Korean J Physiol Pharmacol.  2025 May;29(3):349-358. 10.4196/kjpp.24.320.

Haloperidol, a typical antipsychotic, inhibits 5-HT₃ receptor- mediated currents in NCB-20 cells: a whole-cell patch-clamp study

• Park YS
• Kim GM
• Sung HJ
• Yu JY
• Sung KW

• KMID: 2567192
• DOI: http://doi.org/10.4196/kjpp.24.320

Abstract

Haloperidol is a typical antipsychotic drug effective in alleviating positive symptoms of schizophrenia by blocking dopamine receptor 2 (DR2). However, it is also known to produce neuropsychiatric effects by acting on various targets other than DR. In this study, we investigated effect of haloperidol on function of 5-hydroxytryptamine (5-HT) 3 receptor, a ligand-gated ion channel belonging to the serotonin receptor family using the whole-cell voltage clamp technique and NCB20 neuroblastoma cells. When co-applied with 5-HT, haloperidol inhibited 5-HT₃ receptormediated currents in a concentration-dependent manner. A reduction in maximal effect (E max ) and an increase in EC 50 observed during co-application indicated that haloperidol could act as a non-competitive antagonist of 5-HT₃ receptors. Haloperidol inhibited the activation of 5-HT₃ receptor, while also accelerating their deactivation and desensitization. The inhibitory effect of haloperidol showed no significant difference between pre- and co-application. Haloperidol did not alter the reversal potential of 5-HT₃ receptor currents. Furthermore, haloperidol did not affect recovery from deactivation or desensitization of 5-HT₃ receptors. It did not show a use-dependent inhibition either. These findings suggest that haloperidol can exert its inhibitory effect on 5-HT₃ receptors by allosterically preventing opening of ion channels. This mechanistic insight enhances our understanding of relationships between 5-HT₃ receptors and pharmacological actions of antipsychotics.

Keyword

Antipsychotics; Haloperidol; Patch clamp; Schizophrenia; 5-HT₃ receptor

Figure

• 

  Fig. 1 Inhibition of 5-hydroxytryptamine (5-HT)3 receptor-mediated currents by haloperidol. (A) Representative whole-cell current traces recorded from NCB-20 cells expressing 5-HT3 receptors. Currents were induced by 3 µM 5-HT alone (bottom trace) or in the presence of increasing concentrations (1, 3, 10, 30, 100 µM) of haloperidol. As the concentration of haloperidol increased, there was a progressive reduction in the peak amplitude of the 5-HT3 receptor currents, indicating a concentration-dependent inhibition. (B) Dose-response curve for haloperidol’s effect on normalized peak amplitude of the 5-HT3 receptor currents, with currents normalized to those induced by 3 µM 5-HT alone. (C) Concentration-response curve for the effect of haloperidol on normalized rise slope of the 5-HT3 receptor currents. Data are expressed as mean ± SEM.

• 

  Fig. 2 Modulation of 5-hydroxytryptamine (5-HT)3 receptor currents by haloperidol. (A) Representative whole-cell current traces from NCB-20 cells in response to 1, 3, 10, and 30 µM 5-HT (gray traces) alone or along with the presence of 10 µM haloperidol (black traces). (B) Representative whole-cell current traces as in (A), but with 30 µM haloperidol. Haloperidol inhibits 5-HT-induced currents in a concentration-dependent manner. (C) Averaged concentration-response curves for 5-HT in the absence (open circles) and presence of 10 µM (closed circles) and 30 µM (closed box) haloperidol. The presence of haloperidol caused a rightward shift in the EC50 of 5-HT and a significant reduction in the maximal response (Emax) at 30 µM, suggesting both a decrease in potency and efficacy. Data are expressed as mean ± SEM.

