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INTRODUCTION
Haloperidol is a widely used typical antipsychotic, primarily due to its potent antagonistic effect on dopamine D2 receptors (DR2), which underlies its efficacy in treating schizophrenia and other psychotic disorders [1-3]. However, pharmacological effects of haloperidol are not limited to dopaminergic systems. There is substantial evidence indicating that haloperidol can interact with a range of neurotransmitter systems, including 5-hydroxytryptamine (5-HT)1A receptors [4]. The 5-HT3 receptor is a ligand-gated ion channel belonging to the serotonin receptor family. It can be distinguished from G-protein-coupled 5-HT receptors based on its structural and functional characteristics. The 5-HT3 receptor plays a critical role in modulating synaptic transmission in both central and peripheral nervous systems [5]. The 5-HT3 receptor has been implicated in various physiological and pathological processes, including anxiety, nausea, and schizophrenia [6]. Consequently, the 5-HT3 receptor has been a target of potential therapeutic modulation, particularly in the context of antipsychotic drug action. Previous studies have investigated interactions between antipsychotic agents and 5-HT receptors, focusing on modulation of the 5-HT3 receptor. These studies have demonstrated that specific antipsychotics can either inhibit or potentiate 5-HT3 receptor activity, which may contribute to their therapeutic and side-effect profiles [7,8]. However, precise effects of haloperidol on the 5-HT3 receptor remain poorly understood, particularly in the context of its potential allosteric modulation or direct receptor binding.
The objective of this study was to investigate effects of haloperidol on 5-HT3 receptor activity using the whole-cell patch-clamp technique and NCB-20 cell line expressing the 5-HT3 receptor. By examining changes in ion channel function upon haloperidol exposure, we aimed to determine whether haloperidol could directly modulate 5-HT3 receptor activity and to elucidate potential mechanisms underlying this interaction. An understanding of these interactions could facilitate a deeper insight into complex pharmacological actions of haloperidol, extending beyond its established dopaminergic antagonism. Furthermore, it may contribute to a broader comprehension of how modulation of the 5-HT3 receptor by antipsychotics can influence therapeutic outcomes and adverse effects, thereby informing future drug development and therapeutic strategies.
METHODS
Materials
All chemicals, including haloperidol, serotonin hydrochloride, and other reagents, were purchased from Sigma-Aldrich. Cell culture reagents were obtained from Gibco (Thermo Fisher Scientific).
Cell culture
NCB-20 neuroblastoma cells known to express 5-HT3 receptors were provided by Dr. Lovinger (National Institute on Alcohol Abuse and Alcoholism). Cells were cultured using the method described by Lambert et al. [9], who first characterized 5-HT3 receptor-mediated currents in NCB-20 cells under voltage-clamp conditions. The culture medium consisted of 89% Dulbecco modified Eagle's medium, 10% fetal bovine serum, and 1% hypoxanthine, aminopterin, thymidine supplement. Cells were cultured in an incubator at 37°C with 5% CO₂. When cells reached approximately 70% confluency, they were harvested using 0.25% trypsin-EDTA and seeded into 35 mm culture dishes 24–48 h before being used for recording.
Electrophysiology
The whole-cell voltage-clamp technique was employed to record 5-HT3 receptor-mediated current in NCB-20 cells, following our previous report [10]. The extracellular solution consisted of 150 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 10 mM D-glucose, and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). The pH was adjusted to 7.4 with NaOH. The osmolality was adjusted to 340 mOsm/kg with sucrose. The recording solution was composed of 140 mM CsCl, 2 mM MgCl₂, 5 mM ethylene glycol bis(2-aminoethylether)-N, N, N', N'-tetraacetic acid, and 10 mM HEPES. The pH was adjusted to 7.2 with CsOH and the osmolality was adjusted to 310 mOsm/kg with sucrose. The culture medium was replaced with the extracellular solution prior to harvesting cells from the culture dish. Cells were then transferred to a cover glass two hours before recording. The cover glass was mounted in the recording chamber.
