The mu-opioid receptor is the principal site of action in the brain by which morphine, other opiate drugs of abuse, and endogenous opioid peptides effect analgesia and alter mood. A member of the seven-transmembrane domain (TM) G protein-coupled receptor (GPCR) superfamily, the mu-opioid receptor modulates ion channels and second messenger effectors in an opioid agonist-dependent fashion that is reversible by the classic opiate antagonist naloxone. Mutation of a histidine residue (His297) in TM 6 afforded agonist-like G protein-coupled signal transduction mediated by naloxone and other alkaloid antagonists and enhanced the intrinsic activity of documented alkaloid partial agonists, including buprenorphine. The intrinsic activities of all opioid peptide agonists and antagonists tested were not altered at the His297 mutant receptors. Consistent with a role for the TM 6 histidine in maintaining high affinity binding sites for opioid agonists and antagonists, opioid ligand-dependent protection of this residue from a histidine-specific alkylating agent indicated that the His297 side chain is positioned in or very near the binding cavity. The TM 6 His297 mutants identify a discrete region of the receptor critical for determining whether a specific drug pharmacophore triggers receptor activation. Because many GPCRs possess a similarly positioned TM histidine residue, our findings with the mu-opioid receptor may extend to these receptors and potentially serve as a model for rational design of therapeutic GPCR partial agonists and antagonists.

Cells grown in monolayer culture offer a convenient system for binding and other experiments under conditions that preserve the complexity of the living state. Kinetics experiments, however, may be distorted by the time course of drug penetration into even so simple a "tissue" as the monolayer. The impediments include unstirred layers both above and between the cells, the congregation of receptors within the confined space between cells, and nonspecific binding to membrane components. The contributions of these factors were investigated in cultures of Chinese hamster ovary (CHO) cells either nontransfected or stably transfected with mu opioid receptors. The dissociation of [3H]naloxone was four times faster under displacement than under infinite dilution conditions, clearly demonstrating the "retention effect" of receptors confined in space. Even the penetration of this ligand between nontransfected cells showed salient delays with respect to diffusion into a slab, indicating that nonspecific, low-affinity binding to membrane components was arresting its progress. The optical sectioning capabilities of confocal microscopy demonstrated that the kinetics of two fluorescent antagonists depended on the vertical plane, providing direct evidence for slowed diffusion down a single cell depth. Modeling shows that kinetic errors increase with receptor density, forward rate constant, and the thickness of the unstirred layer.

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