Bupropion Inhibits Serotonin Type 3AB Heteromeric Channels at Clinically Relevant Concentrations

Abstract

Bupropion, a Food and Drug Administration-approved antidepressant and smoking cessation aid, blocks dopamine and norepinephrine reuptake transporters and noncompetitively inhibits nicotinic acetylcholine and serotonin (5-HT) type 3A receptors (5-HT_3ARs). 5-HT₃ receptors are pentameric ligand-gated ion channels that regulate synaptic activity in the central and peripheral nervous system, presynaptically and postsynaptically. In the present study, we examined and compared the effect of bupropion and its active metabolite hydroxybupropion on mouse homomeric 5-HT_3A and heteromeric 5-HT_3AB receptors expressed in Xenopus laevis oocytes using two-electrode voltage clamp experiments. Coapplication of bupropion or hydroxybupropion with 5-HT dose dependently inhibited 5-HT-induced currents in heteromeric 5-HT type 3AB receptors (5-HT_3ABRs) (IC₅₀ = 840 and 526 μM, respectively). The corresponding IC₅₀s for bupropion and hydroxybupropion for homomeric 5-HT_3ARs were 10- and 5-fold lower, respectively (87 and 113 μM). The inhibition of 5-HT_3ARs and 5-HT_3ABRs was non-use dependent and voltage independent, suggesting bupropion is not an open channel blocker. The inhibition by bupropion was reversible and time-dependent. Of note, preincubation with a low concentration of bupropion that mimics therapeutic drug conditions inhibits 5-HT-induced currents in 5-HT_3A and 5-HT_3AB receptors considerably. In summary, we demonstrate that bupropion inhibits heteromeric 5-HT_3ABRs as well as homomeric 5-HT_3ARs. This inhibition occurs at clinically relevant concentrations and may contribute to bupropion's clinical effects. SIGNIFICANCE STATEMENT: Clinical studies indicate that antagonizing serotonin (5-HT) type 3AB (5-HT_3AB) receptors in brain areas involved in mood regulation is successful in treating mood and anxiety disorders. Previously, bupropion was shown to be an antagonist at homopentameric 5-HT type 3A receptors. The present work provides novel insights into the pharmacological effects that bupropion exerts on heteromeric 5-HT_3AB receptors, in particular when constantly present at low, clinically attainable concentrations. The results advance the knowledge on the clinical effects of bupropion as an antidepressant.

Graphical abstract

Fig. 1.

Comparing 5-HT_3ARs to 5-HT_3ABRs. (A) Sample traces of 5-HT_3A (black) and 5-HT_3AB (green) at varying concentrations of 5-HT. (B) Concentration-response curves show a higher potency of 5-HT at 5-HT_3ARs as compared with 5-HT_3AB, as well as a steeper Hill slope. Parameters from these curves: 5-HT_3A: EC₅₀ = 0.8 µM, n_H = 2.53 ± 0.58, n = 5, 5-HT_3AB: EC₅₀ = 4.30 µM, n_H = 1.04 ± 0.02, n = 8. Data are represented as the mean ± S.D. (C) Direct comparison of 5-HT_3A and 5-HT_3AB inward current evoked by 1 μM 5-HT for 30 seconds.

Fig. 2.

Bupropion’s antagonistic activity at homomeric and heteromeric 5-HT₃Rs. (A) Sample traces of oocytes expressing 5-HT_3A or 5-HT_3AB in response to 5-HT (∼EC₃₀) alone and in combination with bupropion. 5-HT–evoked inward currents (gray, 5-HT_3A = 0.3 μM, 5-HT_3AB = 2 μM) were used for the control current. Following, the 5-HT concentration was kept constant and coapplied with increasing concentrations of bupropion (5-HT_3A: 10–1000 μM, 5-HT_3AB: 30–4000 μM). (B) Currents were normalized to the control currents and yielded the following IC₅₀ values: 5-HT_3A: IC₅₀ = 87.1 µM (n_H = 1.28 ± 0.15, n = 5, mean ± S.D.) and 5-HT_3AB: IC₅₀ = 840 µM (n_H = 1.78 ± 0.15, n = 7, mean ± S.D.). (C) Oocytes expressing 5-HT_3A and 5-HT_3AB did not elicit an inward current in response to bupropion alone.

Fig. 3.

Hydroxybupropion is an antagonist for 5-HT_3ARs and 5-HT_3ABRs. (A) Sample traces of oocytes expressing 5-HT_3A or 5-HT_3AB in response to 5-HT (∼EC₃₀) alone and in combination with hydroxybupropion. 5-HT–evoked inward currents (gray, 5-HT_3A = 0.3 μM, 5-HT_3AB = 2 μM) were used for the control current. Following, the 5-HT concentration was kept constant and coapplied with increasing concentrations of hydroxybupropion (5-HT_3A: 10–1000 μM, 5-HT_3AB: 50–2000 μM). (B) Currents were normalized to the control currents and yielded the following IC₅₀ values: 5-HT_3A: IC₅₀ = 113 µM (n_H = 1.17 ± 0.15, n = 5, mean ± S.D.) and 5-HT_3AB: IC₅₀ = 526 µM (n_H = 1.80 ± 0.16, n = 8, mean ± S.D.). (C) Oocytes expressing 5-HT_3A and 5-HT_3AB did not elicit an inward current in response to hydroxybupropion alone.

