Jean Jacques A and D'Avanzo N (2024), Inhibition of HCN1 currents by norquetiapine, a...

XB-ART-61009

Front Pharmacol 2024 Oct 07;15:1445509. doi: 10.3389/fphar.2024.1445509.

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Inhibition of HCN1 currents by norquetiapine, an active metabolite of the atypical anti-psychotic drug quetiapine.

Jean Jacques A , D'Avanzo N .

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Quetiapine is a second-generation atypical antipsychotic drug that has been commonly prescribed for the treatment of schizophrenia, major depressive disorder (depression), and other psychological disorders. Targeted inhibition of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels, which generate Ih, may provide effective resistance against schizophrenia and depression. We investigated if HCN channels could contribute to the therapeutic effect of quetiapine, and its major active metabolite norquetiapine. Two-electrode voltage clamp recordings were used to assess the effects of quetiapine and its active metabolites 7-hydroxyquetiapine and norquetiapine on currents from HCN1 channels expressed in Xenopus laevis oocytes. Norquetiapine, but not quetiapine nor 7-hydroxyquetiapine, has an inhibitory effect on HCN1 channels. Norquetiapine selectively inhibited HCN1 currents by shifting the voltage-dependence of activation to more hyperpolarized potentials in a concentration-dependent manner with an IC50 of 13.9 ± 0.8 μM for HCN1 and slowing channel opening, without changing the kinetics of closing. Inhibition by norquetiapine primarily occurs from in the closed state. Norquetiapine inhibition is not sensitive to the external potassium concentration, and therefore, likely does not block the pore. Norquetiapine inhibition also does not dependent on the cyclic-nucleotide binding domain. Norquetiapine also inhibited HCN4 channels with reduced efficacy than HCN1 and had no effect on HCN2 channels. Therefore, HCN channels are key targets of norquetiapine, the primary active metabolite of quetiapine. These data help to explain the therapeutic mechanisms by which quetiapine aids in the treatment of anxiety, major depressive disorder, bipolar disorder, and schizophrenia, and may represent a novel structure for future drug design of HCN inhibitors.

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Species referenced: Xenopus laevis
Genes referenced: hcn1 hcn2 hcn4 shank3

