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INTRODUCTION
The 5-hydroxytryptamine type3 (5-HT3) receptor is a ligand-gated ion channel primarily found in both central and peripheral nervous systems [1,2]. It plays a crucial role in regulating synaptic transmission, and its dysregulation has been implicated in a variety of neurological and psychiatric disorders, including anxiety, depression, and schizophrenia [3,4]. Due to their role in modulating gastrointestinal motility and the emetic response, 5-HT3 receptors are also targeted in the management of chemotherapy-induced nausea and vomiting, as well as irritable bowel syndrome [5,6]. Quetiapine, an atypical antipsychotic, is commonly used for treating schizophrenia, bipolar disorder, and major depressive disorder [7,8]. While the therapeutic efficacy of quetiapine is largely attributed to its effects on dopamine D2 and serotonin 5-HT2A receptors [9-11], its interaction with the 5-HT3 receptor has recently drawn attention. Studies have suggested that quetiapine might act as an antagonist at the 5-HT3 receptor, influencing serotonin-mediated neurotransmission in both central and peripheral systems [12,13]. However, the precise mechanism by which quetiapine modulates 5-HT3 receptor function, particularly in the context of ion channel kinetics, remains unclear. Previous research on 5-HT3 receptor antagonists, such as ondansetron and granisetron has demonstrated a competitive inhibition, where the antagonist can bind to the same site as the agonist, primarily causing a rightward shift in the concentration-response curve of agonist with little effect on the maximal response (Emax) [14,15]. These findings raise the question of whether quetiapine can exert similar competitive inhibition of the 5-HT3 receptor. Clarifying this could enhance our understanding of the pharmacological profile of quetiapine and its potential impact on serotonergic pathways.
Thus this study aimed to elucidate the mechanism of action of quetiapine on 5-HT3 receptor-mediated currents using whole-cell voltage-clamp recordings in NCB20 neuroblastoma cells. Specifically, we investigated whether quetiapine could act as a competitive antagonist, how it could affect ion channel kinetics, and conditions under which it exerted its inhibitory effects. Gaining insights into these mechanisms is critical not only for clarifying effect of quetiapine on the 5-HT3 receptor, but also for exploring its potential therapeutic applications beyond its established use in psychiatric disorders.
METHODS
Cell culture
NCB-20 neuroblastoma cells known to express the 5-HT3 receptor were provided by Dr. Lovinger (National Institute on Alcohol Abuse and Alcoholism, USA). The expression and functional characteristics of 5-HT3 receptors in NCB-20 cells have been established in previous studies [16,17]. The cells were cultured in Dulbecco's Modified Eagle’s Medium supplemented with 10% fetal bovine serum and 1% hypoxanthine-aminopterin-thymidine supplement. They were maintained at 37°C in an incubator with 5% CO₂. Cells were subcultured every 2–3 days to maintain their exponential growth and seeded into 35 mm culture dishes 24–48 h prior to their use in electrophysiological recordings.
Whole-cell voltage-clamp recording
Currents mediated by 5-HT3 receptor were recorded using the whole-cell voltage-clamp technique in accordance with our previous report [18]. Cells were transferred to a recording chamber mounted on an inverted microscope (DM IRM; Leica) and continuously perfused with an extracellular solution (140 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl₂, 10 mM D-glucose, and 10 mM HEPES, pH adjusted to 7.4 with NaOH). Patch electrodes were fabricated using borosilicate glass (1B150-4; World Precision Instruments). They were filled with an internal pipette solution containing 140 mM CsCl, 2 mM MgCl₂, 10 mM HEPES, 5 mM EGTA, pH adjusted to 7.2 with CsOH (resistance approximately 1.5 MΩ with the internal solution). Once a giga-ohm seal was established, the pipette capacitance was manually compensated using an Axopatch 200B amplifier (Molecular Devices). Upon achieving a whole-cell mode, cell capacitance and series resistance were compensated manually before drug application. Signals were filtered at 2 kHz, digitized at 10 kHz, and stored using DigiData 1440 and pClamp 11.0 software (Molecular Devices). Recordings were conducted at a holding potential of –50 mV, except when current-voltage (I-V) relationships were being measured.
