B5, Canada2Translational Medicine Research Center, China Medical University Hospital,Graduate Institute of Immunology, China Medical University, Taichung 40447,TaiwanFull list of author information is available at the end of the article© 2014 Liu and Wang; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Liu and Wang Molecular Brain 2014, 7:6http://www.molecularbrain.com/content/7/1/6reported [14-16]. Ortinau et al. (2003) reported thatextracellular ATP inhibits the function of N-methyl-D-aspartate (NMDA) glutamate receptors by directly bind-ing to the receptor, suggesting that extracellular ATPmay function as an allosteric modulator for neurotrans-mitter receptors [17].High levels of ATP also exist in the extracellular com-partment under both normal physiological conditions (i.e.as result of synaptic release) [18-20], and pathologicalconditions such as traumatic and ischemic brain insults[21-24]. In addition, previous studies suggest that ATPand GABA are released at GABAergic synapses [18,19,25].Such a co-release suggests that, under certain conditions,ATP could act as an allosteric modulator for postsynapticGABAARs. In the current study, we set out to investigatethis hypothesis by using both cultured hippocampalneurons and HEK293 cells transiently expressing func-tional recombinant GABAARs. We found that both ATPand ADP can potentiate GABAAR-mediated currents.Moreover, this potentiation effect appears to be mediatedby a direct binding of these nucleotides at a putativenucleotide-binding site on the GABAAR.ResultsExtracellular ATP potentiates GABAAR-mediated currentsUnder whole-cell voltage-clamp recordings of hippocam-pal neurons at a holding membrane potential of -60 mV,repetitive short pulses of pressure ejections of GABA(100 μM) to the neuron evoked robust inward currents,and these currents were mediated through GABAAR-gatedCl- channels as they were completely blocked by bath ap-plication of the GABAAR antagonist bicuculline (10 μM;Figure 1A). Bath application of ATP at various concen-trations produced a dose-dependent potentiation of theGABA-evoked currents with an EC50 of 2.1 ± 0.2 mM(n = 8; Figure 1A and B). At a concentration of 4 mM,ATP increased the GABA-evoked currents to 171.9 ±13.8% of the control (n = 7, P < 0.01). The potentiation ofBACFigure 1 Extracellular ATP dose-dependently potentiates GABAAR-mediated currents in neurons. Whole-cell patch-clamp recording weremade from rat cultured hippocampal neurons. GABAAR-mediated currents were evoked by a pulse pressure ejection of GABA (100 μM) from thetip of a pipette positioned close to the neurons under recording. A, Bath application of ATP (4 mM) reversibly potentiates GABA currents (ATP;171.9 ± 13.8% of the Control; P < 0.01; n = 7) and the potentiated currents remained sensitive to bicuculline (10 μM) blockade (ATP + bicuculline).Representative current traces were taken 5 min before and after drug applications or washout as indicated. B, The dose-response curve showingthe dose-dependent potentiation of GABA currents by various concentrations of ATP (n = 8). Each data point represents the mean ± SEM of GABAcurrents (normalized to Control) at the indicated ATP concentrations. The solid line is the best fit of the data to the Hill equation, which yields amean EC50 of 2.1 ± 0.3 mM and H of 1.66. C, ATP increases the amplitude of GABA currents without altering their reversal potential. Left: Superimposedindividual traces of currents evoked by GABA (10 μM) in the absence (Control) and presence of ATP (4 mM) at different holding potentials from -60to +60 mV with a step of 20 mV. Right: current-voltage (I-V) relationships constructed from the data shown in the Left.Liu and Wang Molecular Brain 2014, 7:6 Page 2 of 11http://www.molecularbrain.com/content/7/1/6ATP on GABA currents was reversible upon washout andthe potentiated currents remained sensitive to bicuculliineblockade (Figure 1A). Analysis of the current-voltage(I - V) relationship in the absence and presence of ATPwithin the range of holding potentials from -60 to +60 mVshowed that ATP significantly potentiated the peak am-plitude of GABA currents without altering the reversal po-tential (Figure 1C). Thus, extracellular ATP at mM rangescan significantly potentiate the function of GABAARs.Extracellular ATP potentiates currents mediated by bothsynaptic and extrasynaptic GABAARsCurrents evoked by exogenously applied GABA could bemediated by synaptic and/or extrasynaptic GABAARs.To examine if ATP can modulate GABAARs localizedat the synapse, we examined effects of extracellularlyapplied ATP on whole-cell recordings of miniaturepostsynaptic inhibitory currents (mIPSCs) mediated bysynaptic GABAARs. mIPSCs were recorded at a holdingmembrane potential of -60 mV after blocking ionotropicglutamate receptors with CNQX (20 μM) and AP-5(50 μM), glycine receptor with strychnine (1 μM) andsodium channels with TTX (1 μM). Suramin (100 μM)and BBG (1 μM) were also added to the extracellular so-lution to block P2 receptors. Similar to the observationwith evoked GABA currents above, extracellular appli-cation of ATP at a concentration of 2 mM (but not100 μM) significantly potentiated mIPSCs (Figure 2Aand B). These increased mIPSCs were abolished byaddition of bicuculline (10 μM), indicating that mIPSCsfollowing ATP remained entirely gated through GABAARs(Figure 2A). The ATP enhancement of mIPSCs wasreversible, as it recovered to control levels after ATP andbicuculline wash out (Figure 2A). Consistent with an ef-fect on the postsynaptic GABAAR, but not on presynapticGABA release, ATP potentiation was manifested as aBADCFigure 2 Extracellular ATP potentiates function of both synaptic and extrasynaptic GABAARs in cultured hippocampal neurons.A-C, ATP increases the amplitude (but not the frequency) of mIPSCs mediated by synaptic GABAARs. Pharmacologically isolated GABAAR-mediated mIPSCs were recorded under whole-cell voltage clamp mode at a holding potential of -60 mV. A, Representative continuous recordingtraces taken 5 min before and after drug applications or washout showing dose-dependent effects of ATP on mIPSCs. ATP at a concentration of2 mM, but not 100 μM, potentiated mIPSCs. The potentiated mIPSCs were fully blocked by additional application of bicuculline (10 μM) andreturned to the control level after ATP washout. B, Cumulative amplitude and frequency distribution histograms of mIPSCs from the same cellshown in A before (black lines) and after (dotted lines) ATP application illustrating a specific increase in the mIPSC amplitude without alterationof its frequency. C, Histogram summarizing averaged changes in the amplitude and frequency of mIPSCs before (Control) and after application ofATP (2 mM) from 7 individual neurons. All values were presented as percentage of the controls. D, Extracellular ATP increases the tonic GABAcurrent. Tonic GABA currents were revealed by blocking native GABAARs with addition of bicuculline (20 μM) in the absence (Control) andpresence (ATP 2 mM) of ATP in the extracellular recording solution. Left, Representative tonic GABA current traces in the absence and presence ofATP taken from the same neuron 5 min before and after bath application of ATP (2 mM). Bar graphs on the Right summarizing data obtainedfrom 6 individual neurons. ** P < 0.01.Liu and Wang Molecular Brain 2014, 7:6 Page 3 of 11http://www.molecularbrain.com/content/7/1/6specific increase in the amplitude (132 ± 8.7% of the con-trol; p < 0.01; n = 7; Figure 2E), but not the frequency(96.4 ± 3.3% of the control; p > 0.05; Figure 2E), ofmIPSCs. These results indicate that extracellular ATP po-tentiates function of synaptic GABAARs, thereby increas-ing the synaptic (phasic) currents mediated by thesereceptors.Next, to determine if ATP has a modulatory effect onextrasynaptic GABAARs, thereby affecting tonic GABAcurrents, we examined tonic GABA currents revealed bythe addition of bicuculline (20 μM) in the presence orabsence of ATP (2 mM ATP). As shown in Figure 2D,bath application of bicuculline (20 μM) produced anoutward shift of the baseline current trace, indicatingthat these tonic currents are gated largely through extra-synaptically localized GABAARs activated by ambientGABA under the recording conditions [26]. Followingaddition of ATP (2 mM), bicuculline produced a sig-nificantly larger outward shift of the holding current(Figure 2D; 17.2 ± 2.7 pA in the presence of ATP vs 11.4 ±1.8 pA in the absence of ATP; P < 0.01; n =6). Thus, extra-cellular ATP appears capable of modulating both synapticand extrasynaptic GABAARs, thereby potentiating bothphasic and tonic GABA currents.Extracellular ATP modulates GABAAR function viamechanism of independent of ecto-protein kinases oractivation of P2 receptors.Intracellular ATP is usually maintained at a few milli-molar range (2-5 mM) at which it serves as a phosphatedonor to support regulation of GABAAR by proteinphosphorylation [27-29]. As extracellular (ecto-) proteinkinases have been previously demonstrated to phospho-rylate neuronal membrane proteins such as P2X3 recep-tors [30], we first examined the possibility that the ATPpotentiation of GABAAR function was a result of ecto-protein kinase mediated phosphorylation by using AMP-PNP, a non-hydrolyzable analog of ATP that cannot sup-port the protein phosphorylation process. As illustratedin Figure 3A, AMP-PNP (2 mM) in the bath mimickedATP, increasing the amplitude of GABA currents(Figure 3A; 134.2 ± 11.4%; P < 0.05; n = 5). Similarly, wefound that ADP (2 mM) was also able to significantlypotentiate GABA currents (Figure 3A; 140 ± 14.7;P < 0.05; n = 7). As both AMP-PNP and ADP cannotfunction as a phosphate donor during protein phospho-rylation process, these results suggest that extracellularprotein phosphorylation is not likely involved in theATP potentiation of GABAAR-mediated currents.As a neurotransmitter, at low concentrations (<100 μM)ATP can activate a number of P2 receptors [31]. The factthat extracellular ATP at this concentration has little po-tentiating effect on GABAAR currents induced by eitherexogenous (Figure 1) or endogenous GABA (Figure 2)strongly suggests that activation of purinergic receptors isunlikely to be responsible for the ATP potentiation ofGABA currents observed here. To further rule out thepotential involvement of activation of P2 receptors, P2receptor antagonists were