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	Figure 1. Chemical structures of Quetiapine (QTP) and its active metabolites Norquetiapine (NQTP) and 7-hydroxyquetiapine (7-OH QTP).
	Figure 2. Quetiapine (QTP) does not regulate HCN1 channels. (A) Representative traces from a paired activation experiment following the addition of 30 µM QTP to oocytes expressing full-length HCN1. 30 μM QTP does not affect (B) the voltage-dependence of activation (V1/2 of each cell is shown on the right) (n = 5, P = 0.11) (C) the current-voltage (I-V) relationship (n = 5, P = 0.35) (D) activation kinetics (n = 5, P = 0.56) or (E) deactivation kinetics (n = 5, P = 0.72) of HCN1 channels.
	Figure 3. Norquetiapine (NQTP) inhibits HCN1 channels. (A) Representative traces from a paired activation experiment (control in black) following the addition of 30 µM NQTP (red traces) to oocytes expressing full-length HCN1. (B) NQTP induces a hyperpolarizing shift in the steady-state voltage-dependence of activation (P < 0.05 for V1/2 for 7.5 and 15 min compared to control). (C) Activation time constants (τfast and τslow) are greater in the presence of NQTP (P < 0.05). (D) Deactivation time constants (τdeact) are unchanged in presence of NQTP. (n = 6; P = 0.23). (E) Current-voltage (I-V) relationship in presence of NQTP normalized to maximal current (IControl (−130 mV)). (n = 9; P < 0.05 for 7.5 and 15 min compared to control). (F) Concentration dependence of ΔV1/2 fit with a Hill Equation 3 indicates NQTP inhibits HCN1 channels with an IC50 of 13.9 ± 0.8 μM, a maximum ΔV1/2 of −15.4 ± 1.2 mV and a Hill co-efficient of 4.2 ± 0.1.
	Figure 4. 7-hydroxyquetiapine (7-OH QTP) does not regulate HCN1 channels. (A) Representative traces from a paired activation experiment (control in black) following the addition of 30 µM 7-OH QTP (red traces) to oocytes expressing full-length HCN1. 30 μM 7-OH QTP does not affect (B) the voltage-dependence of activation (V1/2 of each cell is shown on the right) (n = 5, P = 0.64) (C), the current-voltage (I-V) relationship (n = 5, p = 0.47), (D) activation kinetics (n = 5, p = 0.88), or (E) deactivation kinetics (n = 6, P = 0.35) of HCN1 channels.
	Figure 5. Norquetiapine (NQTP) does not inhibit HCN2 channels. (A) Representative traces from a paired activation experiment (control in black) following the addition of 30 µM NQTP (red traces) to oocytes expressing full-length HCN1. Unlike what we observe for HCN1 channels, 30 µM NQTP does not alter (B) the voltage-dependence of activation (V1/2 of each cell is shown on the right) (n = 6, P = 0.09), (C) the current-voltage (I-V) relationship (n = 6, p = 0.79) nor (D) the activation kinetics (n = 6, p = 0.65).
	Figure 6. Norquetiapine (NQTP) inhibits HCN4 channels. (A) NQTP induces a hyperpolarizing shift in the steady-state voltage-dependence of activation (p < 0.05 for V1/2) (V1/2 of each cell is shown on the right) (n = 9, p = 0.001). (B) Current-voltage (I-V) relationship in presence of NQTP normalized to maximal current (IControl (-160 mV)). (n = 9; p = 0.63 for 7.5 min compared to control). (C) Activation time constants (τfast and τslow) are greater in the presence of NQTP (P < 0.05).
	Figure 7. State-dependence of norquetiapine (NQTP) inhibition of HCN1 channels. (A) Open-state block was assessed by a prolonged activation step to −130 mV and applying 30 µM NQTP at steady-state. (B) Relative current (INQTP/Icontrol) to be 0.95 ± 0.01 following NQTP treatment (n = 5; P < 0.05). (C) Closed-state block was assessed using a repetitive −130 mV/+30 mV pulse protocol every 30 s 30 μM NQTP was applied after the stabilization of HCN1 currents, and resulted in a decrease of current by 20.4% ± 1.1% (n = 7). (D) When the repetitive protocol is interrupted and cells are held at VH = −10 mV for 7.5 min during the application of NQTP the amount of inhibition that is induced is 15.8% ± 0.7% (n = 9) indicating NQTP is a closed state blocker of HCN1.
	Figure 8. Norquetiapine (NQTP) inhibition of HCN1 channels does not depend on the CNBD. (A) Representative traces from a paired activation experiment (control in black) following the addition of 30 µM NQTP (red traces) to oocytes expressing HCN1ΔCNBD. (B) Current-voltage (I-V) relationship in presence of NQTP normalized to maximal current (Icontrol (-130 mV)). (n = 6; P < 0.05). (C) NQTP induces a hyperpolarizing shift in the steady-state voltage-dependence of activation (P < 0.05).
	Figure 9. Norquetiapine (NQTP) inhibition of HCN1 channels is not affected by increasing [K+]o. (A) Representative traces from a paired activation experiment following the addition of 30 µM NQTP (red traces) to oocytes expressing full-length HCN1 bathed in high external potassium (control in black). (B) Current-voltage (I-V) relationship in presence of NQTP normalized to maximal current (Icontrol (-130 mV)). (n = 6; P < 0.05). (C) NQTP induces a hyperpolarizing shift in the steady-state voltage-dependence of activation (P < 0.05 for V1/2).