Drug application
Theta glass tubing (2 mm in outer diameter, 1.4 mm in inner diameter, 0.2 mm in septum thickness, Clark Borosilicate Theta, Warner Instruments), a piezoelectric system (P-601 PiezoMove Linear Actuator; Physik Instrumente), and a VC-8 perfusion valve control system (Warner Instruments) were used for rapid application of 5-HT and quetiapine to ensure precise control over drug exposure time with millisecond time resolution as previously reported [18]. Once the whole-cell configuration was established, the cell was manually lifted and positioned in close proximity to the side of the theta tubing using a micromanipulator (QUAD; Sutter Instrument), which was continuously perfused with the extracellular solution alone. The other side of the theta tubing was perfused with an extracellular solution containing 5-HT or quetiapine, as required. For co-application of 5-HT and quetiapine, a mixed solution of 5-HT and quetiapine was perfused. For pretreatment of quetiapine, it was applied for 5 sec before co-application with 5-HT. All chemicals, including quetiapine, 5-HT, and other reagents were purchased from Sigma-Aldrich. Quetiapine and 5-HT were prepared as stock solutions in dimethyl sulfoxide. Their final working concentrations were diluted in extracellular solution and freshly prepared before each experiment.
Data analysis
Concentration-response relationship of 5-HT in the presence or absence of quetiapine was determined by applying increasing concentrations (0.3–100 μM) of 5-HT and recording peak current amplitudes. Peak amplitudes were normalized to 3 or 10 µM 5-HT-induced current. The relationship between normalized peak amplitude and 5-HT concentration was fitted to a four-parameter logistic equation using Prism 10 (GraphPad Software).
To calculate the EC50 of 5-HT, the following equation was used:
where the bottom represented the minimal response and the top indicated the maximal response to 5-HT. X was the logarithm of 5-HT concentrations. EC50 was the concentration of 5-HT producing a half-maximal response and n was the Hill coefficient.
IC50 values for quetiapine were calculated by fitting concentration-inhibition curves using the following equation:
where the bottom denoted the minimal response and the top indicated the maximal response elicited by 5-HT. X was the logarithm of quetiapine concentrations. IC50 was the concentration of quetiapine that produced a 50% reduction in the response between top and bottom values and n was the Hill coefficient.
I–V relationships were determined by holding cells at –50 mV and applying 10 μM 5-HT alone or co-applied with 10 μM quetiapine. Membrane potential was stepped from –50 mV to +30 mV in 20 mV increments. The peak current at each potential was recorded. The reversal potential was calculated using linear regression of the I–V plot.
Desensitization and deactivation kinetics were evaluated by applying 10 μM 5-HT alone or with varying concentrations (1–30 μM) of quetiapine for 15 ms (deactivation) or 20 sec (desensitization). Decay phase of the current was fitted with a single exponential function to determine the time constant (τ). Recovery from desensitization and deactivation was assessed using a two-pulse protocol. For deactivation, the first pulse of 10 μM 5-HT, with or without 10 μM quetiapine was applied for 20 ms, followed by 2.5 sec pulse of 10 μM 5-HT after variable inter-pulse intervals (1, 5, 10, 30, 60 sec). For desensitization, the first pulse duration was 5 sec. Paired-pulse ratio (PPR) was calculated as the peak amplitude of the second response divided by that of the first response. Recovery τ were obtained by fitting PPR data to a one-phase association curve.
