Point mutations in the transmembrane region of GABAB2 facilitate activation by the positive modulator N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) in the absence of the GABAB1 subunit.

Full Abstract

GABA(B) receptors are heterodimers of two subunits, GABA(B1) (GB1) and GABA(B2) (GB2). Agonists such as GABA and baclofen bind to the GB1 subunit only, whereas GB2 is essential for G protein activation. Positive allosteric modulators enhance the potency and efficacy of agonists at GABA(B) receptors and are of particular interest because they lack the sedative and muscle relaxant properties of agonists. In this study, we aimed to characterize the interaction of the positive modulator N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) with the GABA(B) receptor heterodimer. Using functional guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays, we observed positive modulation by GS39783 in different vertebrate species but not in Drosophila melanogaster. However, coexpression of D. melanogaster GB1 with rat GB2 yielded functional receptors positively modulated by GS39783. Together with data from rat/D. melanogaster GB2 subunit chimeras, this pointed to a critical role of the GB2 transmembrane region for positive modulation. We further characterized GS39783 function using point mutations. GS39783 positively modulated GABA responses but also showed considerable agonistic activity at heterodimers containing a mutant rat GB2 subunit with three amino acid substitutions in transmembrane domain VI. It was surprising that in contrast to wild-type rat GB2, this mutant subunit was also activated by GS39783 when expressed without GB1. The mutations of both G706T and A708P are necessary and sufficient for activation and identify a key region for the effect of GS39783 in the GB2 transmembrane region. Our data show that mutations of specific amino acids in GB2 can induce agonism in addition to positive modulation and facilitate GB2 activation in the absence of GB1.

Author information

Author/s: Dupuis, Delphine S (DS); Relkovic, Dinko (D); Lhuillier, Loic (L); Mosbacher, Johannes (J); Kaupmann, Klemens (K);

Affiliation: Neuroscience Research, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.

Grants: U01-MH60962 (Agency:NIMH NIH HHS)

Journal and publication information

Publication Type: Journal Article; Research Support, N.I.H., Extramural

Journal: Molecular pharmacology (Mol Pharmacol), published in United States. (Language: eng)

Reference: 2006-Dec; vol 70 (issue 6) : pp 2027-36

Dates: Created 2006/11/19; Completed 2007/01/30; Revised 2008/11/20;

PMID: 16966477, status: MEDLINE (last retrieval date: 2/18/2009, IMS Date: )

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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MeSH headings (categories)

This article was linked to the MESH Headings shown below.

Drosophila melanogaster
Molecular Sequence Data
Point Mutation
Pyrimidines - pharmacology
Rats
Receptors, GABA-B - agonists; chemistry; genetics; metabolism
Sequence Homology, Amino Acid

Associated Chemicals: Cyclopentanes (0) ; DNA Primers (0) ; N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (0) ; Pyrimidines (0) ; Receptors, GABA-B (0)

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