Allosteric modulation of GABAA receptors by extracellular ATP
© Liu and Wang; licensee BioMed Central Ltd. 2014
Received: 22 July 2013
Accepted: 19 December 2013
Published: 24 January 2014
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.
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.
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.
The γ-aminobutyric acid type A receptor (GABAAR) is a ligand-gated chloride ion channel, activation of which results in membrane hyperpolarization and hence inhibition of the neuronal excitability in the adult mammalian brain. Dysfunction of GABAARs is associated with the pathogenesis of a number of neurological diseases and neuropsychiatric disorders such as epilepsy, Alzheimer disease, and anxiety [1–6]. GABAARs are also targets of many clinically-relevant drugs including benzodiazepine, barbiturates and general anesthetics . Moreover, endogenously produced substances such as neurosteroids  and zinc [9, 10] modulate GABAARs via direct interaction with the putative binding sites on the receptor subunit. Therefore, allosteric modulation is an important mode in regulating GABAAR functions and hence maintaining homeostasis for neuronal excitability.
In the CNS, adenosine 5′-triphosphate (ATP) not only acts as a major intracellular energy source and phosphate donor, but also functions extracellularly as a neurotransmitter via activation of purinergic 2 (P2) receptors. Previous studies demonstrate that extracellular ATP can modulate GABAAR function by activation of P2 receptors [11–13]. In addition, a physical cross-talk between GABAARs and P2 receptors which influencess inhibition of GABAAR-mediated currents has also recently been reported [14–16]. Ortinau et al. (2003) reported that extracellular ATP inhibits the function of N-methyl-D-aspartate (NMDA) glutamate receptors by directly binding to the receptor, suggesting that extracellular ATP may function as an allosteric modulator for neurotransmitter receptors .
High levels of ATP also exist in the extracellular compartment under both normal physiological conditions (i.e. as result of synaptic release) [18–20], and pathological conditions such as traumatic and ischemic brain insults [21–24]. In addition, previous studies suggest that ATP and 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 postsynaptic GABAARs. In the current study, we set out to investigate this hypothesis by using both cultured hippocampal neurons and HEK293 cells transiently expressing functional recombinant GABAARs. We found that both ATP and ADP can potentiate GABAAR-mediated currents. Moreover, this potentiation effect appears to be mediated by a direct binding of these nucleotides at a putative nucleotide-binding site on the GABAAR.
Extracellular ATP potentiates GABAAR-mediated currents
Extracellular ATP potentiates currents mediated by both synaptic and extrasynaptic GABAARs
Next, to determine if ATP has a modulatory effect on extrasynaptic GABAARs, thereby affecting tonic GABA currents, we examined tonic GABA currents revealed by the addition of bicuculline (20 μM) in the presence or absence of ATP (2 mM ATP). As shown in Figure 2D, bath application of bicuculline (20 μM) produced an outward shift of the baseline current trace, indicating that these tonic currents are gated largely through extrasynaptically localized GABAARs activated by ambient GABA under the recording conditions . Following addition of ATP (2 mM), bicuculline produced a significantly 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, extracellular ATP appears capable of modulating both synaptic and extrasynaptic GABAARs, thereby potentiating both phasic and tonic GABA currents.
Extracellular ATP modulates GABAAR function via mechanism of independent of ecto-protein kinases or activation of P2 receptors.
As a neurotransmitter, at low concentrations (<100 μM) ATP can activate a number of P2 receptors . The fact that extracellular ATP at this concentration has little potentiating effect on GABAAR currents induced by either exogenous (Figure 1) or endogenous GABA (Figure 2) strongly suggests that activation of purinergic receptors is unlikely to be responsible for the ATP potentiation of GABA currents observed here. To further rule out the potential involvement of activation of P2 receptors, P2 receptor antagonists were perfused prior to, and during the 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 on P2X7 receptors [32, 33]. BBG is a potent P2X7 receptor antagonist with a very low IC50 (< 10 nM) [34, 35]. As shown in Figure 3C, although application of suramin (100 μM) and BBG (1 μM) resulted in reduction of GABA currents on their own, ATP (2 mM) was still able to potentiate GABA currents in the presence of suramin and BBG (Figure 3C). The mean amplitude of GABA currents was increased to 138.5 ± 16.5% (n = 5; P < 0.05) of the control. This suggests that the ATP potentiation of GABA currents is not due to activation of P2 receptors. Together, our results appear to reveal a previously undescribed mechanism of ATP modulation of GABAARs, one which is not dependent on either ecto-protein kinases or P2 receptor activation.
