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Background and Purpose: Altered glutamatergic neurotransmission is linked to several neurological and psychiatric disorders. Metabotropic glutamate receptor 2 (mGlu₂) plays an important role on the presynaptic control of glutamate release and negative allosteric modulators (NAMs) acting on mGlu₂/₃ receptors are under assessment for their potential as antidepressants, neurogenics and cognitive enhancers. Two new potent mGlu₂/₃ NAMs, RO4988546 and RO5488608, are described in this study and the allosteric binding site in the transmembrane (TM) domain of mGlu₂ is characterized.Experimental Approach: Site directed mutagenesis, functional measurements and β₂-adrenoceptor-based modelling of mGlu₂ were employed to identify important molecular determinants of two new potent mGlu₂/₃ NAMs.Key Results: RO4988546 and RO5488608 affected both [³H]-LY354740 agonist binding at the orthosteric site and the binding of a tritiated positive allosteric modulator (³H-PAM), indicating that NAMs and PAMs could have overlapping binding sites in the mGlu₂ TM domain. We identified eight residues in the allosteric binding pocket that are crucial for non-competitive antagonism of agonist-dependent activation of mGlu₂ and directly interact with the NAMs: Arg³·²⁸, Arg³·²⁹, Phe³·³⁶, His(E2.52) , Leu⁵·⁴³, Trp⁶·⁴⁸, Phe⁶·⁵⁵ and Val⁷·⁴³. The mGlu₂ specific residue His(E2.52) is likely to be involved in selectivity and residues located in the outer part of the binding pocket are more important for [³H]-LY354740 agonist binding inhibition, which is independent of the highly conserved Trp⁶·⁴⁸ residue.Conclusions and Implications: This is the first complete molecular investigation of the allosteric binding pocket of mGlu₂ and Group II mGluRs and provides new information on what determines mGlu₂ NAMs selective interactions and effects. [ABSTRACT FROM AUTHOR]