Abstract
In the present study, the orthosteric GABAA receptor (GABAAR) ligand 4,5,6,7-tetrahydroisothiazolo[5,4-c]pyridin-3-ol (Thio-THIP) was found to possess a highly interesting functional profile at recombinant human GABAARs and native rat GABAARs. Whereas Thio-THIP displayed weak antagonist activity at α1,2,5β2,3γ2S and ρ1 GABAARs and partial agonism at α6β2,3δ GABAARs expressed in Xenopus oocytes, the pronounced agonism exhibited by the compound at α4β1δ and α4β3δ GABAARs was contrasted by its negligible activity at the α4β2δ subtype. To elucidate to which extent this in vitro profile translated into functionality at native GABAARs, we assessed the effects of 100 μm Thio-THIP at synaptic and extrasynaptic receptors in principal cells of four different brain regions by slice electrophysiology. In concordance with its α6β2,3δ agonism, Thio-THIP evoked robust currents through extrasynaptic GABAARs in cerebellar granule cells. In contrast, the compound did not elicit significant currents in dentate gyrus granule cells or in striatal medium spiny neurons (MSNs), indicating predominant expression of extrasynaptic α4β2δ receptors in these cells. Interestingly, Thio-THIP evoked differential degrees of currents in ventrobasal thalamus neurons, a diversity that could arise from differential expression of extrasynaptic α4βδ subtypes in the cells. Finally, whereas 100 μm Thio-THIP did not affect the synaptic currents in ventrobasal thalamus neurons or striatal MSNs, it reduced the current amplitudes recorded from dentate gyrus granule cells, most likely by targeting perisynaptic α4βδ receptors expressed at distal dendrites of these cells. Being the first published ligand capable of discriminating between β2- and β3-containing receptor subtypes, Thio-THIP could be a valuable tool in explorations of native α4βδ GABAARs.
Introduction
The fast signaling of the major inhibitory CNS neurotransmitter GABA is mediated by GABAA receptors (GABAARs), a family of pentameric anion-selective ligand-gated ion channels belonging to the Cys-loop receptor superfamily (Whiting, 2003; Olsen and Sieghart, 2008). Native GABAAR subtypes are assembled from a total of 19 subunits (α1-α6, β1-β3, γ1-γ3, δ, ε, π, θ, ρ1-ρ3), and the differential in vivo distribution of these subunits combined with the distinct functional properties of the assembled subtypes enable the receptors to mediate a wide range of functions throughout the CNS (Pirker et al., 2000; Olsen and Sieghart, 2008; Hörtnagl et al., 2013). The synaptic GABAARs responsible for phasic inhibition are predominantly made up by α1, α2, and/or α3 in combination with β2/β3 and γ2 subunits, whereas the α4βδ, α6βδ, and α5βγ2 subtypes constitute the major perisynaptic/extrasynaptic receptors mediating tonic inhibition of the GABAergic system (Whiting, 2003; Farrant and Nusser, 2005; Olsen and Sieghart, 2008; Belelli et al., 2009; Brickley and Mody, 2012). However, several additional physiologically relevant subtypes exist, and the distinction between synaptic and perisynaptic/extrasynaptic receptors is considerably less clear-cut than outlined above (Mortensen and Smart, 2006; Glykys et al., 2007; Eyre et al., 2012; Marowsky et al., 2012; Milenkovic et al., 2013).
Investigations into the physiological functions governed by different GABAAR subtypes have to a large extent relied on the use of subtype-selective ligands and/or knock-out or knock-in mice (Reynolds et al., 2003; Rudolph and Knoflach, 2011). The availability of allosteric modulators exhibiting functional selectivity for the different α-containing α1,2,3,5βγ subtypes and of the agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and positive allosteric modulators (PAMs) selective for the α4βδ/α6βδ receptors has been instrumental for the explorations of GABAARs defined by their respective α subunits (Stórustovu and Ebert, 2006; Wafford et al., 2009; Hoestgaard-Jensen et al., 2010; Atack, 2011a, b). In contrast, the respective contributions of β1-, β2-, and β3-containing receptors to GABAergic neurotransmission have not been delineated in similar detail, in no small part because of the limited number of β-isoform selective GABAAR ligands available (Sieghart, 2006). Nevertheless, the differential CNS expression patterns of the three β subunits strongly suggest that different β-containing GABAAR subtypes could mediate distinct physiological functions and that selective targeting of cross-sections of receptors other than those defined by α subunits could hold therapeutic potential.
The molecular composition of the orthosteric site located in the extracellular β(+)/α(−) interface of the pentameric GABAAR complex is highly conserved throughout the receptors; thus, orthosteric ligands may not seem obvious candidates in the pursuit of subtype selectivity (Sieghart, 2006). Nonetheless, the functional subtype selectivity displayed by GABA analogs such as THIP and 5-(4-piperidyl)-3-isothiazolol (Thio-4-PIOL) underlines the feasibility of obtaining at least this form of selectivity through the site (Stórustovu and Ebert, 2006; Hoestgaard-Jensen et al., 2013). In the present study, the bicyclic 3-isothiazol GABA analog 4,5,6,7-tetrahydroisothiazolo[5,4-c]pyridin-3-ol (Thio-THIP) is demonstrated to possess a highly interesting functional profile at human GABAARs expressed in Xenopus oocytes; and taking advantage of this functionality, Thio-THIP has been used to elucidate the heterogeneity of native GABAARs in four rat brain regions.
Materials and Methods
Materials.
Thio-THIP and THIP (Fig. 1) were synthesized in-house essentially as previously described (Krogsgaard-Larsen et al., 1977, 1983; Krehan et al., 2003). GABA, ZnCl2, and chemicals used for buffers were purchased from Sigma-Aldrich, and SR95531 (gabazine) was obtained from Alamone Labs. DS2, picrotoxin, CGP 54626, and TTX were obtained from Tocris Bioscience Cookson, and Alexa-488-streptavidin was purchased from Invitrogen. Xenopus laevis oocytes and [3H]GABA were obtained from Lohmann Research Equipment and PerkinElmer, respectively. The cDNAs encoding for the human GABAAR subunits were kind gifts from Drs. P.J. Whiting and D.S. Weiss (ρ1), whereas the cDNAs encoding for the rat GABAB receptors and the chimeric G-protein Gαqi5 were kind gifts from Drs. J. Clark and B.R. Conklin, respectively.
Chemical structures of THIP and Thio-THIP.
Molecular biology.
The subcloning of human α1-α5, β2, γ2S, and ρ1 cDNAs into the pcDNA3.1 vector has been described previously (Jensen et al., 2010), and human β3 cDNA in the pGEMHE vector was used in this study. The human α6, β1, and δ cDNAs were subcloned into pcDNA3.1 using the restriction enzyme pairs XhoI/XbaI (α6 and δ) and NotI/XbaI (β1). The cDNAs for chimeric subunits β2NTD/β3TMD/ICL and β3NTD/β2TMD/ICL were constructed using Splicing by Overlap Extension PCR (Horton et al., 1989) and subcloned into the pcDNA3.1 vector using the restriction enzymes NotI and XbaI. The β2NTD/β3TMD/ICL and β3NTD/β2TMD/ICL cDNAs encode for the mature proteins Gln1-Phe212|Arg213-Asn448 and Gln1-Phe212|Lys213-Asn450, respectively (β2 segments are given in italics). The integrity and the absence of unwanted mutations in all cDNAs created by PCR were verified by DNA sequencing (Eurofins MWG, Operon).
Ca2+/Fluo-4 assay.
The functional properties of Thio-THIP at GABAB receptors were characterized at rat GABAB(1a,2) and GABAB(1b,2) receptors coexpressed with the chimeric G-protein Gαqi5 in tsA201 cells in the Ca2+/Fluo-4 assay. The tsA201 cells were cultured in DMEM supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum. The 2 × 106 cells were split in a 10 cm tissue culture plate and transfected the following day with a total of 8 μg cDNA (GABAB1a-pCDNA3.1 or GABAB1b-pCDNA3.1, GABAB2-pCDNA3.1 and Gαqi5-pCDNAI in a 1:2:1 ratio) using Polyfect Transfection Reagent (QIAGEN). Sixteen to 24 h after transfection, the cells were split into poly-d-lysine-coated black 96-well plates with clear bottom. The following day, the culture medium was aspirated and the cells were incubated in 50 μl assay buffer (Hank's Buffered Saline Solution containing 20 mm HEPES, 1 mm CaCl2, 1 mm MgCl2, and 2.5 mm probenecid, pH 7.4) supplemented with 6 mm Fluo-4/AM (Invitrogen) at 37°C for 1 h. Then the buffer was aspirated, the cells were washed once with 100 μl assay buffer, and 100 μl assay buffer was added to the cells. The 96-well plate was then assayed in a NOVOstar microplate reader measuring emission (in fluorescence units) at 520 nm caused by excitation at 485 nm before and up to 60 s after addition of 33 μl agonist solution in assay buffer. The experiments were performed in duplicate three times for both GABA and Thio-THIP at both GABAB receptors.
