Mutation of a conserved serine in TM4 of opioid receptors confers full agonistic properties to classical antagonists

The involvement of a conserved serine (Ser¹⁹⁶ at the μ-, Ser¹⁷⁷ at the δ-, and Ser¹⁸⁷ at the κ-opioid receptor) in receptor activation is demonstrated by site-directed mutagenesis. It was initially observed daring our functional screening of a μ/δ-opioid chimeric receptor, μδ₂, that classical opioid antagonists such as naloxone, naltrexone, naltriben, and H- Tyr-Tic[ψ,CH₂NH]Phe-Phe-OH (TIPPψ; Tic = 1,2,3,4-tetrahydroisoquinoline- 3-carboxylic acid) could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing the chimeric receptor. Antagonists also activated the G protein-coupled inward rectifying potassium channel (GIRK1) in Xenopus oocytes coexpressing the μδ2 opioid receptor and the GIRK1 channel. By sequence analysis and bach mutation, it was determined that the observed antagonist activity was due to the mutation of a conserved serine to leucine in the fourth transmembrane domain (S196L). The importance of this serine was further demonstrated by analogous mutations created in the μ-opioid receptor (MORS196L) and δ-opioid receptor (DORS177L), in which classical opioid antagonists could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing either MORS196L or DORS177L. Again, antagonists could activate the GIRK1 channel coexpressed with either MORS196L or DORS177L in Xenopus oocytes. These data taken together suggest a crucial role for this serine residue in opioid receptor activation.