The enhancement of GABAAR function by extracellular ATP is likely mediated by an allosteric mechanism
This ATP potentiation is also GABA concentration dependent. As shown in Figure 6C, in the absence of ATP, GABA produced currents in a dose-dependent manner with the EC50 and Hill coefficient (h) being respectively 11.6 ± 3.3 μM and 1.66 ± 0.1 (n = 5). Bath application of ATP (0.5 mM) caused a left shift of the GABA dose-response curve, reducing the EC50 to 5.8 ± 2.1 μM without altering the h (1.62 ± 0.1). Similar to observations obtained in neurons, the ATP potentiation of GABAARs was also mimicked by ADP. We found that bath application of ADP (2 mM) increased GABA currents 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 cells are also subject to potentiation by extracellular ATP. These results therefore provide additional support for the notion that ATP modulation of GABAARs through an allosteric mechanism that does not require other neuronal proteins.
The modulation of GABAAR function by extracellular ATP observed here is unlikely to require activation of P2Y receptors, as was previously reported in rat cerebellar granule cells . First, Saitow and colleagues found that ADP potentiation of postsynaptic GABAAR-mediated currents was long-lasting and could be demonstrated at a much lower ADP concentration (within tens of micromoles). In our study, the potentiating effect of ATP or ADP on GABA currents was transient and reversible, quickly returning to baseline level upon ATP washout. Moreover, ATP potentiation was only observed at concentrations above 100 μM. Second, it is well established that suramin at a concentration of 100 μM can block the majority of P2 receptors (with the exception of P2X7 receptors) and that BBG has a very high affinity for the P2X7 receptor with an IC50 of 10 nM to 0.2 μM . We found that the potentiating effect of ATP or ADP on GABA currents was not blocked by suramin (100 μM) and BBG (1 μM).
It has been speculated that cross-talk between GABAA and P2X receptors may play in a role in ATP regulation of GABAARs. Although underlying mechanisms remain controversial, recent studies suggest that it depends on elevated [Ca2+]i[12, 16] (but, also see ). However, whether [Ca2+]I dependent or not, such a receptor-cross talk is unlikely to be responsible for the ATP potentiation of GABAAR function observed in the present study, as blockade of P2 receptors had little effect, and the BAPTA (10 μM) included in our intracellular recording solution should be sufficient to prevent the [Ca2+]I-dependent processes proposed in these earlier studies.
Ecto-protein kinases have been identified in the CNS and can modulate functions of membrane receptors such as P2X3 receptors . However, in the present study we demonstrated that both AMP-PNP and ADP mimicked the effects of ATP, potentiating the function of GABAARs. As both of AMP-PNP and ADP cannot substitute ATP in supporting the protein phosphorylation reaction, these results can essentially rule out the involvement of ecto-protein kinase mediated ex extracellular protein phosphorylation.
