Abstract:BackgroundGABAA receptor subunit composition has a profound effect on the receptor’s physiological and pharmacological properties. The receptor β subunit is widely recognised for its importance in receptor assembly, trafficking and post-translational modifications, but its influence on extrasynaptic GABAA receptor function is less well understood. Here, we examine the pharmacological properties of a potentially native extrasynaptic GABAA receptor that incorporates the β1 subunit, specifically composed of α4β1δ… Show more
“…However, the potencies of GABA and THIP at α 4 β 1 δ receptors determined in patch clamp electrophysiology were 5–8 times higher than that found using the FMP assay. Although we observed differences in the potency between the two assays, the FMP data are still within the range of what is reported in the literature, ranging from 0.89 to 2.02 μM and the difference between the potency of GABA and THIP is similar for both assays with THIP being 7 and 13 times less potent at α 4 β 1 δ receptors in the FMP and patch clamp assays, respectively. Additionally, no superagonism of THIP was found in the FMP assay at either of the studied ternary δ‐receptors, in contrast to the patch clamp studies on α 4 β 1 δ, where THIP was able to induce higher signals than GABA.…”
Section: Discussionsupporting
confidence: 81%
“…In spite of the number of studies, the precise pharmacology of the δ‐containing GABA A receptors remains uncertain. Although not exclusively, studies performed on α 4 β 1/3 δ expressed in Xenopus laevis oocytes have reported biphasic GABA concentration–response curves with EC 50 values in the low nanomolar and low micromolar range, respectively, whereas monophasic GABA concentration–response curves with EC 50 values in the low micromolar range have typically been observed for receptors expressed in HEK cells . Explanations for these differences may relate to both differences in the biochemistry of the expression system, differences in transfection ratios and the species of the used subunits .…”
mentioning
confidence: 99%
“…Moreover, it is known that δ‐containing GABA A receptors are notoriously difficult to express consistently in recombinant systems, and thus, there is a concern for interfering signals from binary αβ or homomeric β receptors , which may influence the pharmacology. The majority of studies have compared the pharmacology of the δ‐containing receptors to the corresponding γ 2 ‐containing receptors to confirm the incorporation of the δ‐subunit in the receptor complex, but often not ruled out a possible contribution from αβ receptors and or homomeric β receptors , although these have different ligand sensitivity to zinc (Zn 2+ ) and different rates and extents of desensitization compared to ternary receptors . Another way to distinguish α 4 β and α 4 βδ recombinant receptors is by the use of subtype‐selective compounds for δ‐containing receptors; however, only a few have been reported so far, including THIP , DS2 , AA29504 and 3‐OH‐2'MeO6MF .…”
δ-Containing GABA receptors are located extrasynaptically and mediate tonic inhibition. Their involvement in brain physiology positions them as interesting drug targets. There is thus a continued interest in establishing reliable recombinant expression systems for δ-containing GABA receptors. Inconveniently, the recombinant expression of especially α β δ receptors has been found to be notoriously difficult, resulting in mixed receptor populations and/or stoichiometries and differential pharmacology depending on the expression system used. With the aim of developing a facile and robust 96-well format cell-based assay for extrasynaptic α β δ receptors, we have engineered and validated a HEK293 Flp-In™ cell line stably expressing the human GABA δ-subunit. Upon co-transfection of α and β subunits, at optimized ratios, we have established a well-defined system for expressing α β δ receptors and used the fluorescence-based FLIPR Membrane Potential (FMP) assay to evaluate their pharmacology. Using the known reference compounds GABA and THIP, ternary α β δ and binary α β receptors could be distinguished based on potency and kinetic profiles but not efficacy. As expected, DS2 was able to potentiate only δ-containing receptors, whereas Zn had an inhibitory effect only at binary receptors. By contrast, the hitherto reported δ-selective compounds, AA29504 and 3-OH-2'MeO6MF, were non-selective. The expression system was further validated using patch clamp electrophysiology, in which the superagonism of THIP was confirmed. The established FMP assay set-up, based on transient expression of human α and β subunits into a δ-subunit stable HEK293 Flp-In™ cell line, portrays a simple 96-well format assay as a useful supplement to electrophysiological recordings on δ-containing GABA receptors.
