Abstract

The κ-opioid receptor (κOR) represents a promising non-addictive analgesic target due to its critical role in pain and reward pathways. Despite evidence of κOR dimerization, its molecular basis and pharmacological significance remain elusive. Here, we demonstrate stable κOR dimer formation in living cells and present cryo-electron microscopy structures of salvinorin A-bound κOR dimer complexed with two G i proteins, revealing a parallel assembly distinct from previously characterized GPCR dimers that engage only single G protein. Multiple membrane lipids are positioned at the TM1-Helix 8 interface, where they make extensive contacts with both protomers and may contribute to the stability of the κOR dimer. Importantly, dimerization significantly enhances G i protein recruitment to κOR in both potency and efficacy. We also demonstrate that salvinorin A, a non-nitrogenous agonist, binds similarly in monomeric and dimeric κOR, and identify Y312 7.35 as a critical selectivity determinant across opioid receptors. These findings expand our understanding of opioid receptor pharmacology and signaling, providing a foundation for developing superior κOR-targeted therapeutics for pain and related disorders.