To determine association kinetics of quetiapine with opened 5-HT3 receptors, quetiapine (1–30 μM) was co-applied for 8 sec after a 3 sec pre-application of 3 μM 5-HT, as previously reported [18]. Decay of current during quetiapine co-application for 8 sec was fitted to a single exponential function to obtain decay τ. The fitting started from the current observed after quetiapine application and extended through the decay phase until the end of the co-application. Association rate constant (), dissociation rate constant (), and dissociation constant () to opened 5-HT3 receptors were calculated using the following equations:
Statistics
Data are presented as mean ± SEM. Statistical significance was determined using Student’s t-test, one-way or repeated measures ANOVA with post-hoc tests as appropriate. All statistical analyses were conducted with Prism 10 (GraphPad Software). The significance threshold was set at p < 0.05.
RESULTS
Quetiapine shifts 5-HT concentration-response curve for 5-HT3 receptor-mediated currents
To investigate effects of quetiapine on 5-HT3 receptor-mediated currents, we examined the concentration-response relationship of 5-HT in the presence or absence of quetiapine using whole-cell voltage-clamp recordings with NCB20 neuroblastoma cells. Increasing concentrations (0.3, 1, 3, 10, 30, and 100 μM) of 5-HT were applied alone or with quetiapine (3, 10, and 30 μM) co-application. Fig. 1A–C illustrate representative current traces, demonstrating effects of quetiapine on 5-HT3 receptor-mediated currents. In each panel, left traces show currents evoked by 5-HT alone, while right traces show currents upon co-application of 5-HT with quetiapine at concentrations of 3 μM (A), 10 μM (B), and 30 μM (C). For 5-HT at lower concentrations (0.3, 1, 3 μM), quetiapine significantly reduced peak current amplitudes, demonstrating a concentration-dependent inhibition of 5-HT3 receptor-mediated currents. However, for 5-HT at higher concentrations (10, 30, and 100 μM), quetiapine co-application resulted in a minimal reduction of peak current amplitudes. This suggests that while quetiapine can effectively inhibit responses to low concentrations of 5-HT, its inhibitory effect is much less pronounced when higher concentrations of 5-HT are applied. The concentration-response curve for 5-HT shifted to the right in the presence of quetiapine (Fig. 1D). This shift indicated a reduction in the potency of 5-HT in activating the receptor. The EC50 for 5-HT3 receptor-mediated currents increased significantly from 2.64 ± 0.06 μM for 5-HT alone (n = 26) to 4.70 ± 0.23 μM in the presence of 3 μM quetiapine (n = 10), 6.61 ± 0.20 μM in the presence of 10 μM quetiapine (n = 10), and 9.99 ± 1.27 μM in the presence of 30 μM quetiapine (n = 9) (one-way ANOVA, F(3,51) = 48.95, p < 0.001). Despite a rightward shift in EC50, the maximal effect (Emax) remained unchanged across all conditions (one-way ANOVA, F(3,51) = 0.729, p = 0.5394), suggesting that quetiapine acted primarily by reducing the apparent potency of 5-HT rather than its maximal efficacy. Furthermore, the Hill coefficient for 5-HT was not significantly altered by the presence of quetiapine (one-way ANOVA, F(3,51) = 1.747, p = 0.1693), indicating that the cooperativity of 5-HT binding to the receptor remained consistent, with quetiapine primarily modulating receptor sensitivity.