Fig. 4.

Non–use dependent allosteric inhibition of 5-HT_3ARs and 5-HT_3ABRs. (A) Sample traces of oocytes expressing 5-HT_3A (black, left panel) and 5-HT_3AB (green, right panel). The first 5-HT–evoked currents were used for the control currents (gray bars, ∼EC₃₀, 5-HT_3A: 0.5 µM, 5-HT_3AB: 2 µM) that were obtained by coapplication with bupropion (magenta bars, ∼IC₅₀, 5-HT_3A: 100 µM, 5-HT_3AB: 1 mM). Following the stable 5-HT response, bupropion (∼EC₅₀) was perfused for 5 min before another coapplication of 5-HT and bupropion. (B) Same experimental design as in (A) but with hydroxybupropion (blue bars, ∼IC₅₀, 5-HT_3A: 100 µM, 5-HT_3AB: 500 µM). (C) Quantification of fractional inhibition of currents when the oocyte was preincubated in bupropion (magenta) or hydroxybupropion (blue) normalized to the control current (100%). Preincubation reduced current amplitudes for 5-HT_3A (Bup: 76.1% ± 7.16%, n = 5; HydroB: 93.0% ± 6.12%, n = 6) and 5-HT_3AB (Bup: 35.5% ± 5.62%, n = 6; HydroB: 46.1% ± 4.95%, n = 4) as compared with coapplication. Statistical significance was determined with paired t test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001), comparing coapplication (representing 100% of the current) to preapplication + coapplication (Pre+Co) with each drug independently. Data are represented as the mean ± S.D.

Fig. 5.

Recovery times for bupropion. (A and B) Sample traces of bupropion application (magenta bar) and the recovery times for 5-HT_3A (left panel, black) and 5-HT_3AB (right panel, green). (A) In two-electrode voltage clamp experiments, oocytes expressing 5-HT_3A and 5-HT_3AB showed a stable response to repeated applications of 0.8 and 5 μM 5-HT at −60 mV, with an approximate wash time of 2 min. (B) The first 5-HT–evoked response represents the control current for the recovery experiment. Bupropion (400 μM) was applied alone for 60 s at −60 mV, followed by an immediate application of 5-HT. The gray and magenta bars represent the time of application of 5-HT and bupropion, respectively. Moving down the panel, the wash times after bupropion application were 0, 30, and 60 s. (C) Quantitative representation of current amplitudes and results in (B) (n = 3). 5-HT_3A was maximally reduced to 82.4% ± 3.08% and 5-HT_3AB to 38.4% ± 15.8% of the control current when the agonist was applied immediately after bupropion, followed by a stepwise recovery. All currents could be recovered to ∼95% after ∼7.5-min wash. Statistical significance of each wash time as compared with the control current (the 5-HT–induced current response before exposure to Bup or HydroB) was determined with one-way ANOVA, Dunnett’s multiple comparisons test (*P ≤ 0.05; ***P ≤ 0.001). Data are represented as the mean ± S.D.

Fig. 6.

Voltage-independent block of 5-HT₃–mediated currents by bupropion. (A) Sample traces of 5-HT_3A– and 5-HT_3AB–expressing oocytes (5-HT_3A: left, black; 5-HT_3AB: right, green) in response to 5-HT (∼EC₅₀; top and bottom traces, 5-HT_3A: 0.8 µM; 5-HT_3AB: 5.0 µM) in the absence and presence of bupropion (magenta traces, ∼IC₅₀; 5-HT_3A: 100 µM; 5-HT_3AB: 1 mM) at different voltages. (B) Quantification of fractional inhibition, currents were normalized to the control currents at each voltage (n = 4). Data are shown as mean ± S.D. Statistical significance between the inhibition at positive and negative voltages was determined with paired t test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Fig. 7.

Bupropion at clinically achievable concentrations and its effect on 5-HT_3ARs and 5-HT_3ABRs. (A) Sample traces of oocytes expressing 5-HT_3A (black, left panel) and 5-HT_3AB (green, right panel) in response to 0.5, 1.0, and 5.0 μM 5-HT (gray bars) followed by the same concentrations coapplied with 20 μM bupropion. Following the initial exposure to the three 5-HT concentrations (control current), the oocytes were exposed to 20 μM bupropion for at least 2 min before coapplication with the agonist. (B) Quantitative representation of current amplitudes and results in (A) (A: n = 4, AB: n = 5). Data are shown as mean ± S.D. Statistical significance between each 5-HT concentration without and with bupropion at the same 5-HT concentration was determined with paired t test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).