• 

  Fig. 3 Effect of haloperidol on voltage-current relationship of 5-hydroxytryptamine (5-HT)3 receptor-mediated currents. (A) Representative whole-cell current traces of 5-HT3 receptor currents obtained at various holding potentials (–50, –30, –10, +10, +30 mV). The left set of traces shows currents induced by 3 µM 5-HT alone, while the right set of traces shows currents in the presence of 10 µM haloperidol co-applied with 3 µM 5-HT. Haloperidol reduced the amplitude of 5-HT-induced currents across all holding potentials. (B) Averaged data of normalized peak amplitude of 5-HT3 receptor currents as a function of holding potential. Open circles represent data from 3 µM 5-HT alone, and closed circles represent data from 3 µM 5-HT with 10 µM haloperidol. The reversal potential (VRes) was 3.31 ± 0.33 mV for 5-HT alone and 5.22 ± 0.88 mV for 5-HT co-applied with haloperidol. There was no statistically significant difference in reversal potentials between the two conditions (p = 0.0867, paired t-test, n = 13). Data are expressed as mean ± SEM.

• 

  Fig. 4 Effects of haloperidol on deactivation and desensitization kinetics of 5-hydroxytryptamine (5-HT)3 receptor-mediated currents. (A) Deactivation was analyzed by applying 10 µM 5-HT for 35 msec and measuring the decay phase of the current. The left side shows representative current traces for 5-HT alone (gray trace) and for 5-HT co-applied with 10 µM haloperidol (black trace). The right side shows decay time constants (τ) for each condition, with open circles representing 5-HT alone and closed circles representing 5-HT with haloperidol. The average decay τ was significantly reduced from 1,521 ± 98.68 msec for 5-HT alone to 1,437 ± 95.82 msec for co-application with haloperidol (paired t-test, p < 0.001, n = 11), indicating an accelerated deactivation process. (B) Desensitization assessed by applying 10 µM 5-HT for 20 sec and analyzing the decay phase. The left side shows representative current traces under both conditions, and the right graph displays the decay time constants, with open circles for 5-HT alone and closed circles for the combination with haloperidol. The average decay τ decreased from 2,222 ± 150.8 msec to 1,869 ± 124.6 msec with haloperidol (paired t-test, p < 0.001, n = 11), suggesting that haloperidol accelerated the desensitization process of 5-HT3 receptor currents. Data are expressed as mean ± SEM.

• 

  Fig. 5 Effects of haloperidol on recovery from deactivation and desensitization of 5-hydroxytryptamine (5-HT)3 receptor-mediated currents. (A) Recovery from deactivation was assessed using a two-pulse protocol, with a first pulse of 10 µM 5-HT applied for 35 msec (gray arrow) followed by a second pulse of 2.5 sec at varying inter-pulse intervals (5, 10, 20, 30, 60 sec; black arrows). The left trace shows representative currents for 5-HT alone. The middle trace shows currents for 5-HT co-applied with 10 µM haloperidol. The right graph shows a paired pulse ratio (second peak amplitude/first peak amplitude) as a function of inter-pulse interval. Open circles represent 5-HT alone, and closed circles represent 5-HT with haloperidol. Time constants (τ) for recovery were obtained by fitting paired pulse ratios to a one-phase association equation, yielding a value of 3.45 ± 0.20 sec (n = 9) for 5-HT alone and 3.51 ± 0.22 sec (n = 10) for the co-application (unpaired t-test, p = 0.8450). (B) Recovery from desensitization analyzed with a similar protocol, but with a longer first pulse duration of 5 sec (gray bar). The left trace shows representative currents for 5-HT alone. The middle trace shows currents with co-application of 10 µM haloperidol and the right graph depicts paired pulse ratio versus inter-pulse interval. Open circles represent 5-HT alone, and closed circles represent 5-HT with haloperidol. The fitted τ was 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), indicating no significant effect of haloperidol on recovery from desensitization. Data are expressed as mean ± SEM.