[3H]GABA uptake assay.
The functional properties of Thio-THIP at GABA transporters were characterized at the human GAT-1, BGT-1, GAT-2, and GAT-3 subtypes stably expressed in Flp-In-CHO cells in a [3H]GABA uptake assay essentially as previously described (Christiansen et al., 2008; Kvist et al., 2009). The cells were cultured in Ham's F-12 medium with l-glutamine supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 10% FBS, 5 μg/ml plasmocin, and 200 μg/ml hygromycin B. Cells were split into poly-d-lysine-coated white 96-well plates; and the following day, the culture medium was removed and cells were washed with 100 μl assay buffer (Hank's Buffered Saline Solution containing 20 mm HEPES, 1 mm CaCl2, and 1 mm MgCl2, pH 7.4). Then 100 μl assay buffer supplemented with 100 nm [3H]GABA and various concentrations of the test compounds was added onto the cells, and the plate was incubated at 37°C for 3 min. Then the cells were washed with 2 × 100 μl ice-cold assay buffer, and 150 μl Microscint20 scintillation fluid (PerkinElmer) was added to each well. The plate was shaken for at least 1 h, after which the radioactivity in the wells was counted in a Wallac 1450 MicroBeta Trilux scintillation counter (GMI). The experiments were performed in triplicate four times for both GABA and Thio-THIP at all four GABA transporters.
X. laevis oocytes and two-electrode voltage clamp.
The GABAAR subunit cDNAs were linearized, transcribed, and capped in using the mMessage mMachine T7 kit (Ambion). α1,2,5β2,3γ2S cRNAs were mixed and injected into oocytes in a subunit ratio of 1:1:1 (concentration of each subunit: 10 ng/μl; injection volumes: 4–18 nl), whereas α4,6β1,2,3δ cRNAs were mixed and injected in a subunit ratio of 10:1:10 (concentrations of subunits: 1 μg/μl (α), 0.1 μg/μl (β), and 1 μg/μl (δ); injection volume: 46 nl). As for the homomeric receptors, the ρ1 cRNA (concentration: 0.1 μg/μl) was injected in a volume of 4–18 nl, whereas the respective β1, β2, and β3 cRNAs (concentrations: 0.2 μg/μl) were injected in volumes of 32–46 nl. The oocytes were incubated for 1–7 d in modified Barth's saline (88 mm NaCl, 1 mm KCl, 15 mm HEPES, pH 7.5, 2.4 mm NaHCO3, 0.41 mm CaCl2, 0.82 mm MgSO4, 0.3 mm Ca(NO3)2, 100 U/ml penicillin, and 100 μg/ml streptomycin). Oocytes were clamped at −40 mV to −90 mV with an Oocyte Clamp OC-725C amplifier (Warner), and both voltage and current electrodes were agar-plugged with 3 m KCl (resistances: 0.5–2.0 mΩ). In the agonist experiments, GABA, THIP, or Thio-THIP was applied to the perfusate until the peak of the response was observed. To avoid receptor desensitization, a washout period of 2.2 min was executed between drug applications. In the antagonist experiments, the oocyte was preincubated for 30 s with the test compound before coapplication of test compound and GABA. The incorporation of the γ2S subunit into the receptors assembled at the cell surface of α1,2,5β2,3γ2S-expressing oocytes was confirmed with 100 μm ZnCl2 (Karim et al., 2013), and the presence of δ in cell surface-expressed α4βδ and α6βδ receptors was confirmed using the δ-GABAAR selective PAM DS2 (Wafford et al., 2009) and 1 μm ZnCl2 (Stórustovu and Ebert, 2006). All oocyte data were based on recordings performed on oocytes from at least two different batches. The recorded baseline-to-peak current amplitudes were analyzed using Clampfit 10.1 (Molecular Devices) and normalized to the maximum current amplitude elicited by GABA at the individual oocyte. Data were analyzed and fitted to concentration–response or concentration–inhibition curves by use of the nonlinear regression in GraphPad Prism 5 (GraphPad Software), with the fittings of the biphasic and triphasic concentration–response curves for GABA and THIP at α4β1δ and α4β3δ being performed by eye.
Slice electrophysiology: animals.
Young adult male Sprague Dawley rats from Harlan Laboratories, weighing 200–250 g at the time of experiment, were used for the preparation of acute brain slices. Animals were housed at a normal 12 h light/dark cycle at 22°C–25°C in cages of up to 6 rats. The protocols were approved by the Danish Authorities for Animal Experimentation.
Slice preparation.
After decapitation, the brain was rapidly dissected out in ice-cold low-Na artificial CSF (NMDG-ACSF) containing the following (in mm): NaCl (26), N-methyl-d-glucamine-HCl (100), KCl (2.5), CaCl2 (1), MgCl2 (3), NaHCO3 (26), NaH2PO4 (1.25), d-glucose (10), ascorbate (0.3), pyruvic acid (1.0), and kynurenic acid (2). The osmolality of NMDG-ACSF was adjusted to 310 ± 5 mOsm. The brain was glued to the platform of a Leica 1200 VS microtome and cut in ice-cold NMDG-ACSF. Orientation and slice thickness were as follows: striatum (coronal, 250 μm), hippocampus (horizontal, 350 μm), ventrobasal thalamus (coronal 250 μm), and cerebellum (sagittal, 250 μm). After cutting, slices were stored in regular ACSF, which differs from NMDG-ACSF by the omission of kynurenic acid and by NMDG-HCl being replaced by NaCl (total 126 mm NaCl) at 28°C for 1–4 h. All solutions were bubbled with carbogen (95% O2/5% CO2).
Whole-cell recordings.
For recordings of the effects of Thio-THIP at GABAARs at synaptic and extrasynaptic loci, slices were maintained submerged in a 2 ml chamber perfused with ACSF at a flow of 2.5 ml/min at 33°C–34°C and included 2 mm kynurenic acid, 1 μm CGP 54626, and 1 μm TTX. Recordings that included ZnCl2 (15 μm) were performed in phosphate-free ACSF. Cells were visualized using a custom-built infrared video-microscopy system. Somatic whole-cell recordings in voltage-clamp were made using a Multiclamp 700A amplifier (Axon Molecular Devices). Recordings were digitized at 10 kHz on a 1320 digidata digitizer (Axon Molecular Devices) and lowpass filtered (8-pole Bessel) at 3 kHz. The intracellular solution contained the following (in mm): CsCl (135), NaCl (4), MgCl2 (2), EGTA (0.1), HEPES (10), ATP (2), GTP (0.5), TEA (5), and QX-314 (5), with pH adjusted to 7.3 (at 4°C) and osmolality to 292 mOsm. Patch pipettes were pulled with tip diameters required for neurons of the different regions and with resistance ranging from 5 mΩ (hippocampal granule cells) to 10–11 mΩ (cerebellar granule cells) using this solution. All cells were held at −70 mV for the duration of the experiment, and recordings were 70% compensated for series resistance and discarded for further analysis if values for capacitance and series resistance during recordings deviated >30% from initial values. After establishment of whole-cell configuration, cells were held for a minimum of 6 min to allow for perfusion of intracellular solution and stabilize holding current before recording of baseline (3 min) followed by drug perfusion (6 min). Recordings were stopped 1 min after addition of SR95531 directly to the bath resulting in a very fast rise in concentration to ∼100 μm SR95531. A few cells from each region (except cerebellum) were recorded with the intracellular solution containing 1% biocytin for postrecording visualization of cellular architecture by the Alexa-488-coupled streptavidin system as previously described (Hoestgaard-Jensen et al., 2013).
Data analysis.
