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 Lys233(5.39), which is conserved among opioid receptors. Molecular docking of β-FNA showed that K303(6.58) in the MOP receptor and E297(6.58) in the KOP receptor played distinct roles in positioning β-FNA. K303(6.58)E MOP receptor and E297(6.58)K KOP receptor mutants were generated. The mutations did not affect β-FNA affinity or efficacy. K303(6.58)E mutation in the MOP receptor greatly reduced covalent binding of [(3)H]β-FNA; however, E297(6.58)K did not enable the KOP receptor to bind irreversibly to β-FNA. Molecular modeling demonstrated that the ε-amino group of K303(6.58) in the MOP receptor interacted with CO of the acetate group of β-FNA to facilitate covalent bond formation with Lys233(5.39). Replacement of K303(6.58) 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 K233(5.39) 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.