Key residues defining the mu-opioid receptor binding pocket: A site-directed mutagenesis study

Structural elements of the rat mu-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the mu, delta, and kappa receptors (Asn(150) to Ala, His(297) to Ala, and Tyr(326) to Phe) and two designed to test for mu/delta selectivity (Ile(198) to Val and Val(202) to Ile). Mutation of His(297) in transmembrane domain 6 (TM6) resulted in no detectable binding with [H-3] DAMGO (H-3-labeled D-Ala(2), N-Me-Phe(4), Gly-ol(5)-enkephalin), [H-3]bremazocine, or [H-3]ethylketocyclazocine. Mutation of Asn(150) in TM3 produces a three- to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, beta-endorphin(1-31), JOM-13, deltorphin II, dynorphin(1-13), and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor-binaltorphamine. In contrast, the Tyr(326) mutation in TM7 resulted in a decreased affinity for a wide spectrum of mu, delta, and kappa agonists and antagonists. Altering Val(202) to lie in TM4 produced no change on ligand affinity, but Ile(198) to Val resulted in a four- to fivefold decreased affinity for the mu agonists morphine and DAMGO, with no change in the binding affinities of kappa and delta ligands.