All whole-cell recordings were made in pClamp 10 and further analyzed in either Clampfit 10 (Axon Molecular Devices) or for the spontaneous events, in Mini Analysis 6.03 (Synaptosoft) and Origin 9.0 (OriginLab). A two-sample t test with equal variance assumed and p < 0.05 as level of significance was used for assessment of drug effects in individual groups, and a one-way ANOVA (p < 0.05) with post hoc Bonferroni test to assess significance of differences in mean current density across cell types. For detection of mIPSCs, an algorithm in Mini Analysis 6.03 was made to detect baseline deflections of 5 × SD of the trace and sampling 5 ms before and 50 ms after detection. The threshold value for mIPSC detection was determined while drug was perfused. For construction of an average waveform of the synaptic current, events with an interevent interval <50 ms were removed, and the remaining noncontaminated events were aligned with respect to the 50% rising phase. The averaged synaptic current was characterized by the 20%–80% rise time (RT20–80), peak, weight of tau, and area. The weight of tau, τw, was calculated from a fit to a biexponential using τw = (A1/(A1 + A2)) × τ1 + (A2/(A1 + A2)) × τ2, in which A1/A2 and τ1/τ2 are the amplitudes and tau values of the individual exponentials. The current induced by THIP or Thio-THIP was measured as the difference in the holding current of two 30 s windows, the first located 30 s before compound entered the bath and the second immediately before addition of SR95531 to the bath. The holding current in the 30 s window was measured as the center of a Gaussian of 1 ms sampled baseline every 100 ms. The currents are reported relative to the capacitance of the recorded neuron (i.e., current density pA/pF), and data are given as means ± SEM.
Results
Functional characterization of Thio-THIP at recombinant human GABAARs
In a screening program in which a series of GABA analogs synthesized by the Krogsgaard-Larsen group over the years were tested at selected GABAAR subtypes, the compound Thio-THIP was found to exhibit an interesting functional profile (unpublished observations). Thio-THIP has previously been reported to exhibit ∼300-fold lower binding affinity to native GABAARs in rat synaptosomes than the closely related analog THIP, to induce neuronal depression in cat spinal cord neurons, and to be a weak competitive antagonist at human α3β2γ2 and α5β3γ2 GABAARs expressed in Xenopus oocytes (Krogsgaard-Larsen et al., 1983; Brehm et al., 1997). Nevertheless, the findings in the screening prompted us to explore the pharmacology of Thio-THIP in further detail, and the functional properties displayed by the compound in two-electrode voltage-clamp recordings at 13 human GABAAR subtypes expressed in Xenopus oocytes will be presented below. Prior to this, however, the basic functional characteristics exhibiting the three α4βδ subtypes in this study will be outlined, since the functional properties exhibited by α4βδ GABAARs expressed in oocytes in previous studies have varied considerably. Thus, because of the key importance of these receptors for this study, their basic functional properties are important to consider when interpreting the findings for Thio-THIP.
Basic functional characteristics of α4βδ GABAARs expressed in Xenopus oocytes
In agreement with previous studies, GABA displayed distinct monophasic concentration–response curves at the α1β2γ2S, α1β3γ2S, α2β2γ2S, α2β3γ2S, α5β2γ2S, α5β3γ2S, α6β2δ, α6β3δ, and ρ1 GABAAR subtypes expressed in oocytes (Table 1) (Mortensen et al., 2011; Hoestgaard-Jensen et al., 2013; Karim et al., 2013). Analogously, monophasic concentration–response curves were consistently observed for GABA at the α4β2δ GABAAR, whereas the concentration–response relationships exhibited by the agonist at the α4β1δ and α4β3δ receptors were considerably more complex. The GABA concentration–response curves at α4β3δ-expressing oocytes were consistently biphasic and characterized by mid-nanomolar and low-micromolar EC50,1 and EC50,2 values, respectively (Fig. 2A,C; Table 1). Because the relative sizes of the “high-affinity” and “low-affinity” components of the total response varied somewhat between the different oocytes, the biphasic nature of the concentration–response curve is somewhat lost in the averaged data (Fig. 2D). Two divergent profiles were observed for GABA at α4β1δ GABAAR-expressing oocytes: a monophasic concentration–response curve (EC50 of 620 nm), and a multiphasic, possibly triphasic (see Materials and Methods) concentration–response curve with estimated EC50,1 and EC50,2 values of 170 nm and 3.9 μm and a third phase that did not reach saturation at GABA concentrations up to 10 mm (Fig. 2A,C; Table 1). In accordance with previous studies of the α4β3δ GABAAR in oocytes (Stórustovu and Ebert, 2006; Meera et al., 2011), THIP was found to be a superagonist at all three α4βδ receptors, evoking twofold to threefold higher maximal responses than GABA at the receptors (Table 1). THIP displayed monophasic concentration–response relationships at α4β2δ and α4β3δ, whereas the agonist exhibited biphasic or triphasic concentration–response profiles at α4β1δ-expressing oocytes, neither of which reached saturation at THIP concentrations up to 10 mm (Fig. 2B–D). A final substantial difference was the degrees of spontaneous activity exhibited by the three α4βδ subtypes. Whereas pronounced levels of constitutive activity were observed in oocytes expressing α4β1δ and α4β3δ receptors, α4β2δ-expressing oocytes did not display significant levels of spontaneous activity (Fig. 2E).
Functional properties of GABA, THIP, and Thio-THIP at human GABAARs expressed in Xenopus oocytesa
Basic functional characteristics of human α4βδ GABAARs expressed in Xenopus oocytes. A, Representative traces of the currents evoked by various concentrations of GABA at human α4β1δ, α4β2δ, and α4β3δ GABAARs. The different profiles exhibited by GABA at α4β1δ are exemplified as α4β1δ-1 and α4β1δ-2. B, Representative traces of the currents evoked by various concentrations of THIP at human α4β1δ, α4β2δ, and α4β3δ GABAARs. The different profiles exhibited by THIP at α4β1δ are exemplified as α4β1δ-1 and α4β1δ-2. C, Representative concentration–response curves for GABA and THIP at the α4β1δ, α4β2δ, and α4β3δ GABAARs. The different profiles exhibited by GABA and THIP at the α4β1δ GABAAR are given as α4β1δ-1 and α4β1δ-2. The curve fittings of the different phases of the GABA concentration–response relationships at α4β1δ-2 and α4β3δ and of the THIP concentration–response relationships at α4β1δ-1 and α4β1δ-2 are given in hatched lines. D, Averaged concentration–response curves for GABA and THIP at the α4β1δ, α4β2δ, and α4β3δ GABAARs. Data are mean ± SEM (GABA: n = 8, n = 4, and n = 9 for α4β1δ, α4β2δ, and α4β3δ, respectively; THIP: n = 9, n = 4, and n = 4 for α4β1δ, α4β2δ, and α4β3δ, respectively). E, Levels of constitutive activity in oocytes expressing human α4β1δ, α4β2δ, and α4β3δ GABAARs. Left, Representative traces of the currents evoked by 10 mm, 300 μm, and 300 μm GABA followed by application of 10 μm picrotoxin in α4β1δ-, α4β2δ-, and α4β3δ-expressing oocytes, respectively. Right, Averaged levels of constitutive activity in oocytes expressing human α4β1δ, α4β2δ, and α4β3δ GABAARs. Constitutive activity is given as the percentage difference between the basal line and the outward current caused by application of 10 μm picrotoxin. Data are mean ± SEM (n = 6, n = 6, and n = 4 for α4β1δ, α4β2δ, and α4β3δ, respectively).
A number of previous studies have reported a wide range of functional potencies and monophasic as well as biphasic concentration–response profiles for both GABA and THIP at α4β3δ GABAARs expressed in oocytes (Borghese et al., 2006; Stórustovu and Ebert, 2006; Meera et al., 2009, 2011; Hoestgaard-Jensen et al., 2010; Karim et al., 2012, 2013; Hoestgaard-Jensen et al., 2013; Patel et al., 2014), which contrasts the monophasic concentration–response relationships and high-nanomolar and mid-micromolar EC50 values consistently displayed by GABA and THIP, respectively, at α4β3δ receptors expressed in HEK293 cells (Brown et al., 2002; Mortensen et al., 2004; Meera et al., 2009; Mortensen et al., 2010). The formation of both “high-affinity” and “low-affinity” α4β3δ receptors in oocytes has been proposed to arise from the possible formation of multiple α4β3δ stoichiometries or subunit arrangements or from the coexpression of α4β3δ and “pure” α4β3 complexes (Meera et al., 2009; Karim et al., 2012, 2013). To address whether the considerable range in maximal current amplitudes (200–3500 nA) evoked by GABA in α4β3δ-expressing oocytes in this study could arise from mixed α4β3δ/α4β3 receptor populations being expressed in some of the oocytes, we investigated the zinc sensitivities of the cell surface-expressed receptors in oocytes at which GABA exhibited maximal current amplitudes lower or higher than 500 nA (Stórustovu and Ebert, 2006). Zn2+ (1 μm) was found to exert comparable small degrees of inhibition of the currents evoked by 300 μm GABA in these two oocyte populations, reducing the response in “>500 nA” and “<500 nA” α4β3δ-oocytes to 91 ± 5% (n = 7) and 90 ± 1% (n = 4) of that recorded in the absence of zinc, respectively. This strongly suggests that few (if any) α4β3 complexes are assembled in these α4β3δ-oocytes and that the contribution of these receptors to the observed currents thus is negligible. Furthermore, as will be outlined below, the functional properties of the α4β3δ receptors did not seem to arise from the formation of homomeric β3 receptors in the oocytes. On the other hand, we cannot exclude the possibility that different α4β3δ receptor complexes (in terms of positional subunit arrangement or subunit stoichiometry) are assembled in the oocytes. This consideration also applies for the receptors formed in α4β1δ-expressing oocytes, and analogously to α4β3δ both “high-affinity” and “low-affinity” α4β1δ assemblies have been observed in previous studies (Lovick et al., 2005; Karim et al., 2012; Anstee et al., 2013).