• 

  Fig. 6 Use-dependency of haloperidol on 5-hydroxytryptamine (5-HT)3 receptor currents in NCB-20 cells. (A) Representative current traces recorded during the application of twenty 1-sec pulses of 10 µM 5-HT at 30-sec intervals. Upper traces show the control condition with 5-HT alone, while lower traces display the test condition with 10 µM haloperidol co-applied with 5-HT from the 6th pulse (2.5 min) to the 10th pulse (4.5 min), indicated by grey traces. Bars above traces indicate application of drug. (B) Averaged normalized peak amplitude of 5-HT3 receptor currents plotted against time for both control (n = 10, open circles) and test groups (n = 11, closed circles). A gradual decrease in current amplitude was observed in both groups, likely due to current rundown. (C) Comparison of the peak amplitude ratio (10th/6th pulse) between control and test groups to assess use-dependent inhibition. The bar graph shows no significant difference in peak amplitude ratio between 5-HT alone (0.98 ± 0.01) and haloperidol co-application (0.98 ± 0.01), with a p-value of 0.5702 (unpaired t-test). These findings indicated that haloperidol did not induce significant use-dependent inhibition of 5-HT3 receptor currents under conditions tested. Data are expressed as mean ± SEM. ns, non-significant.

• 

  Fig. 7 Comparison of pretreatment and co-application effects of haloperidol on 5-hydroxytryptamine (5-HT)3 receptor currents. (A) Representative current traces recorded under four different experimental conditions: from left, 3 µM 5-HT alone, co-application of 3 µM 5-HT with 10 µM haloperidol, pretreatment with 10 µM haloperidol for 3 sec followed by 3 µM 5-HT alone, and pretreatment with 10 µM haloperidol for 3 sec followed by co-application with 3 µM 5-HT. All haloperidol treatments reduced peak amplitude of 5-HT3 receptor currents compared to 5-HT alone. (B) Averaged normalized peak amplitudes for the three haloperidol conditions, normalized to the peak amplitude of 5-HT alone. Co-application of 5-HT and haloperidol (blank bar) resulted in a normalized peak amplitude of 0.73 ± 0.04, while pretreatment with haloperidol followed by 5-HT alone (doted bar) resulted in a normalized peak amplitude of 0.74 ± 0.02. Pretreatment with haloperidol followed by co-application with 5-HT (hatched bar) resulted in the greatest inhibition, with a normalized peak amplitude of 0.54 ± 0.03. The difference in peak amplitude between co-application and haloperidol pretreatment followed by 5-HT alone was not significant (p = 0.9878), while peak amplitude of all conditions were significantly different from that of 5-HT alone (one-way ANOVA, p < 0.001). These results suggest that haloperidol effectively inhibits 5-HT3 receptor currents under all conditions, with the greatest inhibition observed when haloperidol is pre-applied before co-application. Data are expressed as mean ± SEM. ns, non-significant.

Reference

1. Miyamoto S, Duncan GE, Marx CE, Lieberman JA. 2005; Treatments for schizophrenia: a critical review of pharmacology and mechanisms of action of antipsychotic drugs. Mol Psychiatry. 10:79–104. DOI: 10.1038/sj.mp.4001556. PMID: 15289815.

2. Santa C, Rodrigues D, Coelho JF, Anjo SI, Mendes VM, Bessa-Neto D, Dunn MJ, Cotter D, Baltazar G, Monteiro P, Manadas B. 2023; Chronic treatment with D2-antagonist haloperidol leads to inhibitory/excitatory imbalance in striatal D1-neurons. Transl Psychiatry. 13:312. DOI: 10.1038/s41398-023-02609-w. PMID: 37803004. PMCID: PMC10558446.

3. Jang J, Kim SH, Um KB, Kim HJ, Park MK. 2024; Somatodendritic organization of pacemaker activity in midbrain dopamine neurons. Korean J Physiol Pharmacol. 28:165–181. DOI: 10.4196/kjpp.2024.28.2.165. PMID: 38414399. PMCID: PMC10902590.

4. Gajendiran M. 2007; Differential effects of spinal 5-HT1A receptor activation and 5-HT2A/2C receptor desensitization by chronic haloperidol. Prog Neuropsychopharmacol Biol Psychiatry. 31:1449–1455. DOI: 10.1016/j.pnpbp.2007.06.020. PMID: 17688988.

5. Barnes NM, Hales TG, Lummis SC, Peters JA. 2009; The 5-HT3 receptor--the relationship between structure and function. Neuropharmacology. 56:273–284. DOI: 10.1016/j.neuropharm.2008.08.003. PMID: 18761359. PMCID: PMC6485434.

