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Fig. 1. Comparing 5-HT3ARs to 5-HT3ABRs. (A) Sample traces of 5-HT3A (black) and 5-HT3AB (green) at varying concentrations of 5-HT. (B) Concentration-response curves show a higher potency of 5-HT at 5-HT3ARs as compared with 5-HT3AB, as well as a steeper Hill slope. Parameters from these curves: 5-HT3A: EC50 = 0.8 µM, nH = 2.53 ± 0.58, n = 5, 5-HT3AB: EC50 = 4.30 µM, nH = 1.04 ± 0.02, n = 8. Data are represented as the mean ± S.D. (C) Direct comparison of 5-HT3A and 5-HT3AB inward current evoked by 1 μM 5-HT for 30 seconds.
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Fig. 2. Bupropion’s antagonistic activity at homomeric and heteromeric 5-HT3Rs. (A) Sample traces of oocytes expressing 5-HT3A or 5-HT3AB in response to 5-HT (∼EC30) alone and in combination with bupropion. 5-HT–evoked inward currents (gray, 5-HT3A = 0.3 μM, 5-HT3AB = 2 μM) were used for the control current. Following, the 5-HT concentration was kept constant and coapplied with increasing concentrations of bupropion (5-HT3A: 10–1000 μM, 5-HT3AB: 30–4000 μM). (B) Currents were normalized to the control currents and yielded the following IC50 values: 5-HT3A: IC50 = 87.1 µM (nH = 1.28 ± 0.15, n = 5, mean ± S.D.) and 5-HT3AB: IC50 = 840 µM (nH = 1.78 ± 0.15, n = 7, mean ± S.D.). (C) Oocytes expressing 5-HT3A and 5-HT3AB did not elicit an inward current in response to bupropion alone.
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Fig. 3. Hydroxybupropion is an antagonist for 5-HT3ARs and 5-HT3ABRs. (A) Sample traces of oocytes expressing 5-HT3A or 5-HT3AB in response to 5-HT (∼EC30) alone and in combination with hydroxybupropion. 5-HT–evoked inward currents (gray, 5-HT3A = 0.3 μM, 5-HT3AB = 2 μM) were used for the control current. Following, the 5-HT concentration was kept constant and coapplied with increasing concentrations of hydroxybupropion (5-HT3A: 10–1000 μM, 5-HT3AB: 50–2000 μM). (B) Currents were normalized to the control currents and yielded the following IC50 values: 5-HT3A: IC50 = 113 µM (nH = 1.17 ± 0.15, n = 5, mean ± S.D.) and 5-HT3AB: IC50 = 526 µM (nH = 1.80 ± 0.16, n = 8, mean ± S.D.). (C) Oocytes expressing 5-HT3A and 5-HT3AB did not elicit an inward current in response to hydroxybupropion alone.
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Fig. 4. Non–use dependent allosteric inhibition of 5-HT3ARs and 5-HT3ABRs. (A) Sample traces of oocytes expressing 5-HT3A (black, left panel) and 5-HT3AB (green, right panel). The first 5-HT–evoked currents were used for the control currents (gray bars, ∼EC30, 5-HT3A: 0.5 µM, 5-HT3AB: 2 µM) that were obtained by coapplication with bupropion (magenta bars, ∼IC50, 5-HT3A: 100 µM, 5-HT3AB: 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-HT3A: 100 µM, 5-HT3AB: 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-HT3A (Bup: 76.1% ± 7.16%, n = 5; HydroB: 93.0% ± 6.12%, n = 6) and 5-HT3AB (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.
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Fig. 5. Recovery times for bupropion. (A and B) Sample traces of bupropion application (magenta bar) and the recovery times for 5-HT3A (left panel, black) and 5-HT3AB (right panel, green). (A) In two-electrode voltage clamp experiments, oocytes expressing 5-HT3A and 5-HT3AB 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-HT3A was maximally reduced to 82.4% ± 3.08% and 5-HT3AB 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.
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Fig. 6. Voltage-independent block of 5-HT3–mediated currents by bupropion. (A) Sample traces of 5-HT3A– and 5-HT3AB–expressing oocytes (5-HT3A: left, black; 5-HT3AB: right, green) in response to 5-HT (∼EC50; top and bottom traces, 5-HT3A: 0.8 µM; 5-HT3AB: 5.0 µM) in the absence and presence of bupropion (magenta traces, ∼IC50; 5-HT3A: 100 µM; 5-HT3AB: 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).
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Fig. 7. Bupropion at clinically achievable concentrations and its effect on 5-HT3ARs and 5-HT3ABRs. (A) Sample traces of oocytes expressing 5-HT3A (black, left panel) and 5-HT3AB (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).
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