Functional properties of Thio-THIP at human GABAARs expressed in Xenopus oocytes
In contrast to the concentration-dependent increases in current amplitudes observed for GABA in oocytes expressing α1β2γ2S, α1β3γ2S, α2β2γ2S, and α2β3γ2S GABAARs, Thio-THIP did not elicit significant agonist responses at any of these four receptors at concentrations up to 1 mm (exemplified for α1β3γ2S in Fig. 3A). Instead, the compound was found to be a weak antagonist exhibiting high-micromolar IC50 values at the receptors (Fig. 3A; Table 1). The inhibitory potencies displayed by Thio-THIP at these four receptors concur with the weak antagonism observed for the compound at the α3β2γ2 GABAAR in a previous study (Brehm et al., 1997). Whereas applications of Thio-THIP in concentrations up to 300 μm did not evoke significant responses in oocytes expressing the homomeric ρ1 GABAAR, 1 mm Thio-THIP gave rise to minute but significant currents (Fig. 3B; Table 1). When tested as an antagonist at the receptor using GABA (3 μm) as agonist, Thio-THIP exhibited an IC50 value of ∼150 μm (Fig. 3B; Table 1). This de facto antagonism is in good agreement with the weak competitive antagonism exhibited by its close structural analog THIP at this receptor (Woodward et al., 1993; Vien et al., 2002).
Functional properties of Thio-THIP at various human GABAARs expressed in Xenopus oocytes. A, The α1,2β2,3γ2S GABAARs. Left, Representative traces of the currents evoked by various concentrations of Thio-THIP at the α1β3γ2S GABAAR. Middle, Representative traces of the currents elicited by 30 μm GABA at the α1β3γ2S GABAAR in the presence of various concentrations of Thio-THIP. Right, Averaged concentration–inhibition curves for Thio-THIP at the α1β2γ2S, α1β3γ2S, α2β2γ2S, and α2β3γ2S GABAARs using 30 μm GABA as agonist. Data are mean ± SEM (α1β2γ2S: n = 3; α1β3γ2S: n = 8; α2β2γ2S: n = 4; α2β3γ2S: n = 3). B, The ρ1 GABAAR. Left, Representative traces of the currents evoked by various concentrations of Thio-THIP at the ρ1 GABAAR. Middle, Representative traces of the currents elicited by 3 μm GABA at the ρ1 GABAAR in the presence of various concentrations of Thio-THIP. Right, Averaged concentration–inhibition curve for Thio-THIP at the ρ1 GABAAR using 3 μm GABA as agonist. Data are mean ± SEM (n = 4). C, The α6β2,3δ GABAARs. Left, Representative traces of the currents evoked by various concentrations of Thio-THIP at α6β2δ and α6β3δ GABAARs. Right, Averaged concentration–response curves for Thio-THIP at the α6β2δ and α6β3δ GABAARs. Data are mean ± SEM (α6β2δ: n = 3; α6β3δ: n = 4). D, The α5β2,3γ2S GABAARs. Top, Representative traces of the currents evoked by various concentrations of Thio-THIP at α5β2γ2S and α5β3γ2S GABAARs (left and middle), and representative traces of the currents elicited by 30 μm GABA at the α5β2γ2S GABAAR in the presence of various concentrations of Thio-THIP (right). Bottom, Averaged concentration–inhibition curve for Thio-THIP at the α5β2γ2S GABAAR using 30 μm GABA as agonist (left) and averaged concentration–response curve for Thio-THIP at the α5β3γ2S GABAAR (right). Data are mean ± SEM (α5β2γ2S: n = 6; α5β3γ2S: n = 6).
In contrast to its antagonist activities at the α1,2,3β2,3γ2 and ρ1 GABAARs, Thio-THIP was found to be a partial agonist at the α6β2δ and α6β3δ subtypes displaying high-micromolar EC50 values and maximal responses of ∼50% of those of GABA at the two receptors (Fig. 3C; Table 1). Interestingly, Thio-THIP displayed pronounced functional selectivity between the β2- and β3-containing α5βγ2S subtypes. Whereas the compound was a weak antagonist at α5β2γ2S, eliciting no significant currents through this receptor at concentrations up to 1 mm, it was an agonist, albeit a low-efficacious one, at the α5β3γ2S receptor (Fig. 3D; Table 1). We consider the previously reported antagonist activity of Thio-THIP at α5β3γ2 (Brehm et al., 1997) to be reconcilable with the small intrinsic agonist activity exhibited by the compound at the receptor in this study.
The functional properties displayed by Thio-THIP at the three α4βδ GABAAR subtypes were even more diverse than those at the α5βγ2S receptors. The compound was a moderately potent partial agonist at the α4β3δ receptor exhibiting an EC50 of 13 μm and an Rmax value of 58 ± 7% of that of GABA (Fig. 4A,B; Table 1). Thio-THIP was also a partial agonist at the α4β1δ GABAAR, albeit not as efficacious at this receptor as at α4β3δ. The compound displayed a biphasic concentration–response relationship at the receptor with a second phase that was not always saturated at concentrations up to 1 mm (Fig. 4A,B; Table 1). In contrast, application of Thio-THIP at concentrations up to 100 μm did not evoke significant currents in α4β2δ-expressing oocytes, and 1 mm Thio-THIP only gave rise to a minute response (4 ± 1% of Rmax of GABA). In concordance with this negligible agonist activity, Thio-THIP was a de facto antagonist exerting small but significant inhibition of the GABA EC80-evoked signaling in α4β2δ-oocytes when applied at concentrations of ≥100 μm (Fig. 4C; Table 1). Interestingly, Thio-THIP was a weak low-efficacious partial agonist at the binary α4β3 GABAAR (Table 1). This suggests that the presence of δ in the α4β3δ complex is of key importance for the high-efficacy agonist properties of Thio-THIP at the receptor, a finding that contrasts the comparable agonist efficacies displayed by THIP at α4β3 and α4β3δ GABAARs in a previous study (Stórustovu and Ebert, 2006).
Functional properties of Thio-THIP at human α4βδ GABAARs expressed in Xenopus oocytes. A, Representative traces of the currents evoked by various concentrations of Thio-THIP at human α4β1δ, α4β2δ, and α4β3δ GABAARs. B, Averaged concentration–response curves for Thio-THIP at the α4β1δ, α4β2δ, and α4β3δ GABAARs. Data are mean ± SEM (α4β1δ: n = 8; α4β2δ: n = 7; α4β3δ: n = 13). C, Averaged concentration–inhibition curve for Thio-THIP at the α4β2δ GABAAR using 30 μm GABA as agonist. Data are mean ± SEM (n = 5). D, Correlation between the maximum current amplitude evoked by GABA and the Rmax values of Thio-THIP (in percentage of Rmax of GABA) at α4β3δ-expressing oocytes (n = 25). The vertical hatched line indicates the differentiation between the recordings where GABA exhibited maximal current amplitudes higher than and lower than 500 nA. E, Representative traces of the currents evoked by 1 mm Thio-THIP, 1 mm GABA, and 10 μm picrotoxin at the human β1, β2, and β3 GABAARs (left), and averaged currents evoked by 1 mm Thio-THIP, 1 mm GABA, and 10 μm picrotoxin at the human β1 and β3 GABAARs (right). Data are mean ± SEM in percentage of the response evoked by 10 μm picrotoxin in the oocyte (β1: n = 4; β2: n = 6; β3: n = 5).