perfused prior to, and duringthe application of ATP. At a concentration of 100 μM,suramin (a broad-spectrum antagonist of P2 receptors)blocks almost all P2 receptors, but has almost no effect onP2X7 receptors [32,33]. BBG is a potent P2X7 receptor an-tagonist with a very low IC50 (< 10 nM) [34,35]. As shownin Figure 3C, although application of suramin (100 μM)and BBG (1 μM) resulted in reduction of GABA currentson their own, ATP (2 mM) was still able to potentiateGABA currents in the presence of suramin and BBG(Figure 3C). The mean amplitude of GABA currents wasincreased to 138.5 ± 16.5% (n = 5; P < 0.05) of the control.This suggests that the ATP potentiation of GABA currentsis not due to activation of P2 receptors. Together,our results appear to reveal a previously undescribedA B CFigure 3 Potentiation of GABAAR-mediated currents by ATP and analogs does not require activation of ecto-protein kinase or P2receptors in cultured hippocampal neurons. Representative individual whole-cell GABA current traces were taken 5 min before (Control)and after drug applications (100 μM, pulse-pressure ejection). A and B, AMP-PNP (2 mM; A) or ADP (2 mM; B), two ATP analogues that cannotfunction as phosphate donor for ecto-protein kinase mediated phosphorylation, remain capable of potentiating GABA currents, increasing thepeak currents by 134.2 ± 11.4% (A; P < 0.05; n = 5) and 140 ± 14.7 (B; P < 0.05; n = 7), respectively. C, Non selective P2 receptor antagonist suramin(100 μM) and selective P2X7 receptor antagonist BBG (1 μM) suppressed basal GABA currents on their own, but failed to prevent ATP frompotentiating the currents; ATP increased the peak currents by 138.5 ± 16.5% (n = 5; P < 0.05) in the presence of both antagonists.Liu and Wang Molecular Brain 2014, 7:6 Page 4 of 11http://www.molecularbrain.com/content/7/1/6mechanism of ATP modulation of GABAARs, one whichis not dependent on either ecto-protein kinases or P2 re-ceptor activation.The enhancement of GABAAR function by extracellularATP is likely mediated by an allosteric mechanismATP may also allosterically modulate functions of manyproteins, including neurotransmitter receptors, by directlybinding to these proteins [17,36-38]. As an initial step totest this possibility, we investigated the effect of extracellu-lar ATP on GABAAR-gated single channel activities inexcised membrane patches under various modes of single-channel recordings. Suramin (100 μM) and BBG (1 μM)were added in the extracellular solution to block P2 recep-tors. Under the outside-out mode at a holding potentialof –60 mV (Figure 4), no single channel activities were ob-served in the absence of GABA. ATP alone (1 mM) didnot induce any detectable single channel currents (datanot shown). When 0.5 μM GABA was added to the extra-cellular recording solution, single channel activities oc-curred frequently, but were abolished by the addition ofbicuculline (10 μM) in the extracellular solution, con-firming that the currents were gated through GABAARs(Figure 4A). The mean single channel conductance andopen probability (Po) were 27 ± 0.85 pS and 0.18 ± 0.04(n = 7), respectively. As shown in Figure 4A, co-applica-tion of ATP (1 mM) significantly increased the Po of theGABAAR-gated single-channel activities without alteringeither main channel conductance or reversal potential(Figure 4A and B). The mean Po and single channel con-ductance in the presence of ATP were respectively 0.32 ±0.06 (p < 0.01; n = 7; Figure 4C) and 27 ± 1.02 pS (P > 0.05;n = 7). The changes in mean Po may result from alteredchannel open times and/or frequency. Open time distri-bution histograms revealed that the mean open time inthe presence of ATP was 11.3 ± 1.5 ms, which was sig-nificantly different from the control (6.4 ± 1.1 ms; P < 0.05,n = 7; Figure 4D). Although the frequency in the pre-sence of ATP was slightly increased (control: 2.6 Hz vs.ATP: 3.0 Hz), it was not significantly different from thecontrol (P > 0.05; n = 7). These results suggest that ATPpotentiation of GABA currents is achieved by anAB C DFigure 4 Extracellular ATP increases the open time of GABA single-channel activity in outside-out mode in hippocampal neurons.Outside-out GABAAR gated single channel activities were recorded by bath application of GABA (0.5 μM) to the excised patches through bath applications(A, Left) in absence (GABA; 0.5 μM) or presence of ATP (+ATP 1 mM) or ATP + bicuculline (+bicuculline 10 μM) in the bath or after drug washout (Washout).Suramin (100 μM) and BBG (1 μM) were added in the bath throughout the recording to block P2 receptors. A, Representative GABA current traces wereobtained 5 min before (GABA 0.5 μM) and after drug applications or washout as indicated above traces. In this patch, Po increased from 0.15 in the control(GABA) to 0.28 after addition of ATP. B, Current-voltage relationship of GABA single channel activity in the absence (Control) and presence of ATP (1 mM)at various holding membrane potentials. C and D, Bar graphs summarizing the mean Po (Left) and open times (Right) in the absence (GABA) and presenceof ATP (GABA+ATP) from seven cells. ** P < 0.01.Liu and Wang Molecular Brain 2014, 7:6 Page 5 of 