AbstractSodium ions (Na⁺) allosterically modulate the binding of orthosteric agonists and antagonists to many class A G protein-coupled receptors, including the dopamine D₂ receptor (D₂R). Experimental and computational evidences have revealed that this effect is mediated by the binding of Na⁺ to a conserved site located beneath the orthosteric binding site (OBS). SB269652 acts as a negative allosteric modulator (NAM) of the D₂R that adopts an extended bitopic pose, in which the tetrahydroisoquinoline moiety interacts with the OBS and the indole-2-carboxamide moiety occupies a secondary binding pocket (SBP). In this study, we find that the presence of a Na⁺ within the conserved Na⁺-binding pocket is required for the action of SB269652. Using fragments of SB269652 and novel full-length analogues, we show that Na⁺ is required for the high affinity binding of the tetrahydroisoquinoline moiety within the OBS, and that the interaction of the indole-2-carboxamide moiety with the SBP determines the degree of Na⁺-sensitivity. Thus, we extend our understanding of the mode of action of this novel class of NAM by showing it acts synergistically with Na⁺ to modulate the binding of orthosteric ligands at the D₂R, providing opportunities for fine-tuning of modulatory effects in future allosteric drug design efforts.