It should be mentioned that the agonist efficacies displayed by Thio-THIP at the 25 α4β3δ-expressing oocytes recorded from in this study varied considerably, and a pronounced correlation existed between the Rmax values determined for Thio-THIP and the maximum current amplitudes evoked by GABA at the respective oocytes (Fig. 4D). Because the zinc sensitivities exhibited by receptors in oocytes characterized by both high and low maximal current amplitudes strongly suggest that the receptors in both populations comprise the δ subunit, the differential Thio-THIP efficacies could be speculated to arise from the assembly of different α4β3δ receptor populations in terms of subunit stoichiometry or arrangement in the individual oocytes. Nevertheless, it is important to stress that the averaged Rmax value for Thio-THIP based on all recordings on α4β3δ oocytes (58 ± 7%, n = 25) supports the claim of Thio-THIP exhibiting substantial agonist efficacy at the α4β3δ GABAAR (Table 1).
In a final set of experiments, Thio-THIP was characterized functionally at human homomeric β1, β2, and β3 GABAARs, in part to delineate its pharmacology at these receptors and in part to assess the putative contributions of these homomeric β assemblies in α4β1δ, α4β2δ, and α4β3δ oocytes to the observed functional properties of Thio-THIP. Previous studies of homomeric expression of β2 in mammalian cell lines and in Xenopus oocytes have found the subunit to be retained within the endoplasmic reticulum and thus not being expressed at the cell surface (Connolly et al., 1996; Taylor et al., 1999). In agreement with this, neither 10 μm picrotoxin, nor 1 mm GABA, nor 1 mm Thio-THIP evoked significant responses in β2-expressing oocytes (Fig. 4E). Also in concordance with previous studies (Sanna et al., 1995; Connolly et al., 1996; Krishek et al., 1996; Simeone et al., 2011), the β1 and β3 receptors exhibited high levels of spontaneous activity, as evidenced by the pronounced outward currents produced by application of 10 μm picrotoxin at oocytes expressing these receptors. In contrast, applications of 1 mm GABA did not change the recorded baseline in β1-expressing oocytes and produced a small outward current in β3 oocytes (Fig. 4E). Interestingly, the effects of 1 mm Thio-THIP on the baselines recorded from β1 and β3 oocytes differed, as the compound evoked a small inward current through β1 and a small outward current through β3. In conclusion, the putative formation of β2 homomers clearly does not contribute to the functional properties exhibited by the receptors in α4β2δ-oocytes. As for β1 and β3, the homomeric receptors displayed much more pronounced degrees of constitutive activity than α4β1δ and α4β3δ. More importantly, however, the functional properties exhibited by GABA and Thio-THIP at β1 and β3 were strikingly different from those at their respective α4βδ counterparts. This suggests that, even if homomeric β complexes are assembled in α4β1δ-and α4β3δ-oocytes, their contributions to the observed functional properties of these receptors are negligible.
Investigation of the molecular origin of the β isoform-selectivity of Thio-THIP at α4βδ GABAARs
To elucidate the molecular basis for the functional selectivity exhibited by Thio-THIP at the α4βδ GABAARs, the compound was characterized functionally at α4βδ receptors assembled from the chimeric β2NTD/β3TMD/ICL and β3NTD/β2TMD/ICL subunits. Analogously to the classical chimera between the α7 nicotinic acetylcholine and 5-HT3A receptor subunits (Eiselé et al., 1993), these chimeras consist of the N-terminal domain (NTD) of one β subunit and the transmembrane domain and intracellular loops (TMD/ICL) of the other (Fig. 5A). Thus, receptor complexes formed from α4, β2NTD/β3TMD/ICL, and δ subunits will comprise a “pure” α4β2δ extracellular domain and “pure” α4β3δ ion channel and intracellular domains, and vice versa for α4β3NTD/β2TMD/ICLδ receptors.
Functional properties of Thio-THIP at human α4βδ GABAARs comprising the chimeric β2NTD/β3TMD/ICL and β3NTD/β2TMD/ICL subunits in Xenopus oocytes. A, Schematic representation of the topologies of the β2/3 and β3/2 chimeras. The β2 and β3 segments in the two chimeras are given in blue and red, respectively. B, Averaged levels of constitutive activity in oocytes expressing α4β2NTD/β3TMD/ICLδ and α4β3NTD/β2TMD/ICLδ GABAARs. Constitutive activity is given as the percentage difference between the basal line and the outward current caused by application of 10 μm picrotoxin. Data are mean ± SEM (α4β2NTD/β3TMD/ICLδ: n = 3; α4β3NTD/β2TMD/ICLδ: n = 4). The constitutive activity in oocytes expressing WT α4β2δ and α4β3δ GABAARs (depicted in Fig. 2E) is given in gray for comparison. C, Averaged concentration–response curves for GABA at α4β2NTD/β3TMD/ICLδ and α4β3NTD/β2TMD/ICLδ GABAARs. Data are mean ± SEM (α4β2NTD/β3TMD/ICLδ: n = 6; α4β3NTD/β2TMD/ICLδ: n = 5). The averaged concentration–response curves for GABA at WT α4β2δ and WT α4β3δ (depicted in Fig. 2B) are given in gray-tone hatched curves for comparison. D, Averaged concentration–response curves for Thio-THIP at the α4β2NTD/β3TMD/ICLδ and α4β3NTD/β2TMD/ICLδ GABAARs. Data are mean ± SEM (α4β2NTD/β3TMD/ICLδ: n = 7; α4β3NTD/β2TMD/ICLδ: n = 8). The averaged concentration–response curves for Thio-THIP at WT α4β2δ and WT α4β3δ (depicted in Fig. 4B) are given in gray-tone hatched curves for comparison.
The basic functional characteristics of the α4β2NTD/β3TMD/ICLδ and α4β3NTD/β2TMD/ICLδ receptors assembled in oocytes were found to be very similar to those of the WT α4β2δ GABAAR (Fig. 5B,C). Neither of the chimera-containing receptors exhibited significant levels of constitutive activity, and GABA displayed monophasic concentration–response curves at both receptors with EC50 values not substantially different from its EC50 at WT α4β2δ or its EC50,2 at WT α4β3δ (Fig. 5B,C; Table 1). Thio-THIP was a distinct agonist at both α4β2NTD/β3TMD/ICLδ and α4β3NTD/β2TMD/ICLδ displaying efficacies intermediate of those at the two WT receptors, albeit considerably more similar to that at WT α4β2δ than at WT α4β3δ (Fig. 5C; Table 1).
All in all, the agonist properties exhibited by Thio-THIP at the α4β2NTD/β3TMD/ICLδ and α4β3NTD/β2TMD/ICLδ receptors do not to shed much light on the putative molecular determinants for its α4βδ subtype selectivity. On one hand, the intermediate agonist efficacies displayed by Thio-THIP at these receptors compared with WT α4β2δ and α4β3δ could indicate that the functionality of Thio-THIP at the α4βδ receptor does not arise exclusively from molecular elements in the NTD or the TMD/ICL but from the entire β subunit comprised in the α4βδ complex. On the other hand, the subunit stoichiometries and/or arrangements of the α4β2NTD/β3TMD/ICLδ and α4β3NTD/β2TMD/ICLδ receptors cannot be assumed to be identical to those of WT α4β2δ and α4β3δ, just as the subunit stochiometries and arrangements of the two WT receptors may differ. Thus, the fact that both chimera-containing receptors exhibit basic functionalities (low degrees of constitutive activity and monophasic GABA concentration–response curves) and Thio-THIP efficacies comparable with those at WT α4β2δ could also be a reflection of these three receptors sharing the same α4βδ subunit stoichiometry and/or arrangement, and that this differs from that of WT α4β3δ.
Functional properties of Thio-THIP at other GABAergic targets
The applicability of any ligand as a pharmacological tool in ex vivo or in vivo studies of GABAARs is dependent on its putative off-target effects, and obviously the chance of a compound possessing activity at other GABAergic proteins is particularly prominent when it comes to a GABA analog such as Thio-THIP. To address this issue, we investigated the functional properties of Thio-THIP at the two other classes of membrane-bound GABAergic targets: the GABAB receptors and the GABA transporters.