Borosilicate glass capillaries (1B150-4; World Precision Instruments) were pulled using a horizontal micropipette puller (P-97; Sutter Instrument). The resulting pipette resistance was approximately 1.5 MΩ with the recording solution. Cells were observed using an inverted microscope (DM IRM; Leica). Once the giga-ohm seal was achieved, the pipette capacitance was manually compensated using an Axopatch 200B amplifier (Molecular Devices). Following establishment of the whole-cell mode, compensation was performed for whole-cell capacitance and series resistance prior to 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 made at a holding potential of –50 mV except when measuring the current-voltage relationship.
Drug application and preparation
For fast 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) was pulled to approximately 300 μm of the outer diameter. One side of the theta tube was maintained with a continuous perfusion of an extracellular solution, while the other side was perfused with 5-HT or drugs when necessary. The solution flow was driven by gravity from reservoirs. After whole-cell configuration, cells were manually lifted from cover glasses and positioned on the extracellular solution side of the theta glass tubing using a micromanipulator (QUAD; Sutter Instrument). The theta glass tubing was rapidly displaced with a piezoelectric translator (P-601 PiezoMove Linear Actuator; Physik Instrumenten) and a Piezo Servo Controller (E-625; Physik Instrumenten) in a millisecond time resolution, enabling rapid switching between the extracellular solution and the drug-containing solution. Solution switching was controlled by a perfusion valve control system (VC-8; Warner Instruments). After exposing the cell to a solution containing 5-HT or haloperidol for a specified duration, the cell was returned to the extracellular solution side of the theta tubing through a rapid movement of the piezoelectric translator. Drugs were diluted with the extracellular solution to final concentrations from stock solutions. They were freshly prepared for each experiment.
Data analysis and statistics
Peak current amplitudes were measured using the Clampfit 11.0 software (Molecular Devices). Statistical tools in the Clampfit software were used to analyze time constants and rise slopes of currents. Current traces in desensitization and deactivation were fitted to a first-order exponential function using the Clampfit software. Peak amplitudes were normalized to the 5-HT-induced current at the concentration used in each experiment (e.g., 3 μM or 10 μM, depending on the specific experiment). Relationship between normalized peak amplitude and concentration of 5-HT was fitted to the four-parameter logistic equation using Prism 10 (GraphPad Software).
Calculation of EC50 of 5-HT used equation (1):
RESULTS
Haloperidol inhibits 5-HT3 receptor currents
Fig. 1A illustrates representative whole-cell voltage clamp recordings from NCB-20 cells expressing 5-HT3 receptors. Traces demonstrate inhibitory effect of haloperidol with concentrations ranging from 1 μM to 100 μM on 3 μM 5-HT-induced currents. As the concentration of haloperidol increased, there was a significant reduction in peak amplitude of 5-HT3 receptor-mediated currents, indicating a concentration-dependent inhibition. Fig. 1B shows averaged data, revealing a concentration-dependent inhibition of haloperidol's effect, with currents normalized to those induced by 3 μM 5-HT alone. The IC50 value was determined to be 12.96 ± 0.90 μM, with a Hill slope of –1.07 ± 0.04 (n = 12). Fig. 1C shows a concentration-response relationship for the inhibition of haloperidol on the rise slope of 5-HT3 receptor currents. The calculated IC50 for the rise slope was 13.78 ± 3.52 μM, with a Hill slope of –2.37 ± 0.58. The rise slope analysis is particularly relevant because it provides insight into the kinetics of receptor activation, reflecting how quickly ion channels open in response to ligand binding [11]. IC50 values for both peak amplitude and rise slope were in close agreement. Such close agreement in IC50 values for these parameters suggests that haloperidol consistently affects various aspects of the receptor's kinetic response, from initial channel opening to the maximum current achieved.
