Molecular Mechanism Study Of Agonist Effects Of GABA_B Receptor Positive Allosteric Modulators

GABA_B receptor is the metabotropic receptor of GABA,the main inhibitory neurotransmitter in the central nervous system,and belongs to the class C G protein-coupled receptors?GPCRs?superfamily.It is a heterodimer consisting of the GABA_B1 and GABA_B2 subunits,in which the GABA_B1 subunit is responsible for the binding of orthosteric ligands and the GABA_B2 subunit takes charge for the downstream G protein coupling.GABA_B receptors are broadly expressed and distributed in the central nervous system and their dysfunctions are associated with many neurological and neuropsychiatric disorders.Currently,the only drug developed and marketed for GABA_B receptor is Baclofen,which is an orthosteric agonist of the receptor,and it exerts efficacies against various diseases such as muscle spasms and alcohol dependence in clinical studies.However,Baclofen shows side effects such as sedation,muscle relaxation and decreased body temperature during physiological usage.Allosteric modulators interact with the sites that are topologically different from the orthosteric sites within GABA_B receptor and they can synergistically improve the activities of the receptor induced by orthosteric ligands with better selectivity,which provide new possibilities for drug development and disease treatment.Good progress has been made for the specific positive allosteric modulators?PAMs?of GABA_B receptor and many of them,including CGP7930,GS39783 and rac-BHFF have shown characteristic activities in different functional assays in vitro and animal models in vivo.In addition,some PAMs also exhibit intrinsic agonist effects and provide new options for the regulation of GABA_B receptor.However,the detailed molecular mechanism about how the ago-PAMs with intrinsic agonist effects activate GABA_B receptor alone is largely unknown right now.In this study,three typical PAMs of GABA_B receptor were used as basic tools.In vitro functional assays were performed to investigate the activity characteristics of these compounds and their interaction sites in GABA_B receptor were further verified.The intrinsic agonist activity of rac-BHFF was confirmed by different functional assays as well.Basing on the three-dimensional homology modeling,the potential regions that might involve in the agonist activity of rac-BHFF were predicted,which were further confirmed by site-directed mutagenesis.We found a novel functional region within the seven transmembrane domain of the GABA_B2 subunit,which could significantly reduce the agonist effects of rac-BHFF and participate in the regulation of the constitutive activity of GABA_B receptor.In addition,we also found that the seven transmembrane domain of the GABA_B1 subunit could affect the agonist activity of rac-BHFF directly.The study of the agonist effects of positive allosteric modulators could facilitate the investigation of the GABA_B receptor activation mechanism and the development of novel allosteric modulators.