11http://www.molecularbrain.com/content/7/1/6increase in single channel Po, mainly due to prolongedopen times.The observation that potentiation can be detected inthe excised patches under outside-out mode is consis-tent with the idea that ATP is not dependent on any dif-fusible intracellular signaling molecule. Thus, ATP mayexert its modulating effects by directly acting on theGABAAR itself, or on a protein that is tightly associatedwith the GABAAR. This idea was further supported bysingle-channel recordings under the on-cell attachedconfiguration. In this mode, GABA (0.5 μM) was addedto the recording solution in the pipette to selectivelyactivate GABA single channels within the pipette tip(Figure 5A). Bath perfusion of ATP (1 mM) outside of therecording pipette had little effect on single channel acti-vities (Figure 5A and B; the mean Po and the main channelcurrent amplitude being 0.08 ± 0.02 and 1.66 ± 0.2 pA inthe presence of ATP and 0.07 ± 0.02 and 1.64 ± 0.2 pA inthe absence of ATP; P > 0.05, n = 5). However, applicationof ATP (1 mM) along with GABA (0.5 μM) into the re-cording pipette solution resulted in a significant increasein the mean Po (Po: 0.19 ± 0.04; P < 0.01, compared withthat in the absence of ATP; n = 6; Figure 5A and B) andwas similar to that observed under the outside-out mode,leading to a significant increase in the mean open times(Figure 5C; 7.7 ± 0.8 ms in the presence of ATP vs4.6 ± 0.4 ms in the absence of ATP; P > 0.01, n = 5).Together, single channel results from both outside-outand on-cell modes support the notion that ATP poten-tiates GABA currents, likely via a mechanism of allostericmodulation.To further explore this possible allosteric modulationvia a direct binding to the GABAAR itself, we finallyexamined the ability of extracellular ATP and its analogsto potentiate the function of recombinant GABAARstransiently expressed in HEK293 cells in the absence ofother known neuronal proteins. GABAARs are pentamerichetero-oligmers assembled from various distinct subunitcombinations. Although more than sixteen distinct sub-units have been identified, most native GABAARs in themammalian brain consist of 2α, 2β and 1γ subunits[1,39]. We transiently expressed recombinant rat α1β2γ2GABAARs, the most abundant composition of nativeGABAAR subtypes in the mammalian brain [39]. NeitherBACFigure 5 Extracellular ATP increases GABA single-channel open time in cell attached mode. GABAAR channels in the patch membraneunderneath the tip of the recording pipette were specifically activated by inclusion of GABA (0.5 μM) in the intra-pipette recording solution at aholding membrane potential of 0 mV. A, Representative current traces revealing that ATP (1 mM) increased the GABA single-channel activity onlywhen it was included through the recording pipette. Bar graphs on the Right summarize the mean open probability (Po; B) and open times(C) averaged from 5 individual recordings in the absence (Control) and presence of ATP (1 mM) outside the pipette in the bath solution(ATP outside pipette) or inside the pipette by its inclusion in the pipette solution (ATP inside pipette). ** P < 0.01.Liu and Wang Molecular Brain 2014, 7:6 Page 6 of 11http://www.molecularbrain.com/content/7/1/6ATP nor ADP at a concentration of 2 mM produced anydetectable currents in HEK293 cells. Fast perfusion ofGABA (3 μM) reliably produced inward currents, whichwere blocked by 10 μM bicuculline, indicating the currentswere mediated by bicuculline sensitive GABAAR-mediatedchloride-gated ion channels (Figure 6A). Co-perfusion ofGABA (3 μM) and ATP at various concentrations resultedin a significant increase in the peak amplitudes in a dose-dependent manner, with a minimum concentration of ap-proximately 100 μM and a maximum concentration ofgreater than 2 mM (Figure 6A and B; n = 6). At a con-centration of 0.5 mM, ATP increased the GABA currentsto 138.7 ± 14.2 of the control value (Figure 6A and B;P < 0.05, n = 6). The potentiated currents were completelyabolished by bicuculline (10 μM; Figure 6A), and reco-vered to control levels after washing out ATP (Figure 6A).This ATP potentiation is also GABA concentrationdependent. As shown in Figure 6C, in the absence ofATP, GABA produced currents in a dose-dependentmanner with the EC50 and Hill coefficient (h) beingrespectively 11.6 ± 3.3 μM and 1.66 ± 0.1 (n = 5). Bathapplication of ATP (0.5 mM) caused a left shift of theGABA dose-response curve, reducing the EC50 to 5.8 ±2.1 μM without altering the h (1.62 ± 0.1). Similar toobservations obtained in neurons, the ATP potentiationof GABAARs was also mimicked by ADP. We found thatbath application of ADP (2 mM) increased GABAcurrents by 147.5 ± 15.9% of the control (P < 0.01, n = 5;Figure 5D). Thus, similar to native GABAARs in neurons,recombinant rat GABAARs overexpressed in HEK293 cellsare also subject to potentiation by extracellular ATP.These results therefore provide additional support for thenotion that ATP modulation of GABAARs through anallosteric mechanism that does not require other neuronalproteins.DiscussionThe modulation of GABAAR function by extracellularATP observed here is unlikely to require activation ofP2Y receptors, as