Quetiapine inhibits 5-HT3 receptor-mediated currents in a concentration-dependent manner
To further investigate inhibitory effects of quetiapine on 5-HT3 receptor-mediated currents, we analyzed concentration-dependent inhibition of quetiapine when it was co-applied with 5-HT at two different concentrations (3 μM and 10 μM), as well as when pretreated before co-application. Fig. 2A illustrates representative 5-HT3 receptor currents evoked by 3 μM 5-HT alone (shown in gray) and currents co-applied with quetiapine at concentrations ranging from 0.1 to 30 μM (shown in black). Quetiapine co-application resulted in a concentration-dependent reduction in the amplitude of 5-HT3 receptor-mediated currents. At higher concentrations of quetiapine, the reduction in peak current was more pronounced, indicating potent inhibition. Fig. 2B shows representative 5-HT3 receptor currents evoked by 10 μM 5-HT alone (depicted by gray traces), and those co-applied with quetiapine at concentrations ranging from 0.3 to 100 μM (represented by black traces). A comparable concentration-dependent inhibition was observed (Fig. 2A), with quetiapine effectively reducing the current amplitude across the tested range. However, the magnitude of inhibition was greater with 3 μM 5-HT (2A) compared to that with 10 μM 5-HT (2B), indicating a potential influence of 5-HT concentration on the inhibitory efficacy of quetiapine. In Fig. 2C, quetiapine was pretreated for 5 sec before co-application with 10 μM 5-HT with results shown as black traces alongside gray traces for 10 μM 5-HT alone. Pretreatment with quetiapine enhanced its inhibitory effect compared to co-application alone, as evidenced by a more substantial reduction in peak current at lower concentrations of quetiapine. This suggests that quetiapine preferentially binds to closed states of the 5-HT3 receptor ion channel, enhancing its inhibitory effect when pre-applied before receptor activation by 5-HT. Fig. 2D presents a summary of concentration-response curves for quetiapine’s inhibitory effects under three conditions: co-application with 3 μM 5-HT (gray circle), co-application with 10 μM 5-HT (open circle), and pretreatment with 10 μM 5-HT (filled circle). The IC50 for quetiapine was significantly lower with 3 μM 5-HT (0.58 ± 0.08 μM, n = 10) compared to that with 10 μM 5-HT (25.23 ± 3.28 μM, n = 8) (unpaired t-test, p < 0.001). When quetiapine was pretreated for 5 sec before its co-application with 10 μM 5-HT, its IC50 decreased to 0.20 ± 0.01 μM (n = 10), indicating a significantly enhanced inhibitory effect (unpaired t-test, p < 0.001). The Hill slope did not change significantly across conditions, suggesting that quetiapine could modulate the receptor’s sensitivity without altering the cooperativity of 5-HT binding.
Quetiapine accelerates deactivation and desensitization of 5-HT3 receptor-mediated currents
Effects of quetiapine on deactivation of 5-HT3 receptor-mediated currents were tested by comparing the decay τ of currents recorded after a brief 15 msec application of 5-HT, both with and without co-application of quetiapine. A 15 msec pulse of 5-HT application was used to selectively induce rapid activation and subsequent deactivation of the receptor. Quetiapine was co-applied at concentrations of 1, 3, 10, and 30 μM with 10 μM 5-HT. Fig. 3A shows deactivation of 5-HT3 receptor-mediated currents evoked by 10 μM 5-HT alone (gray trace) or co-applied with increasing concentrations of quetiapine (black traces). Deactivation was concentration-dependently accelerated by co-application with quetiapine. Fig. 3C presents summarized data of deactivation decay τ plotted against quetiapine concentration. The decay τ for 5-HT alone was 1,991 ± 207.4 msec, which decreased to 1,752 ± 190.3 msec in the presence of 1 μM quetiapine, 1,612 ± 170.4 msec in the presence of 3 μM quetiapine, 1,467 ± 164.5 msec in the presence of 10 μM quetiapine, and 1,303 ± 139.7 msec with 30 μM quetiapine. Statistical analysis by repeated measures one-way ANOVA showed a significant effect of quetiapine on deactivation of the 5-HT3 receptor (F(1.253, 10.02) = 47.86, p < 0.001, n = 9). These findings indicate that quetiapine can accelerate the deactivation of 5-HT3 receptor-mediated currents in a concentration-dependent manner.
