Studies on inhibition of mu and delta opioid receptor binding by dithiothreitol and N-ethylmaleimide. His223 is critical for mu opioid receptor binding and inactivation by N-ethylmaleimide

J Biol Chem. 1996 Mar 8;271(10):5505-12. doi: 10.1074/jbc.271.10.5505.


The sensitivity of mu and delta receptor binding to dithiothreitol and N-ethylmaleimide was examined to probe receptor structure and function. Binding to both receptor types was inhibited by dithiothreitol (IC50 values = 250 mM), suggesting the presence of inaccessible but critical disulfide linkages. mu receptor binding was inhibited with more rapid kinetics and at lower N-ethylmaleimide concentrations than delta receptor binding. Ligand protection against N-ethylmaleimide inactivation suggested that alkylation was occurring within, or in the vicinity of, the receptor binding pocket. Sodium ions dramatically affected the IC50 of N-ethylmaleimide toward both receptor types in a ligand-dependent manner. Analysis of receptor chimeras suggested that the site of N-ethylmaleimide alkylation on the mu receptor was between transmembrane domains 3 and 5. Substitution of cysteines between transmembrane domains 3 and 5 and elsewhere had no effect on receptor binding or sensitivity toward N-ethylmaleimide. Serine substitution of His223 in the putative second extracellular loop linking transmembrane domains 4 and 5 protected against N-ethylmaleimide inactivation. The H223S substitution decreased the affinity of bremazocine 25-fold, highlighting the importance of this residue for the formation of the high affinity bremazocine binding site in the mu opioid receptor.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Analgesics / metabolism
  • Base Sequence
  • Binding Sites
  • Cell Line
  • Cell Membrane / metabolism
  • DNA Primers
  • Dithiothreitol / pharmacology*
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
  • Enkephalin, Leucine / analogs & derivatives
  • Enkephalin, Leucine / metabolism
  • Enkephalins / metabolism
  • Ethylmaleimide / pharmacology
  • Histidine*
  • Humans
  • Kidney
  • Kinetics
  • Ligands
  • Models, Structural
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Point Mutation
  • Polymerase Chain Reaction
  • Protein Structure, Secondary*
  • Receptors, Opioid, delta / antagonists & inhibitors*
  • Receptors, Opioid, delta / chemistry*
  • Receptors, Opioid, mu / antagonists & inhibitors*
  • Receptors, Opioid, mu / chemistry*
  • Recombinant Fusion Proteins / antagonists & inhibitors
  • Recombinant Fusion Proteins / chemistry
  • Serine
  • Transfection


  • Analgesics
  • DNA Primers
  • Enkephalins
  • Ligands
  • Receptors, Opioid, delta
  • Receptors, Opioid, mu
  • Recombinant Fusion Proteins
  • Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
  • Serine
  • Histidine
  • Enkephalin, Leucine
  • enkephalin, Ser(2), Leu(5), Thr(6)-
  • Ethylmaleimide
  • Dithiothreitol