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

Abstract

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.

Keywords: Irreversible binding; Kappa opioid receptor; Mu opioid receptor; β-funaltrexamine.

Fig. 1

Chemical structure of β-FNA.

Fig. 2. K303^6.58E mutation in the MOP receptor and E297^6.58K mutation in the KOP receptor did not change affinity of [³H]diprenorphine and β-FNA

(A) ([³H]DIP) saturation binding to wild type and mutant MOP and KOP receptors. Saturation binding of [³H]DIP, a nonselective opioid antagonist, to the receptors was performed using 50 μg of membrane prepared from stably transfected N2A cells. Nonspecific binding was defined using 10 μM naloxone. Shown is a representative saturation curve for each receptor. The mutations did not change [³H]DIP affinity for either KOP or MOP receptor mutant compared to the wild type. K_d and B_max values are shown in Table 1. (B) Inhibition of [³H]DIP binding to the wild type and mutated receptors by unlabeled β-FNA. Inhibition of [³H]DIP (~0.4 nM) binding by β-FNA was performed using 25–50 μg of membrane prepared from stably transfected N2A cells and varying concentrations (0.01 nM – 10 μM) of β-FNA. Nonspecific binding was defined using 10 μM naloxone. The data shown represent the mean ± S.E.M. of three independent experiments performed in duplicate. IC₅₀ values were determined and are shown in Table 1. Mutations in the MOP and KOP receptors did not affect β-FNA affinity for the receptors.

Fig. 3. Irreversible binding of [³H]β-FNA to wild type and mutant MOP and KOP receptors

Irreversible binding of [³H]β-FNA was performed with various concentrations of [³H]β-FNA and 100 μg of membranes prepared from stably transfected N2A cells at 37°C for 75 min. Following incubation, TCA was added to a final concentration of 10% to dissociate non-covalently bound [³H]β-FNA, centrifuged and resuspended in 10% TCA and samples were filtered over GF/B filters. The data shown are the total, nonspecific and specific irreversible binding from one of three experiments performed at each receptor. Wild type MOP receptor shows a high level of irreversible binding while K303^6.58E MOP receptor shows a reduced level of irreversible binding. Wild type KOPR and E297^6.58K KOPR do not show any irreversible binding. Note the difference in the Y-axis scale of the MOP receptor figure. The percentage of irreversible binding as compared to total binding is shown in Table 1.

Fig. 4. Reversible binding of [³H]β-FNA to wild type and mutant MOP and KOP receptors

Total (reversible plus irreversible) binding of [³H]β-FNA was performed alongside irreversible binding on 100 μg of membranes prepared from stably transfected N2A cells. Reversible binding was calculated by subtracting the irreversible binding from the total binding. Representative data from one of three experiments on each construct is shown. All 4 receptors bind [³H]β-FNA with similar affinity, with K_d values shown in Table 1.

Fig. 5. Specific binding of [³H]β-FNA to wild type and mutant MOP and KOP receptors

Data from Fig. 3 and Fig. 4 are replotted to show only specific binding [Total (reversible + irreversible), reversible, irreversible]. Wild type MOP receptor shows an appreciable level of irreversible binding, with the K303^6.58E MOP receptor mutant showing a diminished level of irreversible binding. Wild type and mutant E297^6.58K KOPR do not show any irreversible binding. Representative data from one of three experiments on each receptor is shown.

Fig. 6. E297^6.58K mutation in the KOP receptor and K303^6.58E mutation in the MOP receptor did not change the efficacy of β-FNA

Agonist activity was assessed using [³⁵S]GTPγS (0.08–0.1 nM) binding with different concentrations of β-FNA, 5 μM GDP and 5 μg membrane prepared from stably transfected N2A cells. Nonspecific binding was defined with 1 μM unlabeled GTPγS. Basal levels were subtracted from each datum and the percentage (%) stimulation over basal was calculated. The basal level of binding was 0.4–0.7 fmol/5 μg membrane protein (1100–2000 dpm/tube). Shown here is the mean ± S.E.M. of three independent experiments performed in duplicate at the wild type and mutant receptors. The E_max and EC₅₀ values for wild type KOPR and E297^6.58K KOPR are shown in Table 1. The E_max values were ~64% of those of U50,488H. At the MOP receptor, β-FNA up to 10 μM did not activate either the wildtype or mutant receptor, whereas DAMGO at 10 μM increased [³⁵S]GTPγS binding to 206% and 190% of the basal level, respectively (not shown).

Fig. 7. Proposed interaction between residues in MOP receptor and β-FNA

(A) The methyl acetate group of β-FNA interacts with K303^6.58 in the wild type receptor which positions the double bond in the fumarate in the vicinity of the ε-amino group of K233^5.39 for a covalent bond formation. (B) When K303^6.58 is mutated to a negatively-charged glutamic acid (E303^6.58), there is a repulsion, which changes the position of the double bond in the fumarate in β-FNA preventing a covalent bond formation.