“…However, the potencies of GABA and THIP at α 4 β 1 δ receptors determined in patch clamp electrophysiology were 5–8 times higher than that found using the FMP assay. Although we observed differences in the potency between the two assays, the FMP data are still within the range of what is reported in the literature, ranging from 0.89 to 2.02 μM and the difference between the potency of GABA and THIP is similar for both assays with THIP being 7 and 13 times less potent at α 4 β 1 δ receptors in the FMP and patch clamp assays, respectively. Additionally, no superagonism of THIP was found in the FMP assay at either of the studied ternary δ‐receptors, in contrast to the patch clamp studies on α 4 β 1 δ, where THIP was able to induce higher signals than GABA.…”
Section: Discussionsupporting
confidence: 81%
“…In spite of the number of studies, the precise pharmacology of the δ‐containing GABA A receptors remains uncertain. Although not exclusively, studies performed on α 4 β 1/3 δ expressed in Xenopus laevis oocytes have reported biphasic GABA concentration–response curves with EC 50 values in the low nanomolar and low micromolar range, respectively, whereas monophasic GABA concentration–response curves with EC 50 values in the low micromolar range have typically been observed for receptors expressed in HEK cells . Explanations for these differences may relate to both differences in the biochemistry of the expression system, differences in transfection ratios and the species of the used subunits .…”
mentioning
confidence: 99%
“…Moreover, it is known that δ‐containing GABA A receptors are notoriously difficult to express consistently in recombinant systems, and thus, there is a concern for interfering signals from binary αβ or homomeric β receptors , which may influence the pharmacology. The majority of studies have compared the pharmacology of the δ‐containing receptors to the corresponding γ 2 ‐containing receptors to confirm the incorporation of the δ‐subunit in the receptor complex, but often not ruled out a possible contribution from αβ receptors and or homomeric β receptors , although these have different ligand sensitivity to zinc (Zn 2+ ) and different rates and extents of desensitization compared to ternary receptors . Another way to distinguish α 4 β and α 4 βδ recombinant receptors is by the use of subtype‐selective compounds for δ‐containing receptors; however, only a few have been reported so far, including THIP , DS2 , AA29504 and 3‐OH‐2'MeO6MF .…”
δ-Containing GABA receptors are located extrasynaptically and mediate tonic inhibition. Their involvement in brain physiology positions them as interesting drug targets. There is thus a continued interest in establishing reliable recombinant expression systems for δ-containing GABA receptors. Inconveniently, the recombinant expression of especially α β δ receptors has been found to be notoriously difficult, resulting in mixed receptor populations and/or stoichiometries and differential pharmacology depending on the expression system used. With the aim of developing a facile and robust 96-well format cell-based assay for extrasynaptic α β δ receptors, we have engineered and validated a HEK293 Flp-In™ cell line stably expressing the human GABA δ-subunit. Upon co-transfection of α and β subunits, at optimized ratios, we have established a well-defined system for expressing α β δ receptors and used the fluorescence-based FLIPR Membrane Potential (FMP) assay to evaluate their pharmacology. Using the known reference compounds GABA and THIP, ternary α β δ and binary α β receptors could be distinguished based on potency and kinetic profiles but not efficacy. As expected, DS2 was able to potentiate only δ-containing receptors, whereas Zn had an inhibitory effect only at binary receptors. By contrast, the hitherto reported δ-selective compounds, AA29504 and 3-OH-2'MeO6MF, were non-selective. The expression system was further validated using patch clamp electrophysiology, in which the superagonism of THIP was confirmed. The established FMP assay set-up, based on transient expression of human α and β subunits into a δ-subunit stable HEK293 Flp-In™ cell line, portrays a simple 96-well format assay as a useful supplement to electrophysiological recordings on δ-containing GABA receptors.
“…The concentration of cytosolic and extracellular putrescine has been determined to be 22 nmol/g and 12 nmol/g , respectively [ 339 ]. In contrast, copper may decrease tonic inhibition via acting on delta subunit-containing extrasynaptic GABA A receptors [ 235 , 237 , 246 ], thus adding a new layer to disinhibitory mechanisms in copper-rich brain areas. Fig.…”
Copper-containing enzymes perform fundamental functions by activating dioxygen (O2) and therefore allowing chemical energy-transfer for aerobic metabolism. The copper-dependence of O2 transport, metabolism and production of signalling molecules are supported by molecular systems that regulate and preserve tightly-bound static and weakly-bound dynamic cellular copper pools. Disruption of the reducing intracellular environment, characterized by glutathione shortage and ambient Cu(II) abundance drives oxidative stress and interferes with the bidirectional, copper-dependent communication between neurons and astrocytes, eventually leading to various brain disease forms. A deeper understanding of of the regulatory effects of copper on neuro-glia coupling via polyamine metabolism may reveal novel copper signalling functions and new directions for therapeutic intervention in brain disorders associated with aberrant copper metabolism.
“…Moreover there are some possible effect of species differences in alcohol and alcohol antagonist responses (Wallner, et al, 2014). For example, a recent study suggests that there are some significant differences in the pharmacology of murine and human α4β1δ receptors (Villumsen et al, 2015). Therefore, it is clear that methodological issues should be carefully considered for studies with -GABAARs in general and for the effects of alcohol on these receptors in particular.…”
Section: Alcohol and Extra-synaptic Gabaarsmentioning
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