ATP is also known to function as an allosteric modulator for a number of proteins by directly binding to these proteins, regulating their functions. These proteins include CFTR (Quinton PM, Reddy MM., 1992), GABAARs , InsP3 receptors [37, 41], and capsaicin-activated ion channels . But, in most of these cases, ATP binds to the intracellular domains of these proteins. Whether extracellular ATP can allosterically modulate GABAARs via a direct binding to the extracellular domains of the receptor has not previously been suggested. Here, we provide several pieces of evidence that are consistent with such a mode of regulation. First, using recombinant rat GABAARs transiently expressed in HEK293 cells, we were able to demonstrate that extracellular ATP can potentiate the function of these recombinant GABAARs in a manner similar to observations obtained with native GABAARs in neurons, suggesting that the ATP modulation does not require any additional neuronal specific proteins other than GABAARs themselves. Second, ATP potentiation could be demonstrated in the excised cell-free membrane patches under the outside-out configuration in neurons (Figure 4). These results indicate that the potentiation of GABAARs by extracellular ATP does not require any diffusible second messenger molecule downstream of an unknown metabotropic receptor, thereby providing strong support for a direct binding of ATP or its analog to an extracellular domain of GABAAR itself, or a membrane surface protein tightly associated with the receptor. Finally, this notion is further strengthened by our results obtained with single-channel recordings under the on-cell attached configuration (Figure 5). Under this configuration, currents through single or very few GABAAR channels in the membrane patch underneath of the tip of the recording pipette can be recorded in isolation from GABAARs outside of the pipette tip by applying GABA to the isolated membrane patch through the recording pipette solution. Using this configuration, we were able to demonstrate that the GABAAR single channel activities from the isolated membrane patch inside of the recording pipette can only be potentiated by ATP applied into the patch membrane through the recording pipette solution, but not by that applied extracellularly to the plasma membrane outside of the recording pipette tip (Figure 5). Together, the present study provides strong evidence supporting a novel mode of modulation of GABAARs by extracellular ATP; allosteric modulation likely achieved by either a direct binding of ATP to the receptor itself, or to an unknown protein tightly associated with the receptor. Nonetheless, given that extracellular ATP may potentially penetrate the plasma membrane, the possibility that ATP potentiates GABAAR function by a direct action on an intracellular domain of the receptor remains to be ruled out. Thus, the ultimate evidence for such a novel allosteric modulation will only come from the positive identification/characterization of the novel binding site (s) on GABAAR domain (s) or to identify the receptor associated protein by which ATP acts. In addition, whether the potentiation of GABAARs by ATP is state-dependent remains to be determined.
How ATP binding modulates the GABAAR remains to be determined. ATP binding may cause a conformational change to the GABAAR, thereby affecting its agonist binding affinity, channel gating, or both. Using various modes of single-channel recordings and analysis, in the present study we observed that extracellular ATP can increase GABAAR-gated channel activities by primarily increasing the channel open times without altering its conductance. This may suggest that ATP binding predominantly alters the apparent agonist binding affinity to the receptor, rather than the conformational change of the channel pore. This conjecture is further strengthened by the GABA dose-response relationship analysis from HEK293 cells as extracellular ATP results in a left-shift of the GABA dose-response curves without altering either h coefficient or the maximal responses (Figure 6B). Future site-direct mutations of the putative ATP binding domain along with direct ATP binding assays may provide a better understanding of the detailed mechanisms by which ATP exerts its modulation of the GABAAR.
In the mammalian brain, GABAARs play a key role in regulating the excitation-inhibition balance (and hence the tight control of neuronal excitability), and this function is primarily realized by mediating synaptic (phasic) inhibition and tonic inhibition . In the present study, we demonstrated that application of extracellular ATP at millimolar concentrations not only potentiates GABA currents evoked by exogenously applied GABA, but also both synaptic and tonic currents activated by endogenous GABA. These results suggest that extracellular ATP has significant physiological and/or pathologic impacts on neuronal excitability via modulating GABAARs. To this end, it is relevant to point out that several previous studies have suggested that under normal conditions, the extracellular ATP in the CNS is approximately 1-100 μM . At such a low level, the basal extracellular ATP may have little influence on neuronal excitability via GABAARs. However, it is important to note that ATP has previously been shown to be co-localized with GABA in the same vesicles at certain GABAergic synapses, and more importantly, that these two transmitters can be co-released into the synaptic cleft [18, 19, 25], whereby ATP concentrations can transiently reach the levels above hundreds of micromoles or even millimolar concentrations. In the present work, we demonstrated that at these concentrations, extracellular ATP can potentiate GABAAR-mediated mIPSCs. The fact that ATP potentiation of mIPSCs is primarily manifested as a specific increase in mIPSC amplitude, without altering its frequency, is in good agreement with the allosteric modulation of postsynaptic GABAARs. Thus, this mode of modulation may function at certain GABAergic synapses under physiological conditions. Similarly, the modulation may also occur under pathological conditions (including neuronal overexcitation, epileptic episodes, inflammation, traumatic insults, hypoxia/ischemia), as ATP release from damaged neurons and astrocytes can rapidly increase extracellular ATP concentrations [21–24]. ATP may increase both phasic and tonic GABA currents by acting on both synaptic and extrasynaptic GABAARs. By forming such a homeostatic feedback loop, under pathological conditions extracellular ATP may exert significant impacts on neuronal function and/or dysfunction. However, a related caveat is the potential complication from acidosis that is often associated with high concentrations of extracellular ATP. Given that acidosis is known to reduce GABAAR activity, how it will impact this ATP-induced allosteric potentiation warrants future investigations.
