K3036.58 in the μ opioid (MOP) receptor is important in conferring selectivity for covalent binding of β-funaltrexamine (β-FNA)

β-funaltrexamine (β-FNA) is an irreversible μ opioid (MOP) receptor antagonist and a reversible agonist of κ opioid (KOP) receptor. β-FNA binds covalently to the MOP receptor at Lys2335.39, which is conserved among opioid receptors. Molecular docking of β-FNA showed that K3036.58 in the MOP receptor and E2976.58 in the KOP receptor played distinct roles in positioning β-FNA. K3036.58E MOP receptor and E2976.58K KOP receptor mutants were generated. The mutations did not affect β-FNA affinity or efficacy. K3036.58E mutation in the MOP receptor greatly reduced covalent binding of [3H]β-FNA; however, E2976.58K did not enable the KOP receptor to bind irreversibly to β-FNA. Molecular modeling demonstrated that the ε-amino group of K3036.58 in the MOP receptor interacted with CO of the acetate group of β-FNA to facilitate covalent bond formation with Lys2335.39. Replacement of K3036.58 with Glu in the MOP receptor resulted in repulsion between the COOH of Glu and the CO of β-FNA and increased the distance between K2335.39 and the fumarate group, making it impossible for covalent bond formation. These findings will be helpful for design of selective non-peptide MOP receptor antagonists.