Korean J Physiol Pharmacol.  2022 Jul;26(4):277-285. 10.4196/kjpp.2022.26.4.277.

Inhibitory effects of the atypical antipsychotic, clozapine, on voltage-dependent K⁺ channels in rabbit coronary arterial smooth muscle cells

• 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

• KMID: 2530947
• DOI: http://doi.org/10.4196/kjpp.2022.26.4.277

Abstract

To investigate the adverse effects of clozapine on cardiovascular ion channels, we examined the inhibitory effect of clozapine on voltage-dependent K⁺(Kv) channels in rabbit coronary arterial smooth muscle cells. Clozapine-induced inhibition of Kv channels occurred in a concentration-dependent manner with an halfinhibitory concentration value of 7.84 ± 4.86 µM and a Hill coefficient of 0.47 ± 0.06. Clozapine did not shift the steady-state activation or inactivation curves, suggesting that it inhibited Kv channels regardless of gating properties. Application of train pulses (1 and 2 Hz) progressively augmented the clozapine-induced inhibition of Kv channels in the presence of the drug. Furthermore, the recovery time constant from inactivation was increased in the presence of clozapine, suggesting that clozapineinduced inhibition of Kv channels is use (state)-dependent. Pretreatment of a Kv1.5 subtype inhibitor decreased the Kv current amplitudes, but additional application of clozapine did not further inhibit the Kv current. Pretreatment with Kv2.1 or Kv7 subtype inhibitors partially blocked the inhibitory effect of clozapine. Based on these results, we conclude that clozapine inhibits arterial Kv channels in a concentrationand use (state)-dependent manner. Kv1.5 is the major subtype involved in clozapineinduced inhibition of Kv channels, and Kv2.1 and Kv7 subtypes are partially involved.

Keyword

Clozapine; Coronary artery; Kv1.5 potassium channel; Kv potassium channel; Use-dependent

Figure

• 

  Fig. 1 Inhibitory effect of clozapine on voltage-dependent K+ (Kv) currents in rabbit coronary artery smooth muscle cells. Kv current traces were obtained in response to 600-ms step-depolarizing pulses from a holding potential of −80 mV in the (A) absence or (B) presence of 10 μM clozapine using the same cells. (C) The current–voltage (I–V) curves of the steady-state Kv currents in the absence (○) and presence (●) of 10 μM clozapine. n = 8. The “n” means the number of cells. Only one cell was used from a rabbit to reduce individual differences. Data are mean ± SEM. *p < 0.05 (control vs. clozapine, at each voltage).

• 

  Fig. 2 Concentration dependence of the effects of clozapine on voltage-dependent K+ (Kv) currents. Current recordings of Kv channels were elicited by 600-ms one-step depolarizing pulses of +60 mV from a holding potential of −80 mV. (A) Superimposed current traces of Kv currents with increasing concentration of clozapine. (B) Concentration dependence of the inhibitory effect of clozapine. The inhibition (%) of Kv currents measured at the end of pulses is plotted against the clozapine concentration. The smooth line shows the best fit with a Hill equation. n = 11. Data are mean ± SEM.

• 

  Fig. 3 Lack of effect of clozapine on voltage dependence of steady-state activation and inactivation curve. (A) Activation curves of the voltage-dependent K+ (Kv) currents in the absence (○) and presence (●) of 10 μM clozapine. The tail currents were evoked by a returning potential of −40 mV after 20–50 ms short depolarizing pulses at different voltages. n = 9. (B) Inactivation curves of the Kv currents in the absence (○) and presence (●) of 10 μM clozapine. The curves were elicited by a returning potential of +40 mV after 7-sec depolarizing pulses at different voltages. n = 9. Data are mean ± SEM.

• 

  Fig. 4 Use (state)-dependent inhibitory effect of clozapine on voltage-dependent K+ (Kv) channels. Twenty repetitive depolarizing pulses from −80 to +60 mV with a duration of 150 ms were applied at frequencies of (A) 1 Hz and (B) 2 Hz, in the absence (○) and presence (●) of 10 μM clozapine. The peak currents of the Kv channels were normalized relative to the maximal peak current generated by the first pulse, and then plotted against the pulse numbers. n = 11. Data are mean ± SEM. *p < 0.05 (control vs. clozapine, at each pulse number).

• 

  Fig. 5 Time course of recovery from clozapine-induced inhibition of voltage-dependent K+ (Kv) channels. The recovery time constants were measured by applying an identical twin pulse, as shown in the inset. A 150-ms depolarizing pre-pulse from −80 to +60 mV was followed by an identical second pulse with increasing intervals from 30 ms to 7 sec. Solid lines represent recovery of Kv currents in the absence (○) and presence (●) of 10 μM clozapine. n = 5. Data are mean ± SEM. *p < 0.05 (control vs. clozapine, at each time point).

• 

  Fig. 6 Inhibitory effects of clozapine on voltage-dependent K+ (Kv)1.5, Kv2.1, and Kv7 subtypes. The representative Kv currents were elicited by applying 600-ms one-step depolarizing pulses of +60 mV from a holding potential of −80 mV. (A) Kv currents under control conditions, in the presence of DPO-1, and DPO-1 + clozapine. (B) Summary of the effects of DPO-1 on the inhibitory effect of clozapine on Kv currents. n = 5. NS, not significant (DPO-1 vs. DPO-1 + clozapine). (C) Kv currents under control conditions, in the presence of guangxitoxin, and in the presence of guangxitoxin + clozapine. (D) Summary of the effects of guangxitoxin on the inhibitory effect of clozapine on Kv currents. n = 4. *p < 0.05 (guangxitoxin vs. guangxitoxin + clozapine). (E) Kv currents under control conditions, in the presence of linopirdine, and in the presence of linopirdine + clozapine. (F) Summary of the effects of linopirdine on the inhibitory effect of clozapine on Kv currents. n = 5. *p < 0.05 (linopirdine vs. linopirdine + clozapine). Data are mean ± SEM.

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