Key Residues Defining the μ‐Opioid Receptor Binding Pocket: A Site‐Directed Mutagenesis Study

Abstract:Structural elements of the rat μ‐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 μ, δ, and κ receptors (Asn150to Ala, His297to Ala, and Tyr326to Phe) and two designed to test for μ/δ selectivity (Ile198to Val and Val202to Ile). Mutation of His297in transmembrane domain 6 (TM6) resulted in no detectable binding with [3H]DAMGO (3H‐labeled d‐Ala2,N‐Me‐Phe4,Gly‐ol5‐enkephalin), [3H]bremazocine, or [3H]ethylketocyclazocine. Mutation of Asn150in TM3 produces a three‐ to 20‐fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, β‐endorphin1–31, JOM‐13, deltorphin II, dynorphin1–13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor‐binaltorphamine. In contrast, the Tyr326mutation in TM7 resulted in a decreased affinity for a wide spectrum of μ, δ, and κ agonists and antagonists. Altering Val202to Ile in TM4 produced no change on ligand affinity, but Ile198to Val resulted in a four‐ to fivefold decreased affinity for the μ agonists morphine and DAMGO, with no change in the binding affinities of κ and δ ligands.