	FIGURE 1. Chemical structures of Quetiapine (QTP) and its active metabolites Norquetiapine (NQTP) and 7-hydroxyquetiapine (7-OH QTP).
	FIGURE 2. Quetiapine (QTP) does not regulate HCN1 channels. (A) Representative traces from a paired activation experiment following the addition of 30 µM QTP to oocytes expressing full-length HCN1. 30 μM QTP does not affect (B) the voltage-dependence of activation (V1/2 of each cell is shown on the right) (n = 5, P = 0.11) (C) the current-voltage (I-V) relationship (n = 5, P = 0.35) (D) activation kinetics (n = 5, P = 0.56) or (E) deactivation kinetics (n = 5, P = 0.72) of HCN1 channels.
	FIGURE 3. Norquetiapine (NQTP) inhibits HCN1 channels. (A) Representative traces from a paired activation experiment (control in black) following the addition of 30 µM NQTP (red traces) to oocytes expressing full-length HCN1. (B) NQTP induces a hyperpolarizing shift in the steady-state voltage-dependence of activation (P < 0.05 for V1/2 for 7.5 and 15 min compared to control). (C) Activation time constants (τfast and τslow) are greater in the presence of NQTP (P < 0.05). (D) Deactivation time constants (τdeact) are unchanged in presence of NQTP. (n = 6; P = 0.23). (E) Current-voltage (I-V) relationship in presence of NQTP normalized to maximal current (IControl (−130 mV)). (n = 9; P < 0.05 for 7.5 and 15 min compared to control). (F) Concentration dependence of ΔV1/2 fit with a Hill Equation 3 indicates NQTP inhibits HCN1 channels with an IC50 of 13.9 ± 0.8 μM, a maximum ΔV1/2 of −15.4 ± 1.2 mV and a Hill co-efficient of 4.2 ± 0.1.
	FIGURE 4. 7-hydroxyquetiapine (7-OH QTP) does not regulate HCN1 channels. (A) Representative traces from a paired activation experiment (control in black) following the addition of 30 µM 7-OH QTP (red traces) to oocytes expressing full-length HCN1. 30 μM 7-OH QTP does not affect (B) the voltage-dependence of activation (V1/2 of each cell is shown on the right) (n = 5, P = 0.64) (C), the current-voltage (I-V) relationship (n = 5, p = 0.47), (D) activation kinetics (n = 5, p = 0.88), or (E) deactivation kinetics (n = 6, P = 0.35) of HCN1 channels.
	FIGURE 5. Norquetiapine (NQTP) does not inhibit HCN2 channels. (A) Representative traces from a paired activation experiment (control in black) following the addition of 30 µM NQTP (red traces) to oocytes expressing full-length HCN1. Unlike what we observe for HCN1 channels, 30 µM NQTP does not alter (B) the voltage-dependence of activation (V1/2 of each cell is shown on the right) (n = 6, P = 0.09), (C) the current-voltage (I-V) relationship (n = 6, p = 0.79) nor (D) the activation kinetics (n = 6, p = 0.65).
	FIGURE 6. Norquetiapine (NQTP) inhibits HCN4 channels. (A) NQTP induces a hyperpolarizing shift in the steady-state voltage-dependence of activation (p < 0.05 for V1/2) (V1/2 of each cell is shown on the right) (n = 9, p = 0.001). (B) Current-voltage (I-V) relationship in presence of NQTP normalized to maximal current (IControl (-160 mV)). (n = 9; p = 0.63 for 7.5 min compared to control). (C) Activation time constants (τfast and τslow) are greater in the presence of NQTP (P < 0.05).
	FIGURE 7. State-dependence of norquetiapine (NQTP) inhibition of HCN1 channels. (A) Open-state block was assessed by a prolonged activation step to −130 mV and applying 30 µM NQTP at steady-state. (B) Relative current (INQTP/Icontrol) to be 0.95 ± 0.01 following NQTP treatment (n = 5; P < 0.05). (C) Closed-state block was assessed using a repetitive −130 mV/+30 mV pulse protocol every 30 s 30 μM NQTP was applied after the stabilization of HCN1 currents, and resulted in a decrease of current by 20.4% ± 1.1% (n = 7). (D) When the repetitive protocol is interrupted and cells are held at VH = −10 mV for 7.5 min during the application of NQTP the amount of inhibition that is induced is 15.8% ± 0.7% (n = 9) indicating NQTP is a closed state blocker of HCN1.
	FIGURE 8. Norquetiapine (NQTP) inhibition of HCN1 channels does not depend on the CNBD. (A) Representative traces from a paired activation experiment (control in black) following the addition of 30 µM NQTP (red traces) to oocytes expressing HCN1ΔCNBD. (B) Current-voltage (I-V) relationship in presence of NQTP normalized to maximal current (Icontrol (-130 mV)). (n = 6; P < 0.05). (C) NQTP induces a hyperpolarizing shift in the steady-state voltage-dependence of activation (P < 0.05).
	FIGURE 9. Norquetiapine (NQTP) inhibition of HCN1 channels is not affected by increasing [K+]o. (A) Representative traces from a paired activation experiment following the addition of 30 µM NQTP (red traces) to oocytes expressing full-length HCN1 bathed in high external potassium (control in black). (B) Current-voltage (I-V) relationship in presence of NQTP normalized to maximal current (Icontrol (-130 mV)). (n = 6; P < 0.05). (C) NQTP induces a hyperpolarizing shift in the steady-state voltage-dependence of activation (P < 0.05 for V1/2).