6. Thompson AJ, Lummis SC. 2007; The 5-HT3 receptor as a therapeutic target. Expert Opin Ther Targets. 11:527–540. DOI: 10.1517/14728222.11.4.527. PMID: 17373882. PMCID: PMC1994432.

7. Rammes G, Eisensamer B, Ferrari U, Shapa M, Gimpl G, Gilling K, Parsons C, Riering K, Hapfelmeier G, Bondy B, Zieglgänsberger W, Holsboer F, Rupprecht R. 2004; Antipsychotic drugs antagonize human serotonin type 3 receptor currents in a noncompetitive manner. Mol Psychiatry. 9:846–858. DOI: 10.1038/sj.mp.4001490. PMID: 15024394.

8. Kang M, Heo R, Park S, Mun SY, Park M, Han ET, Han JH, Chun W, Ha KS, Park H, Jung WK, Choi IW, Park WS. 2022; Inhibitory effects of the atypical antipsychotic, clozapine, on voltage-dependent K⁺ channels in rabbit coronary arterial smooth muscle cells. Korean J Physiol Pharmacol. 26:277–285. DOI: 10.4196/kjpp.2022.26.4.277. PMID: 35766005. PMCID: PMC9247706.

9. Lambert JJ, Peters JA, Hales TG, Dempster J. 1989; The properties of 5-HT3 receptors in clonal cell lines studied by patch-clamp techniques. Br J Pharmacol. 97:27–40. DOI: 10.1111/j.1476-5381.1989.tb11920.x. PMID: 2720311. PMCID: PMC1854480.

10. Park YS, Myeong SH, Kim IB, Sung KW. 2018; Tricyclic antidepressant amitriptyline inhibits 5-hydroxytryptamine 3 receptor currents in NCB-20 cells. Korean J Physiol Pharmacol. 22:585–595. DOI: 10.4196/kjpp.2018.22.5.585. PMID: 30181705. PMCID: PMC6115347.

11. Cantor RS. 2018; Understanding anesthetic mechanisms: analysis of the complex kinetics of ligand-gated ion channels. Methods Enzymol. 602:97–110. DOI: 10.1016/bs.mie.2018.02.002. PMID: 29588043.

12. Tillman T, Cheng MH, Chen Q, Tang P, Xu Y. 2013; Reversal of ion-charge selectivity renders the pentameric ligand-gated ion channel GLIC insensitive to anaesthetics. Biochem J. 449:61–68. DOI: 10.1042/BJ20121072. PMID: 22978431. PMCID: PMC3992983.

13. Arias HR, Bhumireddy P, Bouzat C. 2006; Molecular mechanisms and binding site locations for noncompetitive antagonists of nicotinic acetylcholine receptors. Int J Biochem Cell Biol. 38:1254–1276. DOI: 10.1016/j.biocel.2006.01.006. PMID: 16520081.

14. Taly A, Hénin J, Changeux JP, Cecchini M. 2014; Allosteric regulation of pentameric ligand-gated ion channels: an emerging mechanistic perspective. Channels (Austin). 8:350–360. DOI: 10.4161/chan.29444. PMID: 25478624. PMCID: PMC4203737.

15. Olsen RW. 2015; Allosteric ligands and their binding sites define γ-aminobutyric acid (GABA) type A receptor subtypes. Adv Pharmacol. 73:167–202. DOI: 10.1016/bs.apha.2014.11.005. PMID: 25637441.

16. Rammes G, Rupprecht R. 2007; Modulation of ligand-gated ion channels by antidepressants and antipsychotics. Mol Neurobiol. 35:160–174. DOI: 10.1007/s12035-007-0006-1. PMID: 17917105.

17. Cappelli A, Donati A, Anzini M, Vomero S, De Benedetti PG, Menziani MC, Langer T. 1996; Molecular structure and dynamics of some potent 5-HT3 receptor antagonists. Insight into the interaction with the receptor. Bioorg Med Chem. 4:1255–1269. DOI: 10.1016/0968-0896(96)00122-8. PMID: 8879547.