In this study, we demonstrate that extracellular ATP and its analogs such as ADP can potentiate function of GABAARs via a novel mechanism likely involving the direct binding of ATP to a putative ATP-binding site on the GABAAR itself. We demonstrate that through this modulation, extracellular ATP can enhance both phasic (synaptic) and tonic GABAAR-mediated currents. Therefore, the present study reveals a novel means by which extracellular ATP contributes to regulating excitation-inhibition balance and neuronal excitability under certain physiological and pathological conditions. In addition, due to the importance of GABAARs in mediating neuronal inhibition in the brain, they have been a major therapeutic target for the development of many drugs currently used for the clinical treatment of brain disorders. Further identifying the exact putative binding site of ATP on GABAARs may lead to the development of novel GABAAR-based therapeutics for better management of these brain disorders.
Primary culture of hippocampal neurons
Methods for culturing hippocampal neurons have been described previously . Briefly, hippocampi from E18 old Wistar rat embryos were dissected and treated with 0.25% trypsin solution (Invitrogen) for 25 min at 37°C, then mechanically dissociated using fire-polished pasteur pipettes. Cell suspension was centrifuged at 2500 × g for 50 s and the cell pellets were resuspended in DMEM with 10% fetal bovine serum (FBS). Cells were seeded on poly-D-lysine-coated 24-well coverslips at a density of 0.8-1.0 × 105 cells/well. Cultures were maintained in a humidified incubator with 5% CO2 at 37°C. After 24 h, the plating medium was changed to Neurobasal medium supplemented with B-27 and L-glutamine (0.5 mM) and neurons were fed with fresh medium twice weekly. Experiments were done 14-18 days after the plating.
Expression of recombinant GABAA receptors in HEK293 cells
Human embryonic kidney (HEK) 293 cells were cultured as previously described . Briefly, HEK293 cells were cultured in DMEM supplemented with 10% FBS. Cells were harvested weekly and seeded at 10% confluence on poly-L-lysine-coated glass coverslips in 24-mm culture dishes. Cells were transiently co-transfected at 70% confluence with rat cDNAs encoding α1, β2 and γ2-EGFP subunits of the GABAA receptor at a 1:1:1 ratio using Lipofectamine2000 (Invitrogen, Carlsbad, CA). Recordings were made 24-48 h after transfection.
Whole-cell patch clamp recording
Coverslips were transferred to the recording chamber and were continuously perfused with an extracellular solution 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 performed in the voltage-clamp mode using an Axopatch 200B patch-clamp amplifier (Axon Instruments). The cell membrane were held at a potential of -60 mV and signals were filtered at 2 KHz, digitized at 10 KHz using a Digidata 1322A analog-to-digital converter and acquired by Clampex 9.2 (Axon Instruments). Recording electrodes (3–5 M) were fabricated from thin-walled borosilicate glass tubing (World Precision Instruments, USA) with a micropipette puller (Sutter Instruments, model P-97, Novato, CA). Recording pipettes were filled with an intracellular solution containing (in mM): CsCl 140, HEPES 10, 1,2-bis (2-aminophenoxy) ethane-N, N, N, 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 glutamatergic and the voltage-gated sodium channels. All experiments were performed at room temperature.
