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Pharmacological characterisation of murine α4β1δ GABAA receptors expressed in Xenopus oocytes.

Villumsen IS, Wellendorph P, Smart TG - BMC Neurosci (2015)

Bottom Line: GABAA receptor subunit composition has a profound effect on the receptor's physiological and pharmacological properties.The neurosteroid tetrahydro-deoxycorticosterone (THDOC) significantly increased GABA-initiated responses in concentrations above 30 nM for α4β1δ receptors.This study highlights some notable differences in the pharmacology of murine and human α4β1δ receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Physiology & Pharmacology, University College London, Gower Street, London, WC1E 6BT, UK. inge_villumsen@hotmail.com.

ABSTRACT

Background: GABAA 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 and α4β1 subunits.

Results: GABA activated concentration-dependent responses at α4β1δ and α4β1 receptors with EC50 values in the nanomolar to micromolar range, respectively. The divalent cations Zn(2+) and Cu(2+), and the β1-selective inhibitor salicylidine salicylhydrazide (SCS), inhibited GABA-activated currents at α4β1δ receptors. Surprisingly the α4β1 receptor demonstrated biphasic sensitivity to Zn(2+) inhibition that may reflect variable subunit stoichiometries with differing sensitivity to Zn(2+). The neurosteroid tetrahydro-deoxycorticosterone (THDOC) significantly increased GABA-initiated responses in concentrations above 30 nM for α4β1δ receptors.

Conclusions: With this study we report the first pharmacological characterisation of various GABAA receptor ligands acting at murine α4β1δ GABAA receptors, thereby improving our understanding of the molecular pharmacology of this receptor isoform. This study highlights some notable differences in the pharmacology of murine and human α4β1δ receptors. We consider the likelihood that the α4β1δ receptor may play a role as an extrasynaptic GABAA receptor in the nervous system.

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Pharmacological modulation of GABA responses at α4β1δ receptors by various inhibitors and the neurosteroid, THDOC. A, Representative membrane currents showing inhibition of GABA (EC75) by 1 μM Zn2+ at α4β1 (upper) and α4β1δ (lower) receptors. B, Zn2+ concentration-inhibition relationships for α4β1 (n = 6) and α4β1δ (n = 6) receptors. C, Representative currents showing the degree of desensitization when activated by EC75 GABA in the absence (upper) and presence (lower) of 1 μM Cu2+. D, Cu2+ concentration-inhibition relationship for GABA EC75 desensitized responses at α4β1δ receptors by increasing concentrations of Cu2+ (n = 12). E, SCS concentration-inhibition relationship for GABA EC20 peak responses at α4β1δ receptors by increasing concentrations of SCS (n = 4). F, Response of α4β1δ receptors to increasing concentrations of THDOC co-applied with an EC7 GABA concentration. The responses were normalised to a preceding application of GABA EC7 in the absence of THDOC (n = 9). All data shown are means ± SEMs.
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Fig2: Pharmacological modulation of GABA responses at α4β1δ receptors by various inhibitors and the neurosteroid, THDOC. A, Representative membrane currents showing inhibition of GABA (EC75) by 1 μM Zn2+ at α4β1 (upper) and α4β1δ (lower) receptors. B, Zn2+ concentration-inhibition relationships for α4β1 (n = 6) and α4β1δ (n = 6) receptors. C, Representative currents showing the degree of desensitization when activated by EC75 GABA in the absence (upper) and presence (lower) of 1 μM Cu2+. D, Cu2+ concentration-inhibition relationship for GABA EC75 desensitized responses at α4β1δ receptors by increasing concentrations of Cu2+ (n = 12). E, SCS concentration-inhibition relationship for GABA EC20 peak responses at α4β1δ receptors by increasing concentrations of SCS (n = 4). F, Response of α4β1δ receptors to increasing concentrations of THDOC co-applied with an EC7 GABA concentration. The responses were normalised to a preceding application of GABA EC7 in the absence of THDOC (n = 9). All data shown are means ± SEMs.

Mentions: We evaluated Zn2+-inhibition of the GABA response for both α4β1 and α4β1δ receptor isoforms to investigate whether Zn2+ can distinguish between these receptor subtypes. Oocytes were pre-incubated for 1 min with various concentrations of Zn2+ followed by co-application of Zn2+ and GABA at an EC75 (3 μM for α4β1δ and 8 μM for α4β1). As expected, Zn2+ exhibited a higher potency at α4β1 than α4β1δ receptors. For α4β1δ receptors, Zn2+ inhibited the GABA response with an IC50 of 3.3 μM (Figure 2A,B, Table 1). By contrast, for α4β1 receptors, biphasic inhibitory behaviour was observed with a high potency component (IC50A = 0.13 nM) accounting for ~25% of the total inhibition, and a more dominant lower potency component (IC50B = 56 nM) accounting for the remaining (~75%) inhibition (Figure 2A,B, Table 1).Figure 2


Pharmacological characterisation of murine α4β1δ GABAA receptors expressed in Xenopus oocytes.