The effects of Thio-THIP on GABAB receptor signaling were studied in a Ca2+/Fluo-4 assay using tsA201 cells coexpressing either the GABAB(1a,2) or the GABAB(1b,2) receptor with Gαqi5, a chimeric G-protein directing the signaling of the Gαi/o-protein-coupled GABAB receptors into the Gαq-pathway and intracellular Ca2+ mobilization (Conklin et al., 1993). GABA increased fluorescence intensity in this assay in a concentration-dependent manner displaying pEC50 (± SEM) values of 6.42 ± 0.08 and 6.38 ± 0.07 at GABAB(1a,2) and GABAB(1b,2), respectively (n = 3 for both; Fig. 6A). These potencies were comparable with or slightly higher than those previously determined for GABA at GABAB receptors coexpressed with chimeric G-proteins in inositol phosphate accumulation assays (Bräuner-Osborne and Krogsgaard-Larsen, 1999; Duthey et al., 2002). Thio-THIP was found to be a weak GABAB receptor agonist evoking small but significant responses through both of the receptors at concentrations of ≥300 μm (Fig. 6A).
Functional properties of Thio-THIP at GABAB receptors and GABA transporters. A, Concentration–response relationships for GABA and Thio-THIP at rat GABAB(1a,2) and GABAB(1b,2) receptors coexpressed with the chimeric G-protein Gαqi5 in tsA201 cells in the Ca2+/Fluo-4 assay. Data are mean ± SEM (n = 3 for all). B, Concentration–inhibition relationships for GABA and Thio-THIP at human GAT-1, BGT-1, GAT-2, and GAT-3 transporters stably expressed in Flp-In-CHO cells in the [3H]GABA uptake assay. Data are mean ± SEM (n = 4 for all).
The putative activity of Thio-THIP as a substrate or an inhibitor at the four human GABA transporters was investigated in a conventional [3H]GABA uptake assay. The inhibitory potencies displayed by GABA at CHO cell lines stably expressing GAT-1 (pIC50 ± SEM: 4.94 ± 0.07, n = 4), BGT-1 (pIC50 ± SEM: 4.67 ± 0.11, n = 4), GAT-2 (pIC50 ± SEM: 5.04 ± 0.06, n = 4), and GAT-3 (pIC50 ± SEM: 5.18 ± 0.06, n = 4) in this assay were in good agreement with previous findings (Fig. 6B) (Kvist et al., 2009). Thio-THIP did not exert significant inhibition of the [3H]GABA uptake through any of the four transporters at concentrations up to 1 mm (Fig. 6B).
Functional characterization of Thio-THIP at native GABAARs in rat brain slices
We found the functional profile exhibited by Thio-THIP at the recombinant human GABAARs highly interesting, in particular its ability to discriminate between the three α4βδ receptor subtypes. To investigate to which extent this in vitro profile of the compound translated into its functionality at native GABAARs and to probe its potential as a pharmacological tool, we assessed the effects of Thio-THIP on synaptic and extrasynaptic GABAARs in different rat brain regions (Fig. 7A,B). A concentration of 100 μm Thio-THIP was used in these recordings because the in vitro profile of the compound suggests that this concentration will give rise to robust activation of α4β1δ and α4β3δ receptors and negligible inhibition of the α4β2δ GABAAR, and that, besides modest activation of the α6β2,3δ receptors, it will have negligible effects on other mediators of GABAergic neurotransmission (α1,2,3,5β2,3γ2 receptors, GABAB receptors, and GABA transporters).
Effects of Thio-THIP on the GABAARs mediating tonic inhibition in neurons from four rat brain regions: striatal MSNs, principal cells of VBT, DGGCs, and CGCs. A, Schematic representation of the localization of striatum (red), thalamus (green), hippocampus (blue), and cerebellum (brown) in rat brain. B, Micrographs of fluorescence-emitting, biocytin-filled striatal MSNs, VBT cells, and DGGCs. C, Representative traces for the currents induced by 100 μm Thio-THIP in striatal MSNs, VBT cells, DGGCs, and CGCs. D, Representative traces for the currents induced by 0.5 μm THIP in striatal MSNs, VBT cells, DGGCs, and CGCs. C, D, An endogenous current was observed in 10 of 25 VBT neurons and in 6 of 13 CGCs. E, Averaged current densities exhibited 100 μm Thio-THIP and 0.5 μm THIP in striatal MSNs, VBT cells, DGGCs, and CGCs. The current is normalized to the cell capacitance (current density, pA/pF). Groups (bar designated 1–4) were compared for the same compound in different regions: 1,2p < 0.001; 3p = 0.18; 4p = 0.01 (one-way ANOVA, Bonferroni). F, Representative trace exemplifying the lack of effect of 100 μm Thio-THIP on the currents evoked by 2.5 μm THIP in striatal MSNs. G, Correlation between the inward currents induced by 100 μm SR95531 (a) and the currents evoked by 100 μm Thio-THIP (b) in the 15 VBT cells recorded in this study.
Effects of Thio-THIP on the GABAARs mediating tonic inhibition
In these recordings, the capacitance ranges of the neurons used to normalize the respective effects of the test compounds on holding current were 2–4 pF (n = 11) for cerebellar granule cells (CGCs), 8–12 pF (n = 23) for striatal medium spiny neurons (MSNs), 9–13 pF (n = 19) for dentate gyrus granule cells (DGGCs), and 11–18 pF (n = 25) for principal cells of ventrobasal thalamus (VBT). In our recordings from striatal MSNs in the anterior dorsal region of caudate–putamen, we did not differentiate between striatonigral MSNs expressing dopamine D1 receptors (D1 + MSNs) and striatopallidal MSNs expressing dopamine D2 receptors (D2 + MSNs). For all striatal MSNs and DGGCs, addition of SR95531 to the bath was observed to silence all synaptic currents and restore the holding current of the cell (if changed by the applied drug) to its initial value. In contrast, in 10 of 25 VBT neurons and in 6 of 13 CGCs recorded from, the response to SR95531 was a more positive holding current than the baseline, which most likely reflects the baseline GABA-induced tonus on extrasynaptic receptors in these regions (Fig. 7C,D). The use of the functionally selective α4βδ/α6βδ receptor agonist THIP as a reference ligand in these recordings served partly to verify that currents mediated by extrasynaptic δ-GABAARs could indeed be recorded from the patched cells and partly as a reference to which the observed effects of Thio-THIP could be related. In concordance with its extensive use in previous studies of extrasynaptic α4βδ and α6βδ receptors, 0.5 and 2.5 μm THIP were found to elicit robust currents in all four rat brain regions (Fig. 7D; Table 2).
Current densities recorded in four different rat brain regions using THIP or Thio-THIPa
In contrast to the uniform induction of currents by THIP in the four cell types, 100 μm Thio-THIP had highly differential effects on the currents in the four regions. Thio-THIP evoked robust inward currents in CGCs comparable in size with those elicited by 0.5 μm THIP (Fig. 7C–E; Table 2). In contrast, the compound did not induce significant currents in striatal MSNs or in DGGCs (Fig. 7C–E; Table 2). To investigate whether Thio-THIP possesses antagonistic effects at the extrasynaptic receptors in striatal MSNs, we used a scheme of application of 2.5 μm THIP until stable current was obtained followed by coapplication of 2.5 μm THIP and 100 μm Thio-THIP. As can be seen from Figure 7F, the THIP-induced currents in striatal MSNs were not reduced by the presence of Thio-THIP, as the change in holding current upon the coaddition of 100 μm Thio-THIP was negligible (6.4 ± 5.1 pA, n = 4).
In VBT neurons, 100 μm Thio-THIP induced substantial currents characterized by an average current density comparable with those elicited by 0.5 μm THIP (4.6 ± 0.6 pA/pF and 3.8 ± 0.4 pA/pF, respectively) (Fig. 7C–E). One-way ANOVA of the current density for the MSN, DGGC, and VBT groups in 100 μm Thio-THIP gave F = 16.9 and p = 9.9E-6, and post hoc Bonferroni test gave p = 8E-5 (VBT vs DGGC), p = 4E-4 (VBT vs MSN) and p = 1 (DGGC vs MSN). Thus, the average current density observed for 100 μm Thio-THIP in VBT neurons was 7.7- and 15.3-fold higher than those recorded from striatal MSNs or DGGCs, respectively. In comparison, analysis of the differences in current densities observed for 0.5 μm THIP in the three cell types gave F = 6.8 and p = 0.01 (one-way ANOVA) and Bonferroni test gave p = 0.01 (VBT vs DGGC) but p = 0.18 (VBT vs MSN) and p = 0.4 (DGGC vs MSN), indicating a 2.1-fold larger current density in VBT cells than in DGGCs. Interestingly, the current densities evoked by Thio-THIP in the 15 VBT cells recorded from in this study varied greatly and so did the degrees of basal activity observed for the respective neurons (assessed by the inward currents induced by 100 μm SR95531). Interestingly, a pronounced correlation was observed between the sizes of these two parameters from the VBT neuron recordings (Fig. 7G).