Haloperidol changes 5-HT concentration-response curve
Fig. 2 illustrates effects of haloperidol on the concentration-response relationship of 5-HT3 receptor-mediated currents. Representative traces (A and B) showed 5-HT3 receptor currents in response to increasing concentrations of 5-HT (1, 3, 10, 30 μM). Haloperidol clearly inhibited 5-HT-induced currents across all concentrations tested, with greater inhibition observed at a higher concentration of haloperidol. An averaged data are presented in Fig. 2C, where open circles and corresponding curve represent the concentration-response relationship for 5-HT alone. In contrast, closed circles, closed box, and their respective curves show the shift in the 5-HT concentration-response in the presence of 10 µM and 30 µM haloperidol, respectively. The presence of haloperidol induced a rightward shift in the EC50 of 5-HT, indicating a reduction in 5-HT potency due to haloperidol co-application. Notably, 30 µM haloperidol not only shifted the EC50 to the right, from 2.76 ± 0.12 µM (n = 24) for 5-HT alone to 4.85 ± 0.23 µM (n = 12) with 30 µM haloperidol (unpaired t-test, p < 0.001), but also significantly decreased the maximal effect (Emax) of 5-HT. This was evidenced by the top value of closed box (0.98 ± 0.01, n = 12) compared to 5-HT alone (1.05 ± 0.01, n = 24), with p < 0.01 (unpaired t-test) indicating statistical significance. This finding suggests that at 30 µM, haloperidol not only reduces the affinity of the 5-HT3 receptors to 5-HT, as shown by the EC50 shift, but also reduces the Emax, indicating a possible non-competitive inhibition or an allosteric modulation effect. These findings provide important insights into the nature of haloperidol's interaction with 5-HT3 receptors, highlighting its potential to modulate receptor activity beyond simple competitive antagonism.
Effect of haloperidol on the voltage-current relationship of 5-HT3 receptor currents
The voltage-current relationship of 5-HT3 receptor-mediated currents in NCB-20 cells at different holding potentials is shown in Fig. 3. In Fig. 3A, left traces represent 5-HT3 receptor currents induced by 3 µM 5-HT at holding potentials of –50, –30, –10, +10 and +30 mV. Right traces indicate 5-HT3 receptor currents when 3 µM 5-HT is co-applied with 10 µM haloperidol at the same holding potentials. Haloperidol reduced amplitudes of 5-HT3 receptor-mediated currents at all holding potentials tested, suggesting an inhibitory effect that was consistent across the voltage range. Fig. 3B shows averaged normalized peak amplitudes of 5-HT3 receptor currents plotted against holding potentials. Open circles represent data from 3 µM 5-HT alone, while closed circles represent data from 3 µM 5-HT in the presence of 10 µM haloperidol. The current-voltage relationship showed a reversal potential (VRes) of 3.31 ± 0.33 mV for 5-HT alone and 5.22 ± 0.88 mV for 5-HT co-applied with haloperidol. Reversal potentials of 5-HT3 receptor currents induced by 5-HT alone and 5-HT co-applied with haloperidol showed no statistically significant difference (paired t-test, p = 0.0867, n = 13). These results suggest that while haloperidol can effectively reduce the overall amplitude of 5-HT3 receptor currents, it does not significantly alter the reversal potential, indicating that the ion selectivity of the 5-HT3 receptor remains unchanged in the presence of haloperidol.