was previously reported in rat cerebel-lar granule cells [13]. First, Saitow and colleagues foundthat ADP potentiation of postsynaptic GABAAR-me-diated currents was long-lasting and could be demon-strated at a much lower ADP concentration (within tensof micromoles). In our study, the potentiating effect ofATP or ADP on GABA currents was transient and re-versible, quickly returning to baseline level upon ATPwashout. Moreover, ATP potentiation was only observedat concentrations above 100 μM. Second, it is well estab-lished that suramin at a concentration of 100 μM canblock the majority of P2 receptors (with the exception ofP2X7 receptors) and that BBG has a very high affinityfor the P2X7 receptor with an IC50 of 10 nM to 0.2 μM[35]. We found that the potentiating effect of ATP orADP on GABA currents was not blocked by suramin(100 μM) and BBG (1 μM).It has been speculated that cross-talk between GABAAand P2X receptors may play in a role in ATP regulationof GABAARs. Although underlying mechanisms remaincontroversial, recent studies suggest that it depends onelevated [Ca2+]i [12,16] (but, also see [16]). However,AB CDFigure 6 Extracellular ATP and ADP potentiates function ofrecombinant GABAARs. Whole-cell voltage-clamp recordings wereperformed in HEK293 cells transiently expressing rat recombinant α1β2γ2GABAARs at a holding membrane potential of -60 mV. GABA currentswere induced by fast perfusion of GABA at various concentrations.A, Representative current traces showing the potentiation of GABA(3 μM) currents by co-perfusion of ATP (0.5 mM). The increased currentwas blocked in the presence of bicuculline (10 μM) and returned to thecontrol level after ATP washout. B, The dose-response curve showing thedose-dependent potentiation of the GABA (3 μM) currents by variousconcentration of ATP. All values were normalized to the maximal GABAcurrents (Imax) induced at 3 mM of ATP. Each data point is the mean ±SEM of normalized GABA currents at the indicated ATP concentrations(n = 6). The solid line is the best fit of the data to the Hill equation. TheEC50 and H were 390 ± 48 μM and 1.66 ± 0.1, respectively. C, ATPpotentiation of GABAAR activity is GABA dose-dependent. GABA dose-response curves were constructed in the absence (Control) and presence(ATP) of ATP (0.5 mM) from five cells. ATP shifted the curve to the leftand reduced EC50 from 11.6 ± 3.3 μM to 5.8 ± 2.1 (P < 0.01) withoutchange in H (control: 1.57 ± 0.1; ATP: 1.62 ± 0.1; P > 0.05). All values werenormalized to the Imax induced by GABA (300 μM). D, ADP mimics ATP,potentiating the activity of recombinant GABAARs. Representative currenttraces showed that application of ADP (0.5 mM) reversibly increasedcurrents induced by GABA (3 μM). On average, it increased the currentsby 147.5 ± 15.9% (P < 0.01; n = 5).Liu and Wang Molecular Brain 2014, 7:6 Page 7 of 11http://www.molecularbrain.com/content/7/1/6whether [Ca2+]I dependent or not, such a receptor-crosstalk is unlikely to be responsible for the ATP poten-tiation of GABAAR function observed in the presentstudy, as blockade of P2 receptors had little effect,and the BAPTA (10 μM) included in our intracellularrecording solution should be sufficient to prevent the[Ca2+]I-dependent processes proposed in these earlierstudies.Ecto-protein kinases have been identified in the CNSand can modulate functions of membrane receptorssuch as P2X3 receptors [30]. However, in the presentstudy we demonstrated that both AMP-PNP and ADPmimicked the effects of ATP, potentiating the functionof GABAARs. As both of AMP-PNP and ADP cannotsubstitute ATP in supporting the protein phosphory-lation reaction, these results can essentially rule out theinvolvement of ecto-protein kinase mediated ex extracel-lular protein phosphorylation.ATP is also known to function as an allostericmodulator for a number of proteins by directly bindingto these proteins, regulating their functions. These pro-teins include CFTR (Quinton PM, Reddy MM., 1992),GABAARs [40], InsP3 receptors [37,41], and capsaicin-activated ion channels [42]. But, in most of these cases,ATP binds to the intracellular domains of these proteins.Whether extracellular ATP can allosterically modulateGABAARs via a direct binding to the extracellular do-mains of the receptor has not previously been suggested.Here, we provide several pieces of evidence that areconsistent with such a mode of regulation. First, using re-combinant rat GABAARs transiently expressed in HEK293cells, we were able to demonstrate that extracellularATP can potentiate the function of these recombinantGABAARs in a manner similar to observations obtainedwith native GABAARs in neurons, suggesting that theATP modulation does not require any additional neuronalspecific proteins other than GABAARs themselves.Second, ATP potentiation could be demonstrated in theexcised cell-free membrane patches under the outside-outconfiguration in neurons (Figure 4). These results indicatethat the potentiation of GABAARs by extracellular ATPdoes not require any diffusible second messenger mol-ecule downstream of an unknown metabotropic receptor,thereby providing strong support for a direct binding ofATP or its analog to an extracellular