The effect of quetiapine on desensitization of the 5-HT3 receptor was evaluated by extending the application time of 10 μM 5-HT to 20 sec, allowing receptor desensitization to occur. Quetiapine was co-applied at 1, 3, 10, or 30 μM with 10 μM 5-HT. Decay τ was then calculated from currents recorded during the prolonged application. Fig. 3B shows desensitization of 5-HT3 receptor-mediated currents evoked by 10 μM 5-HT alone for 20 sec (gray trace) or co-applied with quetiapine (black traces). Quetiapine accelerated the decay of currents, with faster desensitization observed at higher concentrations of quetiapine. Fig. 3C presents averaged data for desensitization decay τ plotted against quetiapine concentration. The τ for 5-HT alone was 3.865 ± 511.5 msec, which decreased to 2.247 ± 316.6 msec in the presence of 1 μM quetiapine, 1.312 ± 157.7 msec in the presence of 3 μM quetiapine, 507.9 ± 48.02 msec in the presence of 10 μM quetiapine, and 214.1 ± 20.44 msec in the presence of 30 μM quetiapine. Statistical analysis by repeated measures one-way ANOVA revealed a significant effect of quetiapine on desensitization of the 5-HT3 receptor (F(1.019, 8.152) = 51.23, p < 0.001, n = 9). These findings demonstrate that quetiapine can significantly accelerate the desensitization of 5-HT3 receptor-mediated currents in a concentration-dependent manner. This suggests that quetiapine can promote faster transitions to inactive states during both deactivation and desensitization, supporting the hypothesis that quetiapine can stabilize closed or desensitized states of the 5-HT3 receptor.
Quetiapine modulates recovery from deactivation and desensitization of 5-HT3 receptor-mediated currents
To evaluate the recovery from deactivation and desensitization of 5-HT3 receptor-mediated currents, we used a two-pulse protocol of 5-HT application with variable inter-pulse intervals (1, 5, 10, 30, and 60 sec). The PPR, defined as the ratio of the second peak amplitude to the first, was plotted as a function of inter-pulse interval. The data were fitted to a one-phase association equation to determine the time constant (τ) for recovery. It should be noted that the recovery kinetics were analyzed based on the PPR values rather than directly analyzing decay τ from individual current traces. Experiments were conducted with 10 μM 5-HT alone or co-applied with 10 μM quetiapine.
In the recovery from deactivation experiment, the first pulse was a brief 20 msec application of 10 μM 5-HT, followed by a 2.5 sec application as the second pulse. Fig. 4A shows representative superimposed traces, with gray arrows indicating the first pulse and black arrows indicating the second pulse. Co-application of 10 μM quetiapine (right) did not significantly affect the recovery from deactivation compared to 5-HT alone (left). Fig. 4B presents averaged values of paired pulse ratio PPR plotted against inter-pulse interval. The τ for recovery from deactivation was 5.53 ± 0.28 sec for 5-HT alone (n = 10) and 5.43 ± 0.39 sec for co-application with quetiapine (n = 9). Statistical analysis revealed no significant difference between the two conditions (unpaired t-test, p = 0.8437), indicating that quetiapine did not alter recovery kinetics from deactivation of 5-HT3 receptor-mediated currents.
For recovery from desensitization experiment, the first pulse was a 5 sec application of 10 μM 5-HT, followed by a 2.5 sec second pulse. Fig. 4C shows representative superimposed traces, with gray arrows marking the first pulse and black arrows indicating the second pulse. The left panel shows currents evoked by 10 μM 5-HT alone, while the right panel displays currents co-applied with 10 μM quetiapine. In contrast to the recovery from deactivation, quetiapine co-application notably affected recovery from desensitization. Fig. 4D shows summarized PPR plotted against inter-pulse intervals, with data fitted to a one-phase association equation. In the presence of quetiapine, the PPR decreased significantly at shorter inter-pulse intervals (e.g., 1, 5, 10, and 30 sec), indicating delayed recovery from desensitization due to quetiapine stabilizing the receptor in a desensitized state. At 60 sec, the PPR approached 1.0, reflecting full recovery of receptor availability. This suggests that quetiapine slows the recovery process in a time-dependent manner but does not exert a lasting inhibitory effect when sufficient recovery time is allowed. The τ was 6.88 ± 0.41 sec for 5-HT alone (n = 9) and 9.85 ± 0.75 sec when quetiapine was co-applied (n = 9). Statistical analysis revealed a significant difference between the two conditions (unpaired t-test, p < 0.01), indicating that quetiapine prolonged the recovery from desensitization of 5-HT3 receptor-mediated currents. These results suggest that while quetiapine does not affect the recovery from deactivation, it can significantly alter the recovery from desensitization, potentially by stabilizing the desensitized state of the 5-HT3 receptor.