Effects of haloperidol on deactivation and desensitization kinetics of 5-HT3 receptor currents
Fig. 4 shows effects of haloperidol on deactivation and desensitization kinetics of 5-HT3 receptor-mediated currents. In Fig. 4A, deactivation of 5-HT3 receptor currents was induced by application of 10 µM 5-HT for 35 msec. Representative current traces are shown on the left. The grey line represents currents induced by 5-HT alone, while the black line represents currents recorded with co-application of 10 µM haloperidol. Co-application of haloperidol resulted in a significant reduction in peak amplitude of 88.21 ± 1.63% (paired t-test, p < 0.001, n = 11). The right panel shows decay time constants (τ), with open circles representing 5-HT alone and closed circles representing co-application of haloperidol. The averaged decay τ decreased from 1,521 ± 98.68 msec to 1,437 ± 95.82 msec with co-application of haloperidol (paired t-test, p < 0.001, n = 11), indicating accelerated deactivation. Fig. 4B examines desensitization after application of 10 µM 5-HT for 20 sec. Left side shows traces, with grey line indicating 5-HT alone and black line indicating co-application with haloperidol. Co-application of haloperidol reduced the peak amplitude to 90.95 ± 0.63% of the control value (paired t-test, p < 0.001, n = 11). On the right, the decay τ was plotted with open circle for 5-HT alone and closed circle for co-application. The mean decay τ was reduced from 2,222 ± 150.8 msec to 1,869 ± 124.6 msec with co-application of haloperidol (paired t-test, p < 0.001, n = 11), indicating a faster desensitization process. Taken together, these results demonstrate that haloperidol can significantly affect both deactivation and desensitization kinetics of 5-HT3 receptor-mediated currents, revealing a potential mechanism by which haloperidol modulates receptor function.
Effects of haloperidol on recovery from deactivation and desensitization of 5-HT3 receptor currents
Fig. 5 shows effects of haloperidol on recovery from deactivation and desensitization of 5-HT3 receptor-mediated currents in NCB-20 cells using a two-pulse protocol with varying inter-pulse intervals of 5, 10, 20, 30, and 60 sec. Fig. 5A illustrates recovery from deactivation. The first pulse of 5-HT (10 µM) was applied for 35 msec to induce activation and then deactivation, followed by a second pulse of 2.5 sec after varying inter-pulse intervals. Left current traces show representative traces to 5-HT alone, while middle traces show response to the co-application of 5-HT and 10 µM haloperidol. The right graph in Fig. 5A plots paired pulse ratio (the ratio of the peak amplitude of the second pulse to the first pulse) as a function of the inter-pulse interval. Open circles represent data for 5-HT alone, while closed circles represent data for the co-application of 5-HT and haloperidol. The τ for recovery from deactivation was obtained by fitting paired pulse ratios to a one-phase association equation. Results showed similar time constants for the two conditions: 3.45 ± 0.20 sec (n = 9) for 5-HT alone and 3.51 ± 0.22 sec (n = 10) for the co-application with haloperidol (unpaired t-test, p = 0.8450). This indicates that haloperidol does not significantly affect the recovery from deactivation. Fig. 5B illustrates recovery from desensitization. In this experiment, the first pulse of 5-HT (10 µM) was applied for 5 sec to induce desensitization, followed by a second pulse of 2.5 sec after varying inter-pulse intervals. Left current traces show responses to 5-HT alone and middle traces show responses to co-application of 10 µM haloperidol. The right graph presents a plot of the paired pulse ratio against the inter-pulse interval, with open circles representing data for 5-HT alone and closed circles representing data for co-application of 5-HT with haloperidol. The recovery τ was also similar between the two conditions: 6.98 ± 0.45 sec (n = 10) for 5-HT alone and 6.80 ± 0.36 sec (n = 9) for the co-application (unpaired t-test, p = 0.7669). These results indicate that haloperidol does not significantly alter the recovery from desensitization of 5-HT3 receptor currents either. Overall, these findings suggest that while haloperidol can influences both deactivation and desensitization kinetics of 5-HT3 receptors, it does not significantly affect the recovery from these states under conditions tested.