domain of GABAARitself, or a membrane surface protein tightly associatedwith the receptor. Finally, this notion is further streng-thened by our results obtained with single-channel recor-dings under the on-cell attached configuration (Figure 5).Under this configuration, currents through single or veryfew GABAAR channels in the membrane patch under-neath of the tip of the recording pipette can be recordedin isolation from GABAARs outside of the pipette tip byapplying GABA to the isolated membrane patch throughthe recording pipette solution. Using this configuration,we were able to demonstrate that the GABAAR singlechannel activities from the isolated membrane patch in-side of the recording pipette can only be potentiated byATP applied into the patch membrane through the re-cording pipette solution, but not by that applied extracel-lularly to the plasma membrane outside of the recordingpipette tip (Figure 5). Together, the present study providesstrong evidence supporting a novel mode of modulationof GABAARs by extracellular ATP; allosteric modulationlikely achieved by either a direct binding of ATP to the re-ceptor itself, or to an unknown protein tightly associatedwith the receptor. Nonetheless, given that extracellularATP may potentially penetrate the plasma membrane, thepossibility that ATP potentiates GABAAR function by adirect action on an intracellular domain of the receptorremains to be ruled out. Thus, the ultimate evidence forsuch a novel allosteric modulation will only come fromthe positive identification/characterization of the novelbinding site (s) on GABAAR domain (s) or to identifythe receptor associated protein by which ATP acts. Inaddition, whether the potentiation of GABAARs by ATP isstate-dependent remains to be determined.How ATP binding modulates the GABAAR remains tobe determined. ATP binding may cause a conformationalchange to the GABAAR, thereby affecting its agonistbinding affinity, channel gating, or both. Using variousmodes of single-channel recordings and analysis, in thepresent study we observed that extracellular ATP canincrease GABAAR-gated channel activities by primarilyincreasing the channel open times without altering itsconductance. This may suggest that ATP binding pre-dominantly alters the apparent agonist binding affinityto the receptor, rather than the conformational changeof the channel pore. This conjecture is further streng-thened by the GABA dose-response relationship analysisfrom HEK293 cells as extracellular ATP results in a left-shift of the GABA dose-response curves without alteringeither h coefficient or the maximal responses (Figure 6B).Future site-direct mutations of the putative ATP bindingdomain along with direct ATP binding assays may pro-vide a better understanding of the detailed mechanismsby which ATP exerts its modulation of the GABAAR.In the mammalian brain, GABAARs play a key role inregulating the excitation-inhibition balance (and hencethe tight control of neuronal excitability), and this func-tion is primarily realized by mediating synaptic (phasic)inhibition and tonic inhibition [26]. In the present study,we demonstrated that application of extracellular ATP atmillimolar concentrations not only potentiates GABAcurrents evoked by exogenously applied GABA, but alsoboth synaptic and tonic currents activated by endo-genous GABA. These results suggest that extracellularATP has significant physiological and/or pathologicLiu and Wang Molecular Brain 2014, 7:6 Page 8 of 11http://www.molecularbrain.com/content/7/1/6impacts on neuronal excitability via modulating GABAARs.To this end, it is relevant to point out that several previousstudies have suggested that under normal conditions, theextracellular ATP in the CNS is approximately 1-100 μM[25]. At such a low level, the basal extracellular ATP mayhave little influence on neuronal excitability via GABAARs.However, it is important to note that ATP has previouslybeen shown to be co-localized with GABA in the samevesicles at certain GABAergic synapses, and more impor-tantly, that these two transmitters can be co-released intothe synaptic cleft [18,19,25], whereby ATP concentrationscan transiently reach the levels above hundreds of micro-moles or even millimolar concentrations. In the presentwork, we demonstrated that at these concentrations, extra-cellular ATP can potentiate GABAAR-mediated mIPSCs.The fact that ATP potentiation of mIPSCs is primarilymanifested as a specific increase in mIPSC amplitude,without altering its frequency, is in good agreement withthe allosteric modulation of postsynaptic GABAARs.Thus, this mode of modulation may function at certainGABAergic synapses under physiological conditions.Similarly, the modulation may also occur under patho-logical conditions (including neuronal overexcitation,epileptic episodes, inflammation, traumatic insults, hyp-oxia/ischemia), as ATP release from damaged neuronsand astrocytes can rapidly increase extracellular ATPconcentrations [21-24]. ATP may increase both phasicand tonic GABA currents by acting on both synapticand extrasynaptic GABAARs. By forming such a ho-meostatic feedback loop, under pathological conditionsextracellular ATP may