Villumsen IS, Wellendorph P, Smart TG - BMC Neurosci (2015)

Pharmacological modulation of GABA responses at α4β1δ receptors by various inhibitors and the neurosteroid, THDOC. A, Representative membrane currents showing inhibition of GABA (EC75) by 1 μM Zn2+ at α4β1 (upper) and α4β1δ (lower) receptors. B, Zn2+ concentration-inhibition relationships for α4β1 (n = 6) and α4β1δ (n = 6) receptors. C, Representative currents showing the degree of desensitization when activated by EC75 GABA in the absence (upper) and presence (lower) of 1 μM Cu2+. D, Cu2+ concentration-inhibition relationship for GABA EC75 desensitized responses at α4β1δ receptors by increasing concentrations of Cu2+ (n = 12). E, SCS concentration-inhibition relationship for GABA EC20 peak responses at α4β1δ receptors by increasing concentrations of SCS (n = 4). F, Response of α4β1δ receptors to increasing concentrations of THDOC co-applied with an EC7 GABA concentration. The responses were normalised to a preceding application of GABA EC7 in the absence of THDOC (n = 9). All data shown are means ± SEMs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359537&req=5

Fig2: Pharmacological modulation of GABA responses at α4β1δ receptors by various inhibitors and the neurosteroid, THDOC. A, Representative membrane currents showing inhibition of GABA (EC75) by 1 μM Zn2+ at α4β1 (upper) and α4β1δ (lower) receptors. B, Zn2+ concentration-inhibition relationships for α4β1 (n = 6) and α4β1δ (n = 6) receptors. C, Representative currents showing the degree of desensitization when activated by EC75 GABA in the absence (upper) and presence (lower) of 1 μM Cu2+. D, Cu2+ concentration-inhibition relationship for GABA EC75 desensitized responses at α4β1δ receptors by increasing concentrations of Cu2+ (n = 12). E, SCS concentration-inhibition relationship for GABA EC20 peak responses at α4β1δ receptors by increasing concentrations of SCS (n = 4). F, Response of α4β1δ receptors to increasing concentrations of THDOC co-applied with an EC7 GABA concentration. The responses were normalised to a preceding application of GABA EC7 in the absence of THDOC (n = 9). All data shown are means ± SEMs.
Mentions: We evaluated Zn2+-inhibition of the GABA response for both α4β1 and α4β1δ receptor isoforms to investigate whether Zn2+ can distinguish between these receptor subtypes. Oocytes were pre-incubated for 1 min with various concentrations of Zn2+ followed by co-application of Zn2+ and GABA at an EC75 (3 μM for α4β1δ and 8 μM for α4β1). As expected, Zn2+ exhibited a higher potency at α4β1 than α4β1δ receptors. For α4β1δ receptors, Zn2+ inhibited the GABA response with an IC50 of 3.3 μM (Figure 2A,B, Table 1). By contrast, for α4β1 receptors, biphasic inhibitory behaviour was observed with a high potency component (IC50A = 0.13 nM) accounting for ~25% of the total inhibition, and a more dominant lower potency component (IC50B = 56 nM) accounting for the remaining (~75%) inhibition (Figure 2A,B, Table 1).Figure 2

Bottom Line: GABAA receptor subunit composition has a profound effect on the receptor's physiological and pharmacological properties.The neurosteroid tetrahydro-deoxycorticosterone (THDOC) significantly increased GABA-initiated responses in concentrations above 30 nM for α4β1δ receptors.This study highlights some notable differences in the pharmacology of murine and human α4β1δ receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Physiology & Pharmacology, University College London, Gower Street, London, WC1E 6BT, UK. inge_villumsen@hotmail.com.

ABSTRACT

Background: GABAA 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 and α4β1 subunits.

Results: GABA activated concentration-dependent responses at α4β1δ and α4β1 receptors with EC50 values in the nanomolar to micromolar range, respectively. The divalent cations Zn(2+) and Cu(2+), and the β1-selective inhibitor salicylidine salicylhydrazide (SCS), inhibited GABA-activated currents at α4β1δ receptors. Surprisingly the α4β1 receptor demonstrated biphasic sensitivity to Zn(2+) inhibition that may reflect variable subunit stoichiometries with differing sensitivity to Zn(2+). The neurosteroid tetrahydro-deoxycorticosterone (THDOC) significantly increased GABA-initiated responses in concentrations above 30 nM for α4β1δ receptors.

Conclusions: With this study we report the first pharmacological characterisation of various GABAA receptor ligands acting at murine α4β1δ GABAA receptors, thereby improving our understanding of the molecular pharmacology of this receptor isoform. This study highlights some notable differences in the pharmacology of murine and human α4β1δ receptors. We consider the likelihood that the α4β1δ receptor may play a role as an extrasynaptic GABAA receptor in the nervous system.

Show MeSH
Related in: MedlinePlus