Effects of quetiapine on 5-HT3 receptor-mediated currents at different holding potentials
Effects of quetiapine on 5-HT3 receptor-mediated currents were investigated by analyzing I-V relationships at varying holding potentials. Fig. 5A shows representative current traces recorded at holding potentials of –50, –30, –10, +10, and +30 mV. The left panel depicts currents evoked by 10 μM 5-HT alone, while the right panel shows currents with co-application of 10 μM quetiapine. Fig. 5B presents averaged normalized peak amplitudes of 5-HT3 receptor-mediated currents plotted against the holding potential (VHolding). The reversal potential was determined by extrapolating the I-V curve to the point where the current amplitude crossed zero. The reversal potential was 2.38 ± 0.20 mV for 10 μM 5-HT alone and 2.51 ± 0.19 mV for co-application with 10 μM quetiapine. Statistical analysis using a paired t-test revealed no significant difference between the two conditions (n = 9, p = 0.1617), indicating that quetiapine did not alter the reversal potential of 5-HT3 receptor-mediated currents. Fig. 5C shows peak amplitude ratios (defined as the ratio of the peak current amplitude for 5-HT with quetiapine co-application to that for 5-HT alone) plotted across different holding potentials. Peak amplitude ratios remained consistent across all tested holding potentials. Statistical analysis by repeated measures one-way ANOVA confirmed no significant effect of holding potential on the peak amplitude ratio (F(2.046, 16.37) = 1.045, p = 0.3756).
These findings suggest that the inhibitory action of quetiapine on 5-HT3 receptor-mediated currents is consistent across holding potentials tested, indicating a voltage-independent block. Additionally, there were no significant changes in reversal potential, suggesting no significant impact of quetiapine on channel gating mechanisms.
Quetiapine binding kinetics on open 5-HT3 receptor
Association kinetics of quetiapine on open state of 5-HT3 receptor was investigated using a protocol in which a 16 sec application of 3 μM 5-HT was used to maintain the open state of channels and quetiapine at varying concentrations (1, 3, 10, 30 μM) was co-applied with 5-HT for 8 sec, starting at 3 sec after the onset of 5-HT application. Fig. 6A shows superimposed current traces, demonstrating that quetiapine accelerated the 5-HT3 receptor current decay during co-application in a concentration-dependent manner. At concentrations higher than 3 μM, currents reappeared upon discontinuation of quetiapine co-application. Decay of the current during quetiapine co-application was fitted to a single exponential function to obtain decay τ and analyzed for each concentration of quetiapine. Fig. 6B presents τ values plotted against the concentration of quetiapine, showing that the τ for decay of the current decreased as the concentration of quetiapine increased. The τ values was 3.11 ± 0.19 sec for 5-HT alone, 1.97 ± 0.12 sec in the presence of 1 μM quetiapine, 1.26 ± 0.10 sec in the presence of 3 μM quetiapine, 0.71 ± 0.06 sec in the presence of 10 μM quetiapine, and 0.36 ± 0.03 sec in the presence of 30 μM of quetiapine (n = 10). Statistical analysis using repeated measures one-way ANOVA showed a significant effect of quetiapine concentration on the τ (F(1.436, 12.92) = 191.7, p < 0.001). Fig. 6C shows reciprocal of time constant (1/τ) as a function of quetiapine concentration. According to the first-order blocking scheme, the slope of the linear fit obtained from the data in Fig. 6C represents the association rate constant (k+1), while the y-intercept corresponds to the dissociation rate constant (k-1) for the open 5-HT3 receptor. Linear fit yielded a slope of 0.08776 and a y-intercept of 0.4539. The apparent dissociation constant (Kd) of quetiapine for the open 5-HT3 receptor was calculated from the ratio of k-1 to k+1. Based on the linear fit shown in Fig. 6C, the apparent Kd calculated for quetiapine was 5.17 μM. Further details on the calculation of the apparent dissociation constant can be found in the Methods section. These results indicate that quetiapine can effectively bind to the open state of the 5-HT3 receptor and modulates its activity by accelerating current decay in a concentration-dependent manner.