exert significant impacts onneuronal function and/or dysfunction. However, a relatedcaveat is the potential complication from acidosis that isoften associated with high concentrations of extracellularATP. Given that acidosis is known to reduce GABAARactivity, how it will impact this ATP-induced allostericpotentiation warrants future investigations.ConclusionsIn this study, we demonstrate that extracellular ATP andits analogs such as ADP can potentiate function ofGABAARs via a novel mechanism likely involving thedirect binding of ATP to a putative ATP-binding site onthe GABAAR itself. We demonstrate that through thismodulation, extracellular ATP can enhance both phasic(synaptic) and tonic GABAAR-mediated currents. There-fore, the present study reveals a novel means by whichextracellular ATP contributes to regulating excitation-inhibition balance and neuronal excitability under certainphysiological and pathological conditions. In addition, dueto the importance of GABAARs in mediating neuronal in-hibition in the brain, they have been a major therapeutictarget for the development of many drugs currently usedfor the clinical treatment of brain disorders. Furtheridentifying the exact putative binding site of ATP onGABAARs may lead to the development of novelGABAAR-based therapeutics for better management ofthese brain disorders.MaterialsPrimary culture of hippocampal neuronsMethods for culturing hippocampal neurons have beendescribed previously [43]. Briefly, hippocampi from E18old Wistar rat embryos were dissected and treated with0.25% trypsin solution (Invitrogen) for 25 min at 37°C,then mechanically dissociated using fire-polished pasteurpipettes. Cell suspension was centrifuged at 2500 × g for50 s and the cell pellets were resuspended in DMEMwith 10% fetal bovine serum (FBS). Cells were seeded onpoly-D-lysine-coated 24-well coverslips at a density of0.8-1.0 × 105 cells/well. Cultures were maintained in ahumidified incubator with 5% CO2 at 37°C. After 24 h,the plating medium was changed to Neurobasal mediumsupplemented with B-27 and L-glutamine (0.5 mM) andneurons were fed with fresh medium twice weekly.Experiments were done 14-18 days after the plating.Expression of recombinant GABAA receptors in HEK293cellsHuman embryonic kidney (HEK) 293 cells were culturedas previously described [44]. Briefly, HEK293 cells werecultured in DMEM supplemented with 10% FBS. Cellswere harvested weekly and seeded at 10% confluence onpoly-L-lysine-coated glass coverslips in 24-mm culturedishes. Cells were transiently co-transfected at 70% con-fluence with rat cDNAs encoding α1, β2 and γ2-EGFPsubunits of the GABAA receptor at a 1:1:1 ratio usingLipofectamine2000 (Invitrogen, Carlsbad, CA). Recor-dings were made 24-48 h after transfection.Whole-cell patch clamp recordingCoverslips were transferred to the recording chamberand were continuously perfused with an extracellularsolution containing the following (in mM): NaCl 140,KCl 5.4, MgCl2 1.3, HEPES 25, CaCl2 1.3, glucose 20,pH 7.35-7.45, 305-315 mOsm. Recordings were per-formed in the voltage-clamp mode using an Axopatch200B patch-clamp amplifier (Axon Instruments). Thecell membrane were held at a potential of -60 mV andsignals were filtered at 2 KHz, digitized at 10 KHz usinga Digidata 1322A analog-to-digital converter and ac-quired by Clampex 9.2 (Axon Instruments). Recordingelectrodes (3–5 M) were fabricated from thin-walledborosilicate glass tubing (World Precision Instruments,USA) with a micropipette puller (Sutter Instruments,model P-97, Novato, CA). Recording pipettes were filledwith an intracellular solution containing (in mM): CsCl140, HEPES 10, 1,2-bis (2-aminophenoxy) ethane-N, N,Liu and Wang Molecular Brain 2014, 7:6 Page 9 of 11http://www.molecularbrain.com/content/7/1/6N, N-tetraacetic acid (BAPTA-Cs) 10, Mg-ATP 4, QX-314 5, pH 7.20; osmolarity, 290-295 mOsm. CNQX(20 μM), AP-5 (50 μM) and tetrodotoxin (TTX, 0.5 μM)were included in the external solution to block gluta-matergic and the voltage-gated sodium channels. Allexperiments were performed at room temperature.Induction of GABAAR-mediated currentsGlass pipettes were filled with GABA (100 μM) dissolvedin the extracellular recording solution. The pipette tipwas placed in the vicinity of recorded neurons. GABAwas applied via pressure ejection using a Picospritzer(General Valve Corporation, Fairfield, NJ) at 60 sec in-tervals. For recording of GABA currents in HEK293cells, fast perfusion of GABA and/or other ligands wereemployed using a computer-controlled multibarrel fastperfusion system (Warner Instruments). For some of theexperiments using bath perfusion of ATP or ADP, 5 mMEGTA was added into Ca2+-free extracellular solution tofurther reduce residual Ca2+ in extracellular solution,thereby minimizing any potential effect of ATP/ADP-induced extracellular Ca2+ influx. Under our experi-mental conditions in cultured neurons, the Ca2+-freesolution neither produced any observable current on itsown nor significantly affected GABAAR-gated currents(1 μM GABA; -648 ± -245pA in control solutionvs -677 ± -256pA in Ca2+-free solution supplementedwith 5 mM EGTA; n = 7; p < 0.05).mIPSCs and GABA tonic current recording