Elsevier

Neuropharmacology

Volume 88, January 2015, Pages 145-154
Neuropharmacology

Pharmacological characterization of GABAB receptor subtypes assembled with auxiliary KCTD subunits

https://doi.org/10.1016/j.neuropharm.2014.08.020Get rights and content
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Highlights

  • High affinity of native GABABRs for GABA is not caused by auxiliary KCTD subunits.

  • GS39783 increases potency and efficacy of GABA at GABABRs with and without KCTDs.

  • GS39783 is not selective for GABABR subtypes owing to KCTDs.

  • KCTD8 reduces tonic G-protein activation in the presence of GABABRs.

  • Overall, KCTDs exert marginal allosteric influences on principal GABABR subunits.

Abstract

GABAB receptors (GABABRs) are considered promising drug targets for the treatment of mental health disorders. GABABRs are obligate heteromers of principal GABAB1 and GABAB2 subunits. GABABRs can additionally associate with auxiliary KCTD8, 12, 12b and 16 subunits, which also bind the G-protein and differentially regulate G-protein signaling. It is unknown whether the KCTDs allosterically influence pharmacological properties of GABABRs. Here we show that KCTD8 and KCTD16 slightly but significantly increase GABA affinity at recombinant receptors. However, KCTDs clearly do not account for the 10-fold higher GABA affinity of native compared to recombinant GABABRs. The positive allosteric modulator (PAM) GS39783, which binds to GABAB2, increases both potency and efficacy of GABA-mediated G-protein activation ([35S]GTPγS binding, BRET between G-protein subunits), irrespective of whether KCTDs are present or not. Of note, the increase in efficacy was significantly larger in the presence of KCTD8, which likely is the consequence of a reduced tonic G-protein activation in the combined presence of KCTD8 and GABABRs. We recorded Kir3 currents to study the effects of GS39783 on receptor-activated G-protein βγ-signaling. In transfected CHO cells and cultured hippocampal neurons GS39783 increased Kir3 current amplitudes activated by 1 μM of baclofen in the absence and presence of KCTDs. Our data show that auxiliary KCTD subunits exert marginal allosteric influences on principal GABABR subunits. PAMs at principal subunits will therefore not be selective for receptor subtypes owing to KCTD subunits. However, PAMs can differentially modulate the responses of receptor subtypes because the KCTDs differentially regulate G-protein signaling.

This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’.

Keywords

GABA-B
G-protein-coupled receptor
GS93783
Baclofen
Kir3
KCTD

Abbreviations

3KO
Kctd8/12/16−/− triple knockout mice
A1
adenosine receptor 1
ANOVA
analysis of variance
BRET
bioluminescence resonance energy transfer
CHO cells
Chinese hamster ovary K1 cells
CHO-GABAB
CHO cells stably expressing GABAB(1b,2) receptors
CGP7930
2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol
ΔBRET
changes in BRET
EC50
half maximal effective concentration
EGFP
enhanced green fluorescent protein
Emax
maximum stimulatory effect
GABABR
GABAB receptor
GDP
guanosine 5'-diphosphate
GPCR
G-protein-coupled receptor
GS39783
N,N'-Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine
[35S]GTPγS
[35S]guanosine 5′-O-(3-thio)triphosphate
HEK293T cells
Human Embryonic Kidney 293T cells
HIV-1
human immunodeficiency virus-1
IC50
half maximal inhibitory concentration
KCTD
Potassium Channel Tetramerisation Domain
KH buffer
Krebs-Henseleit buffer
Kir
K+ inwardly rectifying
mBU
milli BRET units
PAM
positive allosteric modulator
Rluc
Renilla reniformis luciferase

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1

These authors contributed equally to this work.