Effects of Thio-THIP on the GABAARs mediating phasic inhibition
To characterize the effects of Thio-THIP on the GABAARs mediating phasic currents in VBT neurons, DGGCs and striatal MSNs, we analyzed the mIPSCs in the same neurons used for the analysis described in the previous section. A similar analysis of mIPSCs in CGCs could not be performed because the few spontaneous events (<20 during a 3 min period) that could be resolved during baseline conditions in these cells were lost in the noise caused by the Thio-THIP application (unpublished observations). Furthermore, in 3 of the 15 VBT neurons patched, very low frequencies of mIPSCs (<0.1 Hz) were detected, even though the cells displayed similar levels of extrasynaptic GABAAR-mediated currents and similar responses to SR95531 as the 12 other cells (unpublished observations). Thus, these three cells were excluded from the analysis of the synaptic currents.
The properties of the averaged synaptic current in VBT neurons, DGGCs, and striatal MSNs during baseline conditions and in the presence of 100 μm Thio-THIP are given in Table 3, and representative recordings from the cells are depicted in Figure 8A. The shape of the averaged synaptic current in the striatal MSN was not affected by the presence of 100 μm Thio-THIP. In the VBT neuron, the presence of Thio-THIP prolonged the decay time of the current significantly (Fig. 8A; Table 3). Although the detection limit in these neurons is based upon the noise level (SD) in the drug period, an increased noise will in particular affect the measurement of the decay time of small mIPSCs. Using a detection limit of 10 × SD, we observed a decay tau of 6.3 ± 0.8 in the absence and 7.3 ± 0.7 in the presence of Thio-THIP (p = 0.12), indicating that increased baseline noise interfere with the measurement of decay time, and this effect is larger for small events.
Properties of mIPSCs in regional principal cells recorded under basal conditions and in the presence of 100 μm Thio-THIPa
Effects of Thio-THIP on the GABAARs mediating phasic inhibition in neurons from three rat brain regions: striatal MSNs, principal cells of VBT, and DGGCs. A, The average noncontaminated waveforms of the mIPSCs recorded from striatal MSNs, VBT cells, and DGGCs in the absence (Control) or in the presence of 100 μm Thio-THIP. B, The average noncontaminated waveforms of the mIPSCs recorded from DGGCs in the absence (Control) or in the presence of 15 μm Zn2+ or 15 μm Zn2+ and 100 μm Thio-THIP. C, Examples of the average mIPSC waveform of DGGCs exposed to 25 μm Thio-THIP (left), 1 μm DS2 (middle), or the combination (right). The decay time constants are the weighted time constants from fits to a biexponential function.
The most pronounced effect of 100 μm Thio-THIP on synaptic currents observed in this study was the significantly reduced peaks of the mIPSCs recorded in DGGCs in the presence of the compound. In contrast, the compound had only very little effect on the baseline noise (i.e., the detection limit) in these cells (Fig. 8A; Table 3). We tested whether it was possible to reverse the effect of Thio-THIP on mIPSC peak in DGGCs after washout, and we found that peak values of 42.9 ± 3.6 pA (n = 5) during baseline were reduced to 34.6 ± 3.3 pA during 100 μm Thio-THIP, and again increased to 40.0 ± 3.9 pA, 8 min after washout begin (p = 0.03, Thio-THIP vs washout). In a study of mouse DGGCs, Wei et al. (2003) have found δ-containing GABAARs (most likely α4βδ assemblies) to be localized perisynaptically, particularly on the distal part of the apical dendrite (Soltesz et al., 1995). To assess whether the observed effects of Thio-THIP on the recorded mIPSCs could be ascribed to Thio-THIP targeting an analogous receptor population in rat DGGCs, we first tested the sensitivity of mIPSCs to 15 μm Zn2+ in phosphate-free buffer. In these recordings (n = 5), Thio-THIP had no effect on the RT20–80, peak, or decay characteristics of the averaged mIPSC (Fig. 8B). Specifically, the average mIPSC peaks (± SEM) were 41.2 ± 6.8 pA at baseline, 28.9 ± 4.7 pA in the presence of 15 μm Zn2+ (p = 0.01), and 29.0 ± 4.8 pA in the concomitant presence of 15 μm Zn2+ and 100 μm Thio-THIP (p = 0.9, Zn2+ vs Zn2+/Thio-THIP, paired t test). Furthermore, we tested the effects of the addition of the δ-GABAAR-selective PAM DS2 (1 μm) alone and in combination with a submaximal dose of Thio-THIP (25 μm) on these currents. When tested alone, DS2 displayed no effects on peak, decay, or RT20–80 (n = 5), whereas Thio-THIP (25 μm) prolonged the decay only (6.1 ± 0.2 ms in baseline and 6.9 ± 0.3 ms in 25 μm Thio-THIP, n = 5, p = 0.01; Fig. 8C). Coapplication of 1 μm DS2 and 25 μm Thio-THIP significantly diminished the mIPSC peak (31.4 ± 2.6 in baseline vs 27.4 ± 2.2 in drug combination, n = 6, p = 0.001) and prolonged the decay (5.7 ± 0.1 ms in baseline vs 6.9 ± 0.4 in drug combination, n = 6, p = 0.01). Together, this suggests that the reduction in the peak of the average mIPSC observed in the presence of 100 μm Thio-THIP can be ascribed to the compound targeting δ-containing GABAARs responsible for a component of the synaptic current in rat DGGCs, possibly perisynaptically localized α4βδ receptors analogous to those proposed to be expressed in mice DGGC dendrites (Wei et al., 2003). In a final series of experiments, we recorded from dentate hilar subgranular interneurons characterized by large soma (capacitance range 17–26 pF, n = 3). Perfusion of 100 μm Thio-THIP to the bath had no effect on the holding current in these cells (2.1 ± 3.6 μA), and peak of the average events was unchanged (28.6 ± 2.4 pA in the absence vs 27.7 ± 2.3 pA in the presence of 100 μm Thio-THIP). Although the absence of an effect from Thio-THIP on one hand could indicate that α4β1,3δ receptors are not expressed in the cells, activation of the receptors, if indeed expressed, could also be insufficient to trigger significant currents. Thus, we shall refrain from drawing solid conclusions about whether or not these interneurons express α4β1,3δ receptors based on these data. On the other hand, the lack of effect from Thio-THIP could indicate that the compound does not activate the α1βδ receptors shown to mediate tonic currents in these interneurons (Glykys et al., 2007), although this will be important to verify at recombinant α1βδ receptors in heterologous expression systems.
Discussion
The abundant expression and the essential roles of α4βδ GABAARs as mediators of tonic conductance in the CNS make them important contributors to numerous physiological processes and pathophysiological states (Belelli et al., 2009; Brickley and Mody, 2012), and the therapeutic potential of the receptors are substantiated by the in vivo effects of THIP and δ-GABAAR selective PAMs (Ebert et al., 2006; Wafford et al., 2009; Hoestgaard-Jensen et al., 2010). Considering the heterogeneous nature of native α4βδ receptor populations and the likely differential regulation of the β1-, β2-, and β3-containing subtypes by post-translational processes such as phosphorylation (Kittler and Moss, 2003; Houston et al., 2009), the possibility that the α4βδ subtypes could mediate distinct physiological functions has attracted surprisingly little attention. In the present study, Thio-THIP has been shown to be a potentially useful tool for investigations of the α4βδ subtypes and their respective roles in GABAergic neurotransmission.
