CONVERSION OF ANTAGONIST-BINDING SITE TO METAL-ION SITE IN THE TACHYKININ NK-1 RECEPTOR

MUTATIONAL analysis of the tachykinin NK-1 (refs 1-7), NK-2 (ref. 8) a nd angiotensin AT-1 (refs 9, 10) receptors indicates that non-peptide antagonists act through residues located between the seven transmembra ne segments, whereas natural peptide agonists bind to residues scatter ed in the exterior part of the receptor(1-4,11-13). The presumed conta ct points for the prototype NK-1 antagonist CP96,345 cluster on opposi ng faces of the outer portions of transmembrane helices V and VI (refs 1-5). Here we show that systematic introduction of histidyl residues at this antagonist-binding site in the human NK-1 receptor gradually c onverts it into a high-affinity metal-ion-binding site without affecti ng agonist binding. In a double mutant with histidine residues substit uted at the top of transmembrane segments V and VI, respectively, Zn2 inhibits binding of radiolabelled agonist peptide and efficiently blo cks phosphoinositol turnover induced by substance P. We propose that Z n2+ and CP96,345 act as 'allosteric competitive' antagonists by stabil izing inactive conformations of the mutant and the wild-type receptor respectively. Introduction of metal-ion-binding sites could be used as a general tool in the structural and functional characterization of h elix-helix interactions in G-protein-coupled receptors, as well as in other membrane proteins.