Use-dependence of quetiapine’s inhibitory effects on 5-HT3 receptor currents
To evaluate whether inhibition of 5-HT3 receptor-mediated currents by quetiapine might be use-dependent, 10 μM 5-HT was applied in 2 sec pulses at 30 sec intervals for a total of 16 applications. Quetiapine (10 μM) was co-applied during the 4th to 8th pulses, corresponding to 1.5 to 3.5 min from the start of the experiment. Fig. 7A shows representative current traces, with gray traces indicating currents evoked by 5-HT alone and black traces representing currents during co-application of 5-HT and quetiapine. Gray bar above each trace indicates the period of 5-HT application and the black bar marks the period of quetiapine co-application. Fig. 7B shows average normalized peak amplitudes plotted over time. The peak current amplitude decreased during quetiapine co-application, but recovered upon discontinuation of quetiapine co-application, suggesting that the effect of quetiapine was reversible. Fig. 7C presents a bar graph comparing normalized peak amplitudes at the 5th (2 min) and 8th (3.5 min) applications. The normalized peak amplitude was 0.69 ± 0.01 for the 5th application and 0.67 ± 0.02 for the 8th application, showing no significant difference between them (p = 0.1340, n = 8, paired t-test). These findings suggested that the inhibitory effect of quetiapine on 5-HT3 receptor-mediated currents was not use-dependent, since there was no progressive increase in inhibition with repeated co-application of quetiapine over the tested interval.
DISCUSSION
The present study demonstrates that quetiapine functions as a competitive antagonist of 5-HT3 receptor-mediated currents in NCB20 neuroblastoma cells. The rightward shift of EC50 by quetiapine in the 5-HT concentration-response curve with no significant change in Emax indicates that quetiapine can decrease the apparent potency of 5-HT without affecting the maximal response. This pattern is characteristic of a competitive inhibition, where antagonists bind at the same site as agonists and shift the EC50 to higher concentrations without reducing maximal efficacy [19,20]. IC50 values obtained in this study demonstrate that quetiapine can exert its inhibitory effects in a conformation-dependent manner. The IC50 for quetiapine when co-applied with 10 μM 5-HT was 25.23 μM. However, pre-application of quetiapine before 5-HT administration significantly enhanced its inhibitory potency, reducing the IC50 to 0.20 μM. This considerable discrepancy suggests that quetiapine can bind more efficiently to the receptor in its inactive state before agonist binding, likely stabilizing the receptor in a less active conformation. When quetiapine was co-applied with 3 μM 5-HT, the IC50 was 0.58 μM, more than 40 times lower than the IC50 of 10 μM 5-HT. This difference in IC50 value indicates that quetiapine’s inhibitory potency is inversely correlated with the concentration of 5-HT. At a higher concentration of 5-HT (10 μM), quetiapine might have to compete with more available agonist molecules for the same binding sites, resulting in a weaker inhibitory effect and a higher IC50. Conversely, at a lower concentration of 5-HT (3 μM), there was less competition for binding, allowing quetiapine to inhibit the receptor more effectively, leading to a lower IC50. This pattern of concentration-dependent inhibition is the characteristic of competitive antagonism, where the effect of antagonist diminishes as the agonist concentration increases [19-21]. Furthermore, the IC50 derived from interaction kinetics experiments, calculated from association (k+1) and dissociation (k−1) rate constants, was 5.17 μM for quetiapine binding to the open state of the receptor. This calculated IC50 value was approximately nine-fold greater than the IC50 of quetiapine for 3 μM 5-HT, which was 0.58 μM. This higher IC50 value suggests that quetiapine competes less effectively with 5-HT when the receptor is in an open state and that quetiapine has a stronger inhibitory effect when the receptor is transitioning between close and open conformations, as observed in co-application experiments with 3 μM 5-HT [19]. These observed differences in IC50 values across experimental conditions suggest that quetiapine could preferentially bind to the receptor in its closed state, stabilizing it in a conformation less favorable to opening. This pattern is consistent with the behavior of other 5-HT3 receptor antagonists such as ondansetron and granisetron known to similarly induce a rightward shift in the dose-response curve without significantly altering the Emax [15]. Furthermore, this variability in IC50 is a common feature of ligand-gated ion channels, where receptor conformation influences antagonist binding affinity [19,22]. While our findings suggest that the inhibitory mechanism of quetiapine is similar to ondansetron and granisetron, further studies, such as molecular docking analysis, are needed to confirm whether quetiapine binds to the same receptor site as these antagonists and to elucidate its detailed binding interactions.