The distinct subtype selectivity profile of Thio-THIP at recombinant GABAARs
The different functionalities exhibited by THIP and Thio-THIP at recombinant GABAARs are intriguing. Introduction of the sulfur into the heteroaromatic ring of THIP not only transforms its agonism and superagonism at α1,2,3,5βγ2 and α4,6βδ receptors, respectively, into antagonism and partial agonism, it also converts its nonselective agonist activity at α4βδ receptors into pronounced α4β1δ/α4β3δ selectivity. Extensive use of 3-isoxazolol and 3-isothiazolol as carboxylate bioisosteres in the glutamate receptor field has shown that it is virtually impossible to rationalize pharmacological differences arising from this seemingly small structural difference solely by the physicochemical properties of the two ring systems (Matzen et al., 1997; Hermit et al., 2004). Moreover, considering the conserved nature of the orthosteric GABAAR site, in particular when it comes to the principal β(+) binding component (Sieghart, 2006), the β selectivity of Thio-THIP is unlikely to be rooted in substantially different binding interactions with the three α4βδ subtypes. Instead, it may arise from different energy barriers associated with allosteric transitions between ligand binding and gating in the receptors or from the assembly of different α4βδ complexes in α4β1δ-, α4β2δ-, and α4β3δ-oocytes. α4βδ GABAARs have been reported to assemble as both βαβαδ and/or βαβδα pentamers (anticlockwise, viewed from extracellular space) in heterologous expression systems (Barrera et al., 2008; Shu et al., 2012; Eaton et al., 2014; Patel et al., 2014), and concatameric α1,6β3δ receptors with several different subunit arrangements have also been shown to be functional (Baur et al., 2009; Kaur et al., 2009). Whether all α4βδ pentamers assembled in vitro are expressed in neurons is obviously another matter, and the possible implications of this for Thio-THIP as a pharmacological tool will be addressed below.
The effects of Thio-THIP at native rat GABAARs
All in all, the observed effects of Thio-THIP on the currents mediated by synaptic and extrasynaptic GABAARs in four rat brain regions are in good agreement with its properties at the recombinant human receptors. Moreover, the observations in the slice recordings not only concurs with previous findings about native α4βδ GABAARs but also sheds further light on the heterogeneity of these receptors.
Extrasynaptic α6βδ GABAARs are the key mediators of tonic inhibition in CGCs (Jones et al., 1997; Brickley et al., 2001), and the robust currents evoked by Thio-THIP in these cells thus concur with its agonist activity at recombinant α6β2,3δ receptors. α4βδ GABAARs are equally well established as major extrasynaptic receptors in rodent DGGCs (Nusser and Mody, 2002; Stell et al., 2003; Chandra et al., 2006; Zhang et al., 2007). In an elegant study using β2 knock-out (β2−/−) and etomidate-insensitive β2 knock-in (β2/N265S) mice, α4β2δ has been identified as the key mediator of tonic inhibition in these cells, albeit with significant contributions from α5β1/3γ2, αxβ1/3, and/or αxβ1/3δ receptors (Herd et al., 2008). The inability of 100 μm Thio-THIP to activate extrasynaptic receptors in rat DGGCs substantiates the notion of α4β2δ being the major extrasynaptic GABAAR in rodent DGGCs.
The current insight into the compositions of extrasynaptic GABAARs in striatal MSNs is largely based on mice studies. α2, α4, β3, and δ are the predominant subunits in adult mouse MSNs, but surprisingly the α4δ-containing receptors mediating tonic currents here do not appear to comprise β3 (Janssen et al., 2009; Luo et al., 2013). Single-cell RT-PCR analysis has detected widespread expression of β1 and β3 but not of β2 in adult rat D1+ MSNs (Flores-Hernandez et al., 2000); and although a concomitant abundant β1 and β3 mRNA expression and absence of the two subunits in extrasynaptic α4βδ assemblies in rat MSNs would be analogous to the observations for β3 in mouse MSNs, it does call for caution when interpreting our findings. Thus, although the inability of 100 μm Thio-THIP to elicit currents and to block THIP-induced currents in MSNs seems to point to α4β2δ as the major extrasynaptic α4βδ subtype in adult rodent striatal MSNs, it will be important to challenge this conclusion in future studies.
α4βδ GABAARs are the key extrasynaptic/perisynaptic receptors in VBT neurons (Sur et al., 1999; Jia et al., 2005; Chandra et al., 2006; Herd et al., 2009). In concordance with higher expression levels of β2 than β1 and β3 (Laurie et al., 1992; Wisden et al., 1992; Hörtnagl et al., 2013), a study using β2−/− and β2/N265S mice has identified α4β2δ as the predominant extrasynaptic α4βδ subtype in these neurons (Belelli et al., 2005). The significant currents evoked by 100 μm Thio-THIP in VBT neurons do not necessarily challenge this conclusion, but the observation suggests that α4β1δ and/or α4β3δ receptors contribute to tonic inhibition in these cells as well, at least in rats. The differential amplitudes of Thio-THIP-induced currents in the 15 VBT neurons could potentially arise from differential expression of the three α4βδ subtypes in the cells. In line with this, the correlation between basal activity levels and Thio-THIP-induced current amplitudes in these neurons could be a reflection of the higher GABA sensitivities exhibited by α4β1δ and α4β3δ compared with α4β2δ in the oocytes (Figs. 2D and 7G).
The negligible effects of 100 μm Thio-THIP at synaptic currents in rat striatal MSNs and VBT cells concur with its properties at recombinant α1,2,3β2,3γ2 receptors (Figs. 3 and 8). Interestingly, the significantly reduced peak amplitudes of mIPSCs recorded from DGGCs in the presence of the compound seem to be attributable to δ-containing receptors (Fig. 8). Since 100 μm Thio-THIP is not expected to affect α4β2δ receptors substantially, we speculate that the effect arises from α4β1/3δ receptors expressed analogously to the proposed perisynaptic α4βδ receptors in mouse DGCCs (Wei et al., 2003). Occupancy of these receptors by Thio-THIP would counteract the effects of synaptically released GABA, and conversely agonist binding to the receptors would not trigger measurable tonic currents, presumably due to their small magnitude evoked from a distal localization (Soltesz et al., 1995). Finally, as for the concern whether the functionality determined for Thio-THIP at α4βδ complexes assembled in oocytes is truly representative of its properties at the native receptors, the robust currents induced by the compound in VBT neurons and its effect on mIPSCs in DGCCs certainly suggest that at least some α4β1/3δ receptors are targeted by the compound, just as its inability to induce currents through the extrasynaptic GABAARs in the DGGCs seems to concur with its negligible activity at the recombinant α4β2δ receptors.
Thio-THIP: a novel pharmacological tool for explorations of α4βδ GABAARs
In conclusion, we propose that Thio-THIP could be a valuable addition to the rather limited selection of β-isoform-selective GABAAR ligands currently available. Whereas β-selectivity in previously published ligands has come in the form of β2/β3- or β1-selectivity (Wafford et al., 1994; Belelli et al., 1997; Hill-Venning et al., 1997; Halliwell et al., 1999; Thompson et al., 2004; Absalom et al., 2012), Thio-THIP is to our knowledge the first GABAAR ligand exhibiting pronounced β1/β3-over-β2-selectivity and thus being able to differentiate between β2- and β3-containing subtypes, at least when it comes to α4βδ receptors. Although Thio-THIP admittedly has not been characterized functionally at all physiologically relevant GABAAR subtypes, the present data suggest that Thio-THIP (100 μm) will act fairly selectively at α4β1,3δ and α6βδ receptors; thus, the compound would be expected to be selective for α4β1,3δ receptors in most CNS regions, except for cerebellum. Also, in this respect, Thio-THIP differs from the previously published allosteric modulators, which target a wide range of β2/β3- or β1-containing GABAAR subtypes. On the other hand, the use of Thio-THIP in combination with one of these modulators or in transgenic mice could be an attractive approach to delineate the exact compositions of native α4βδ receptors, although such an endeavor potentially could face another level of complexity in the form of the putative assembly of “mixed” α4βδ receptors (α4β1β2δ, α4β1β3δ, α4β2β3δ or even α4β1β2β3δ) in vivo. Finally, perhaps the most interesting perspective arising from this study is the ability of an β-isoform-selective α4βδ agonist to affect tonic inhibition in some CNS regions but not in others. Although its moderate potency makes Thio-THIP an unlikely therapeutic candidate, it would nevertheless be interesting to explore whether the discrete modulation of tonic GABAergic signaling in specific brain regions exerted by it could hold some therapeutic advantages compared with drugs acting nonselectively at α4βδ receptors, such as its close structural analog THIP.
Footnotes
This study was supported by the Novo Nordisk Foundation and the Lundbeck Foundation. We thank Drs. P.J. Whiting, D.S. Weiss, J. Clark, and B.R. Conklin for generous gifts of cDNAs; and Dr. H. Bräuner-Osborne for granting us access to the GAT-CHO cell lines.
The authors declare no competing financial interests.
- Correspondence should be addressed to Dr. Anders A. Jensen, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen OE, Denmark. aaj{at}sund.ku.dk