Analysis of use-dependent inhibition of 5-HT3 receptor currents by haloperidol
Fig. 6 shows results of an experiment designed to test whether the inhibition of 5-HT3 receptor currents by haloperidol is use-dependent. In this experiment, twenty 1-s pulses of 5-HT were applied at 30-s intervals. 5-HT3 receptor currents with or without haloperidol co-application were then recorded. Fig. 6A shows representative current traces from both control and test groups. Upper traces represent the control experiment with 5-HT (10 µM) was administered alone. Lower traces show the test group with haloperidol (10 µM) co-applied with 5-HT starting from the 6th (2.5 min) to the 10th (4.5 min) pulse, as indicated by gray traces. Fig. 6B presents averaged normalized peak amplitude of 5-HT3 receptor currents over time for both control (n = 10) and test groups (n = 11). Both groups showed a gradual decrease in current amplitude over time, likely due to rundown of currents. To further evaluate use dependence, ratios of peak amplitudes between the 10th and 6th pulses for both groups were compared in Fig. 6C. The bar graph showed that the 10th/6th peak amplitude ratio was similar between the 5-HT alone (0.98 ± 0.01, n = 10) and the haloperidol co-application (0.98 ± 0.01, n = 11), with a p-value of 0.5702 (unpaired t-test), indicating no significant difference between the two groups. These results suggest that haloperidol does not exhibit use-dependent inhibition on 5-HT3 receptor currents, as the reduction in current amplitude with repeated pulses is comparable between control and test conditions.
Comparison of pretreatment and co-application effects of haloperidol on 5-HT3 receptor currents
Fig. 7 illustrates effects of haloperidol pretreatment versus co-application on 5-HT3 receptor-mediated currents in NCB-20 cells, exploring the possibility of a closed-channel block mechanism. Fig. 7A shows representative current traces from four different drug application conditions: 5-HT (3 µM) alone, co-application of 5-HT (3 µM) with haloperidol (10 µM), pretreatment with haloperidol (10 µM) for 3 sec followed by 5-HT alone, and pretreatment with haloperidol (10 µM) for 3 sec followed by co-application with 5-HT. These traces showed that all conditions involving haloperidol reduced 5-HT3 receptor current compared to 5-HT alone. Fig. 7B presents averaged normalized peak amplitudes from these experiments. Each condition was normalized to the peak amplitude of 5-HT alone to assess the extent of inhibition. Results demonstrated that all three haloperidol application conditions significantly inhibited 5-HT3 receptor currents: co-application (0.73 ± 0.04, n = 9), haloperidol pretreatment followed by 5-HT alone (0.74 ± 0.02, n = 9), and haloperidol pretreatment followed by co-application (0.54 ± 0.03, n = 9) with p < 0.001 (one-way ANOVA F(3, 32) = 70.89). The difference between co-application and haloperidol pretreatment followed by 5-HT alone was not significant (p = 0.9878, one-way ANOVA and Tukey’s multiple comparisons test). These findings indicated that haloperidol had a significant inhibitory effect on 5-HT3 receptor currents under all conditions tested, with the greatest inhibition observed when haloperidol was pre-treated before co-application with 5-HT. These results suggest that while haloperidol does not exhibit a classic closed-channel block effect, its inhibitory potency might be enhanced through a combination of pretreatment and co-application.
DISCUSSION
The present study aimed to elucidate the mechanism by which haloperidol, a typical antipsychotic, inhibits 5-HT3 receptor-mediated currents in NCB-20 cells, focusing on competitive vs. non-competitive inhibition and orthosteric vs. allosteric binding modes. Our findings provide valuable insights into the pharmacodynamics of haloperidol, with implications for its therapeutic and adverse effects.
