Received for publication June 22, 2006.
Revised September 10, 2006.
Accepted for publication September 11, 2006.

Point mutations in the transmembrane region of GABA_B2 facilitate activation by the positive modulator GS39783 in the absence of the GABA_B1 subunit

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 as they lack the sedative and muscle relaxant properties of agonists. In this study we aimed to characterize the interaction of the positive modulator GS39783 (N,N'-Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine) with the GABA_B receptor heterodimer. Using functional GTPS binding assays we observed positive modulation by GS39783 in different vertebrate species but not in drosophila. However, co-expression of drosophila GB1 with rat GB2 yielded functional receptors positively modulated by GS39783. Together with data from rat/drosophila 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 acids substitutions in transmembrane domain VI. Surprisingly, 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.

Key words: GABAB, Gi family, Structure-activity relationships and modeling