18. Jiang C, He X, Wang Y, Chen CJ, Othman Y, Hao Y, Yuan J, Xie XQ, Feng Z. 2023; Molecular modeling study of a receptor-orthosteric ligand-allosteric modulator signaling complex. ACS Chem Neurosci. 14:418–434. DOI: 10.1021/acschemneuro.2c00554. PMID: 36692197. PMCID: PMC10032570.

19. Brömstrup T, Howard RJ, Trudell JR, Harris RA, Lindahl E. 2013; Inhibition versus potentiation of ligand-gated ion channels can be altered by a single mutation that moves ligands between intra- and intersubunit sites. Structure. 21:1307–1316. DOI: 10.1016/j.str.2013.06.018. PMID: 23891290. PMCID: PMC3787718.

20. Hess GP, Ulrich H, Breitinger HG, Niu L, Gameiro AM, Grewer C, Srivastava S, Ippolito JE, Lee SM, Jayaraman V, Coombs SE. 2000; Mechanism-based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and MK-801. Proc Natl Acad Sci U S A. 97:13895–13900. DOI: 10.1073/pnas.240459497. PMID: 11095713. PMCID: PMC17672.

21. Zaitsev AV, Kim KK, Fedorova IM, Dorofeeva NA, Magazanik LG, Tikhonov DB. 2011; Specific mechanism of use-dependent channel block of calcium-permeable AMPA receptors provides activity-dependent inhibition of glutamatergic neurotransmission. J Physiol. 589:1587–1601. Erratum. DOI: 10.1113/jphysiol.2010.204362. PMID: 21486838. PMCID: PMC3099017.

22. Bali M, Akabas MH. 2007; The location of a closed channel gate in the GABAA receptor channel. J Gen Physiol. 129:145–159. DOI: 10.1085/jgp.200609639. PMID: 17227918. PMCID: PMC2154354.

23. Tikhonov DB, Mellor IR, Usherwood PN. 2004; Modeling noncompetitive antagonism of a nicotinic acetylcholine receptor. Biophys J. 87:159–170. DOI: 10.1529/biophysj.103.037457. PMID: 15240454. PMCID: PMC1304339.

24. Thompson AJ, Lummis SC. 2006; 5-HT3 receptors. Curr Pharm Des. 12:3615–3630. DOI: 10.2174/138161206778522029. PMID: 17073663. PMCID: PMC2664614.

25. Juza R, Vlcek P, Mezeiova E, Musilek K, Soukup O, Korabecny J. 2020; Recent advances with 5-HT₃ modulators for neuropsychiatric and gastrointestinal disorders. Med Res Rev. 40:1593–1678. DOI: 10.1002/med.21666. PMID: 32115745.

26. Raudenska M, Gumulec J, Babula P, Stracina T, Sztalmachova M, Polanska H, Adam V, Kizek R, Novakova M, Masarik M. 2013; Haloperidol cytotoxicity and its relation to oxidative stress. Mini Rev Med Chem. 13:1993–1998. DOI: 10.2174/13895575113136660100. PMID: 24160710.

27. Gerace E, Salomone A, Pellegrino S, Vincenti M. 2012; Evidence of Haldol (haloperidol) long-term intoxication. Forensic Sci Int. 215:121–123. DOI: 10.1016/j.forsciint.2011.01.038. PMID: 21371832.

28. Lee HJ, Sung KW, Hahn SJ. 2014; Effects of haloperidol on Kv4.3 potassium channels. Eur J Pharmacol. 740:1–8. DOI: 10.1016/j.ejphar.2014.06.043. PMID: 24998874.

29. Sah DW, Bean BP. 1994; Inhibition of P-type and N-type calcium channels by dopamine receptor antagonists. Mol Pharmacol. 45:84–92. DOI: 10.1016/S0026-895X(25)09907-9.

30. Zink M, Schmitt A, May B, Müller B, Demirakca T, Braus DF, Henn FA. 2004; Differential effects of long-term treatment with clozapine or haloperidol on GABAA receptor binding and GAD67 expression. Schizophr Res. 66:151–157. DOI: 10.1016/S0920-9964(03)00088-4. PMID: 15061247.