The mechanism by which quetiapine inhibits 5-HT3 receptor-mediated currents involves modulation of the ion channel kinetics of receptor. Quetiapine accelerated both deactivation and desensitization of 5-HT3 receptor-mediated currents, indicating that it could stabilize the receptor in inactive states. The faster deactivation observed with increasing concentrations of quetiapine indicates that it promotes a more rapid closure of the receptor channel, preventing sustained ion flow. Similarly, quetiapine significantly accelerated desensitization, implying that it could drive the receptor into a non-conductive state more efficiently. These effects are characteristic of an antagonist that binds preferentially to closed or desensitized states of the receptor [22]. The I–V relationship analysis further supports quetiapine as a voltage-independent antagonist. Inhibitory effects of quetiapine were consistent across the range of holding potentials without significant change in the reversal potential, indicating that quetiapine did not affect channel gating. This suggests that quetiapine primarily inhibits the receptor by competing with 5-HT at the binding site rather than modulating intrinsic ion channel properties [23,24]. In terms of recovery kinetics, quetiapine did not significantly alter recovery from deactivation. This suggests that quetiapine does not preferentially bind to receptors that are in the process of closing after brief activation. This finding further supports its role as a competitive antagonist, whereby the primary effect is to prevent 5-HT from activating the receptor without affecting receptor recovery. However, quetiapine significantly prolonged recovery from desensitization, indicating that it could stabilize the desensitized state of the receptor and delays its return to a fully responsive condition [22]. Inhibitory effect of quetiapine was also shown to be independent of use, as there was no cumulative increase in inhibition with repeated co-application of quetiapine and 5-HT. This lack of use-dependence is a characteristic of competitive antagonism, wherein binding affinity remains consistent regardless of repeated exposure [19,23].
Taken together, these results show that quetiapine primarily inhibits the 5-HT3 receptor through competitive antagonism, preventing binding of 5-HT, thereby preventing receptor activation, and stabilizing it in non-conductive conformations. Its effects on deactivation, desensitization, voltage-independence, and lack of use-dependence provide compelling evidence that quetiapine is a competitive antagonist of the 5-HT3 receptor. Additional structural studies using techniques such as X-ray crystallography or cryo-electron microscopy could further clarify specific interactions between quetiapine and the 5-HT3 receptor [14].
The ability of quetiapine to competitively inhibit 5-HT3 receptor-mediated currents has considerable clinical implications. The 5-HT3 receptor plays a role in various physiological and pathological processes, including nausea, vomiting, anxiety, and certain neuropsychiatric disorders [3,4,25]. The action of quetiapine on these receptors may contribute to its therapeutic effects on conditions such as schizophrenia and bipolar disorder, where serotonergic dysfunction is thought to play a role [26,27]. Due to limitations of this study using NCB-20 cells, which are not native neurons, further studies using isolated neurons or slices from brain will be necessary to validate these findings in a physiologically or pathologically relevant context. A deeper understanding of its mechanism of action may also help optimize its clinical use and guide the development of more targeted therapies with fewer side effects.
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