Construction of a high affinity zinc switch in the kappa-opioid receptor.

Very limited structural information is available concerning the superfamily of G-protein-coupled receptors with their seven-transmembrane segments. Recently a non-peptide antagonist site was structurally and functionally replaced by a metal ion site in the tachykinin NK-1 receptor. Here, this Zn(II) site is transferred to the kappa-opioid receptor by substituting two residues at the outer portion of transmembrane V (TM-V), Asp223 and Lys227, and one residue at the top of TM-VI, Ala298, with histidyl residues. The histidyl residues had no direct effect on the binding of either the non-peptide antagonist [3H]diprenorphine or the non-peptide agonist, [3H]CI977, just as these mutations/substitutions did not affect the apparent affinity of a series of other peptide and non-peptide ligands when tested in competition binding experiments. However, zinc ions in a dose-dependent manner prevented binding of both agonist and antagonist ligands with an apparent affinity for the metal ion, which gradually was built up to 10(-6) M. This represents an increase in affinity for the metal ion of about 1000-fold as compared with the wild-type kappa receptor and is specific for Zn(II) as the affinity for e.g. Cu(II) was almost unaffected. The direct transfer of this high affinity metal ion switch between two only distantly related receptors indicates a common overall arrangement of the seven-helix bundle among receptors of the rhodopsin family.