Induction of GABAAR-mediated currents
Glass pipettes were filled with GABA (100 μM) dissolved in the extracellular recording solution. The pipette tip was placed in the vicinity of recorded neurons. GABA was applied via pressure ejection using a Picospritzer (General Valve Corporation, Fairfield, NJ) at 60 sec intervals. For recording of GABA currents in HEK293 cells, fast perfusion of GABA and/or other ligands were employed using a computer-controlled multibarrel fast perfusion system (Warner Instruments). For some of the experiments using bath perfusion of ATP or ADP, 5 mM EGTA was added into Ca2+-free extracellular solution to further reduce residual Ca2+ in extracellular solution, thereby minimizing any potential effect of ATP/ADP-induced extracellular Ca2+ influx. Under our experimental conditions in cultured neurons, the Ca2+-free solution neither produced any observable current on its own nor significantly affected GABAAR-gated currents (1 μM GABA; -648 ± -245pA in control solution vs -677 ± -256pA in Ca2+-free solution supplemented with 5 mM EGTA; n = 7; p < 0.05).
mIPSCs and GABA tonic current recording and analysis
GABAA receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) were recorded at a holding potential of −60 mV. CNQX (20 μM), APV (50 μM) and TTX (1 μM) were added to the extracellular solution to isolate GABAergic mIPSCs. Before drug application, a 3-5 min period of baseline recording (control) was obtained. The recordings were low-pass filtered (Clampfit software) at 2 kHz, digitized at 10 KHz using a Digidata 1322A analog-to-digital converter and acquired by Clampex 9.2. Detection and analysis of mIPSCs were performed using Mini Analysis Program (Synaptosoft, Decatur, GA). Any spurious noise was rejected. The mIPSC kinetics were obtained from analysis of the averaged single events. To facilitate analysis, decay time constant (τD) was obtained by fitting the decay phase to a single exponential equation. Tonic GABA current was estimated as the change in baseline current produced by a 2 min application of bicuculline (20 μM).
Single channel analysis
Patch pipettes for cell-attached and outside-out single channel recordings were pulled from thick-wall borosilicate glass (GC150F, Harvard Apparatus), fire polished, and coated with Sylgard 184 (Dow Corning) with a resistance of 6-10 MΩ. For outside-out recordings, the recording pipette was filled with the intracellular solution 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 pipette solution was (in mM): KCl 120, TEA-Cl 20, 1.3 MgCl2, HEPES 10, pH 7.40, 290-300 mOms. GABA (0.5 μM) or GABA (0.5 μM) and ATP (1 mM) were included in the pipette solution. Signals were filtered at 1 kHz, sampled at 10 kHz and analyzed off-line using Clampfit 9.2. An idealized recording of the durations and amplitudes of detectable events of the single-channel data was generated using 50% threshold crossing criteria. Events with a duration less than 300 μs were ignored. Single channel activities were expressed as the product of the number of channels × the open probability (P o ); i.e. NPo = ∑[(open time × number of channels open) ∕ total time of record].
Drugs used in the present study were purchased from the following sources: nucleotides (ATP, UTP, GTP, ADP, UDP, GDP, AMP, UMP, GMP), GABA and bicuculline (Sigma-Aldrich); suramin, CNQX, APV, PPADS (Tocris); and TTX (Alomone Labs).
Data is presented as means ± SEM, where n represents the number of tested cells. One-way ANOVA or the two-tailed Student’s test was used for statistical analysis and P values less than 0.05 were considered statistically significant. Dose–response curves were constructed by fitting data to the Hill equation: I = I max /(1 + EC50 / [A]n), where I is the current, I max is the maximum current, [A] is a given concentration of agonist, n is Hill coefficient (H).
We thank Y.P. Li for preparation and maintenance of hippocampal neuronal cultures and L. W. Oschipok for his excellent editorial assistance. This work was supported by the Canadian Institutes for Health Research and the Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH102-TD-B-111-004). J.L. was supported by postdoctoral fellowships from National Sciences and Engineering Research Council, the Michael Smith Foundation for Health Research and the British Columbia Epilepsy Society. Y.T.W. is the holder of the Heart and Stroke Foundation of British Columbia and Yukon chair in stroke research.
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