Our results demonstrated that haloperidol inhibited 5-HT3 receptor-mediated currents in a concentration-dependent manner, with consistent IC50 values across different kinetic parameters, such as peak amplitude and rise slope. Haloperidol inhibited 5-HT3 receptor without altering the reversal potential, suggesting that the ion selectivity of the 5-HT3 receptor remained unchanged by haloperidol. Therefore, although haloperidol inhibits 5-HT3 receptor currents, it does not affect intrinsic properties of the channel itself [12]. The rightward shift in the 5-HT concentration-response curve in the presence of haloperidol, along with a significant reduction in the maximal response (Emax), indicates that the inhibition mechanism is non-competitive. Non-competitive inhibitors often bind to sites distinct from the orthosteric agonist binding site, thereby exerting an allosteric modulation of receptor activity. This hypothesis is supported by previous studies suggesting that non-competitive inhibition can involve allosteric sites that modulate receptor conformation and function [13-15]. However, some study has reported conflicting findings regarding the binding site of haloperidol and inhibitory mechanism on ligand-gated ion channels [16]. Therefore, while our data support a non-competitive, allosteric inhibition mechanism, the possibility of additional or alternative mechanisms cannot be entirely ruled out without further molecular and structural studies. While haloperidol shares common structural features with known 5-HT3 receptor antagonists, such as a basic nitrogen atom that may facilitate its interaction with the ion channel, its distinct structure from typical antagonists such as ondansetron likely accounts for its non-competitive inhibition and broader effects on other neurotransmitter systems [17]. In our experiments examining deactivation and desensitization kinetics, haloperidol significantly accelerated both processes, suggesting that it could stabilize the receptor in a deactivated or desensitized state. This is consistent with the idea of allosteric modulation, whereby binding to a site distinct from the orthosteric site ca induce conformational changes that result in altered receptor activity [18]. Additionally, our recovery experiments indicated that haloperidol did not significantly affect the recovery from deactivation or desensitization, suggesting that binding of haloperidol would not prevent the receptor from returning to a resting state after activation. This supports the notion of a non-competitive inhibition mechanism, as competitive inhibitors are likely to show a more pronounced effect on recovery kinetics of ion channels [19,20]. In the use-dependency experiment, the absence of a significant difference between the 10th and 6th peak amplitude ratios in the presence of haloperidol suggested that its inhibitory effect was consistent across repeated exposures, supporting the idea that haloperidol would not preferentially bind to open or activated receptor states [21]. The comparison of pretreatment and co-application effects revealed that haloperidol pretreatment followed by co-application resulted in the greatest inhibition. However, the absence of a significant difference between co-application and pretreatment alone suggests that haloperidol does not act as a classical closed-channel blocker, which would imply its preferential binding to the receptor in a closed state [22,23]. Given the complex nature of this interaction, further investigation using advanced techniques, such as cryo-electron microscopy or mutagenesis studies, is necessary to elucidate the precise binding sites and conformational states involved.
Therapeutic implications of 5-HT3 receptor inhibition by haloperidol are noteworthy, as this receptor is involved in modulating neurotransmitter pathways associated with nausea, anxiety, and psychosis [5,24,25]. Inhibiting 5-HT3 receptors may contribute to antipsychotic effects of haloperidol beyond its primary action as a dopamine D2 receptor antagonist. However, this inhibition could also underlie some adverse effects associated with haloperidol, such as gastrointestinal disturbances and potential cardiac effects due to its interactions with ion channels [26,27]. Beyond its effects on 5-HT3 receptors, haloperidol has been shown to modulate several other ion channels, including voltage-gated potassium channels, calcium channels, and GABAA receptors. These interactions contribute to its diverse pharmacological profile and may underlie some of its off-target effects, such as its role in modulating neuronal excitability and influencing synaptic transmission [28-30]. Due to the limitations of this study using NCB-20 cells, which are not native neurons, further studies with isolated neurons or brain slices will be necessary to better understanding of haloperidol's action on 5-HT3 receptors in a physiological setting.
In conclusion, findings of this study provide evidence that haloperidol can inhibit 5-HT3 receptor function primarily through a non-competitive, allosteric mechanism. An understanding of these interactions is crucial for elucidating both therapeutic and adverse effects of haloperidol and other typical antipsychotics.
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