ANALYSIS OF SELECTIVE BINDING EPITOPES FOR THE KAPPA-OPIOID RECEPTOR ANTAGONIST NOR-BINALTORPHIMINE

The structural determinants for the selective binding of the nonpeptid e opioid receptor antagonist nor-binaltorphimine (nor-BNI) to the kapp a-opioid receptor were characterized using a systematic series of chim eras between the kappa receptor and the homologous mu-opioid receptor. All 10 chimeric constructs bound the nonselective antagonists (-)-nal oxone and diprenorphine with similar affinities, as did the two wild-t ype receptors. Introduction of amino-terminal segments of increasing l ength, extending to and including transmembrane segment VI, from the m u receptor into the kappa receptor did not impair the high affinity bi nding of nor-BNI, and neither did introduction of the intracellular ca rboxyl-terminal extension of the mu receptor. In contrast, nor-BNI bin ding was impaired greater than or equal to 600-fold in constructs in w hich extracellular loop 3 and transmembrane segment VII originated fro m the mu receptor. The exchange of a single residue within this region , Glu(297), for lysine, the corresponding residue from the mu receptor , reduced the binding affinity of nor-BNI 142-fold, without affecting the binding of the nonselective compounds (-)-naloxone and diprenorphi ne. It is concluded that the selective binding of nor-BNI to the kappa -opioid receptor is determined by nonconserved residues located in ext racellular loop 3 and transmembrane segment VII and that Glu(297), loc ated just outside transmembrane segment VI, plays a major role in the kappa-selective binding characteristics of nor-BNI.