The action of a negative allosteric modulator at the dopamine D receptor is dependent upon sodium ions.

Sodium ions (Na) allosterically modulate the binding of orthosteric agonists and antagonists to many class A G protein-coupled receptors, including the dopamine D receptor (DR). 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 DR 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 DR, providing opportunities for fine-tuning of modulatory effects in future allosteric drug design efforts.