and analysisGABAA receptor-mediated miniature inhibitory post-synaptic currents (mIPSCs) were recorded at a holdingpotential of −60 mV. CNQX (20 μM), APV (50 μM) andTTX (1 μM) were added to the extracellular solution toisolate GABAergic mIPSCs. Before drug application, a3-5 min period of baseline recording (control) was ob-tained. The recordings were low-pass filtered (Clampfitsoftware) at 2 kHz, digitized at 10 KHz using a Digidata 1322A analog-to-digital converter and acquired byClampex 9.2. Detection and analysis of mIPSCs wereperformed using Mini Analysis Program (Synaptosoft,Decatur, GA). Any spurious noise was rejected. ThemIPSC kinetics were obtained from analysis of the aver-aged single events. To facilitate analysis, decay time con-stant (τD) was obtained by fitting the decay phase to asingle exponential equation. Tonic GABA current wasestimated as the change in baseline current produced bya 2 min application of bicuculline (20 μM).Single channel analysisPatch pipettes for cell-attached and outside-out singlechannel recordings were pulled from thick-wall borosili-cate glass (GC150F, Harvard Apparatus), fire polished,and coated with Sylgard 184 (Dow Corning) with aresistance of 6-10 MΩ. For outside-out recordings, therecording pipette was filled with the intracellular solu-tion at a holding potential of – 60 mV. GABA (0.5 μM)was added to the external solution to activate GABAARs.In cell-attached patch mode, the composition of the pip-ette solution was (in mM): KCl 120, TEA-Cl 20, 1.3MgCl2, HEPES 10, pH 7.40, 290-300 mOms. GABA(0.5 μM) or GABA (0.5 μM) and ATP (1 mM) were in-cluded in the pipette solution. Signals were filtered at1 kHz, sampled at 10 kHz and analyzed off-line usingClampfit 9.2. An idealized recording of the durationsand amplitudes of detectable events of the single-channel data was generated using 50% threshold cros-sing criteria. Events with a duration less than 300 μswere ignored. Single channel activities were expressed asthe product of the number of channels × the open prob-ability (Po); i.e. NPo = ∑[(open time × number of channelsopen) ∕ total time of record].DrugsDrugs used in the present study were purchased fromthe following sources: nucleotides (ATP, UTP, GTP,ADP, UDP, GDP, AMP, UMP, GMP), GABA and bicu-culline (Sigma-Aldrich); suramin, CNQX, APV, PPADS(Tocris); and TTX (Alomone Labs).Statistical analysisData is presented as means ± SEM, where n represents thenumber of tested cells. One-way ANOVA or the two-tailed Student’s test was used for statistical analysis andP values less than 0.05 were considered statistically signifi-cant. Dose–response curves were constructed by fittingdata to the Hill equation: I = Imax/(1 + EC50 / [A]n), whereI is the current, Imax is the maximum current, [A] is agiven concentration of agonist, n is Hill coefficient (H).Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsJL and YTW designed the experiments. JL performed and analyzed allexperiments. JL and YTW wrote the manuscript. Both authors read andapproved the final manuscript.AcknowledgementsWe thank Y.P. Li for preparation and maintenance of hippocampal neuronalcultures and L. W. Oschipok for his excellent editorial assistance. This workwas supported by the Canadian Institutes for Health Research and theTaiwan Department of Health Clinical Trial and Research Center of Excellence(DOH102-TD-B-111-004). J.L. was supported by postdoctoral fellowships fromNational Sciences and Engineering Research Council, the Michael SmithFoundation for Health Research and the British Columbia Epilepsy Society.Y.T.W. is the holder of the Heart and Stroke Foundation of British Columbiaand Yukon chair in stroke research.Author details1Brain Research Centre and Department of Medicine, Vancouver CoastalHealth Research Institute, University of British Columbia, Vancouver, BC V6T2B5, Canada. 2Translational Medicine Research Center, China MedicalUniversity Hospital, Graduate Institute of Immunology, China MedicalLiu and Wang Molecular Brain 2014, 7:6 Page 10 of 11http://www.molecularbrain.com/content/7/1/6University, Taichung 40447, Taiwan. 3Current address: Medical ResearchCenter, Qianfoshan Hospital, Shandong University, Jinan, Shandong 250014,China.Received: 22 July 2013 Accepted: 19 December 2013Published: 24 January 2014References1. Macdonald RL, Olsen RW: GABAA receptor channels. 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Molecular Brain 2014 7:6.Liu and Wang Molecular Brain 2014, 7:6 Page 11 of 11http://www.molecularbrain.com/content/7/1/6"@en ; edm:hasType "Article"@en ; edm:isShownAt "10.14288/1.0132616"@en ; dcterms:language "eng"@en ; ns0:peerReviewStatus "Reviewed"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "BioMed Central"@en ; ns0:publisherDOI "10.1186/1756-6606-7-6"@en ; dcterms:rights "Attribution 4.0 International (CC BY 4.0)"@en ; ns0:rightsURI "http://creativecommons.org/licenses/by/4.0/"@en ; ns0:scholarLevel "Faculty"@en ; dcterms:subject "GABAA receptor"@en, "ATP"@en, "Allosteric potentiation"@en, "Neuronal cultures"@en ; dcterms:title "Allosteric modulation of GABAA receptors by extracellular ATP"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/55001"@en .