CONSTRUCTION OF A HIGH-AFFINITY ZINC SWITCH IN THE KAPPA-OPIOID RECEPTOR

Very limited structural information is available concerning the superf amily of G-protein-coupled receptors with their seven-transmembrane se gments. Recently a non-peptide antagonist site was structurally and fu nctionally replaced by a metal ion site in the tachykinin NK-1 recepto r. 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), Asp(223) and Lys(227), and one residue at the top of TM-VI, Ala (298), with histidyl residues. The histidyl residues had no direct eff ect on the binding of either the non-peptide antagonist [H-3]diprenorp hine or the non-peptide agonist, [H-3]CI977, just as these mutations/s ubstitutions did not affect the apparent affinity of a series of other peptide and non-peptide ligands when tested in competition binding ex periments. However, zinc ions in a dose-dependent manner prevented bin ding of both agonist and antagonist ligands with an apparent affinity for the metal ion, which gradually was built up to 10(-6) M. This repr esents 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 tra nsfer of this high affinity metal ion switch between two only distantl y related receptors indicates a common overall arrangement of the seve n-helix bundle among receptors of the rhodopsin family.