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Effects of neferine on Kv4.3 channels expressed in HEK293 cells and ex vivo electrophysiology of rabbit hearts.

Wang C, Chen YF, Quan XQ, Wang H, Zhang R, Xiao JH, Wang JL, Zhang CT, Xiang JZ, Tang Q - Acta Pharmacol. Sin. (2015)

Bottom Line: Furthermore, neferine (10 μmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation.Neferine-induced blocking of Kv4.3 currents was frequency-dependent.Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD10 at Epi sites of rabbit heart.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

ABSTRACT

Aim: Neferine is an isoquinoline alkaloid isolated from seed embryos of Nelumbo nucifera (Gaertn), which has a variety of biological activities. In this study we examined the effects of neferine on Kv4.3 channels, a major contributor to the transient outward current (I(to)) in rabbit heart, and on ex vivo electrophysiology of rabbit hearts.

Methods: Whole-cell Kv4.3 currents were recorded in HEK293 cells expressing human cardiac Kv4.3 channels using patch-clamp technique. Arterially perfused wedges of rabbit left ventricles (LV) were prepared, and transmembrane action potentials were simultaneously recorded from epicardial (Epi) and endocardial (Endo) sites with floating microelectrodes together with transmural electrocardiography (ECG).

Results: Neferine (0.1-100 μmol/L) dose-dependently and reversibly inhibited Kv4.3 currents (the IC50 value was 8.437 μmol/L, and the maximal inhibition at 100 μmol/L was 44.12%). Neferine (10 μmol/L) caused a positive shift of the steady-state activation curve of Kv4.3 currents, and a negative shift of the steady-state inactivation curve. Furthermore, neferine (10 μmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation. Neferine-induced blocking of Kv4.3 currents was frequency-dependent. In arterially perfused wedges of rabbit LV, neferine (1, 3, and 10 μmol/L) dose-dependently prolonged the QT intervals and action potential durations (APD) at both Epi and Endo sites, and caused dramatic increase of APD10 at Epi sites.

Conclusion: Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD10 at Epi sites of rabbit heart.

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Effects of neferine on recovery from the inactivation of Kv4.3 channels. (A) Representative traces show the recovery of Kv4.3 channels from inactivation evoked by the double pulse protocol in the absence or presence of neferine. The cells were depolarized to +50 mV for 500 ms (P1) from a holding potential of −80 mV, then returned to the holding potential for a variable time interval between 50 and 5000 ms followed by a second 500-ms pulse to +50 mV (P2). (B) The time courses of recovery from inactivation in the absence or presence of 10 μmol/L neferine. Summarized data are plotted as the relative of amplitude of IKv4.3 (IP2/P1) against the inter-pulse interval. The recovery curves fitted a single-exponential function well. (C) The data show the average recovery time constant (τ) in the absence or presence of 10 μmol/L neferine. The data are expressed as the mean±SEM. n=6. bP<0.05 vs control.
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fig6: Effects of neferine on recovery from the inactivation of Kv4.3 channels. (A) Representative traces show the recovery of Kv4.3 channels from inactivation evoked by the double pulse protocol in the absence or presence of neferine. The cells were depolarized to +50 mV for 500 ms (P1) from a holding potential of −80 mV, then returned to the holding potential for a variable time interval between 50 and 5000 ms followed by a second 500-ms pulse to +50 mV (P2). (B) The time courses of recovery from inactivation in the absence or presence of 10 μmol/L neferine. Summarized data are plotted as the relative of amplitude of IKv4.3 (IP2/P1) against the inter-pulse interval. The recovery curves fitted a single-exponential function well. (C) The data show the average recovery time constant (τ) in the absence or presence of 10 μmol/L neferine. The data are expressed as the mean±SEM. n=6. bP<0.05 vs control.

Mentions: The increase in blocking by neferine at faster rates suggests a time-dependent recovery after repolarization. The time course of recovery from inactivation was determined using a double-pulse protocol. The cells were depolarized to +50 mV for 500 ms (P1) from a holding potential of −80 mV, then returned to the holding potential for a variable time interval between 50 and 5000 ms, followed by a second 500-ms pulse to +50 mV (P2) (Figure 6A). The peak amplitude of the second pulse (P2) was normalized to the corresponding first peak (P1) amplitude. The normalized data were plotted against the inter-pulse interval. The recovery curve fitted a single-exponential function well: y=y0+A1[1-exp(-t/τ)], where τ represents the time course of the recovery from inactivation. A dose of 10 μmol/L neferine significantly slowed the recovery from the inactivation of Kv4.3 current and increased the τ from 278.08± 30.52 ms to 331.40±30.82 ms (n=6, P<0.05) (Figure 6B and 6C).


Effects of neferine on Kv4.3 channels expressed in HEK293 cells and ex vivo electrophysiology of rabbit hearts.

Wang C, Chen YF, Quan XQ, Wang H, Zhang R, Xiao JH, Wang JL, Zhang CT, Xiang JZ, Tang Q - Acta Pharmacol. Sin. (2015)

Effects of neferine on recovery from the inactivation of Kv4.3 channels. (A) Representative traces show the recovery of Kv4.3 channels from inactivation evoked by the double pulse protocol in the absence or presence of neferine. The cells were depolarized to +50 mV for 500 ms (P1) from a holding potential of −80 mV, then returned to the holding potential for a variable time interval between 50 and 5000 ms followed by a second 500-ms pulse to +50 mV (P2). (B) The time courses of recovery from inactivation in the absence or presence of 10 μmol/L neferine. Summarized data are plotted as the relative of amplitude of IKv4.3 (IP2/P1) against the inter-pulse interval. The recovery curves fitted a single-exponential function well. (C) The data show the average recovery time constant (τ) in the absence or presence of 10 μmol/L neferine. The data are expressed as the mean±SEM. n=6. bP<0.05 vs control.
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Related In: Results  -  Collection

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fig6: Effects of neferine on recovery from the inactivation of Kv4.3 channels. (A) Representative traces show the recovery of Kv4.3 channels from inactivation evoked by the double pulse protocol in the absence or presence of neferine. The cells were depolarized to +50 mV for 500 ms (P1) from a holding potential of −80 mV, then returned to the holding potential for a variable time interval between 50 and 5000 ms followed by a second 500-ms pulse to +50 mV (P2). (B) The time courses of recovery from inactivation in the absence or presence of 10 μmol/L neferine. Summarized data are plotted as the relative of amplitude of IKv4.3 (IP2/P1) against the inter-pulse interval. The recovery curves fitted a single-exponential function well. (C) The data show the average recovery time constant (τ) in the absence or presence of 10 μmol/L neferine. The data are expressed as the mean±SEM. n=6. bP<0.05 vs control.
Mentions: The increase in blocking by neferine at faster rates suggests a time-dependent recovery after repolarization. The time course of recovery from inactivation was determined using a double-pulse protocol. The cells were depolarized to +50 mV for 500 ms (P1) from a holding potential of −80 mV, then returned to the holding potential for a variable time interval between 50 and 5000 ms, followed by a second 500-ms pulse to +50 mV (P2) (Figure 6A). The peak amplitude of the second pulse (P2) was normalized to the corresponding first peak (P1) amplitude. The normalized data were plotted against the inter-pulse interval. The recovery curve fitted a single-exponential function well: y=y0+A1[1-exp(-t/τ)], where τ represents the time course of the recovery from inactivation. A dose of 10 μmol/L neferine significantly slowed the recovery from the inactivation of Kv4.3 current and increased the τ from 278.08± 30.52 ms to 331.40±30.82 ms (n=6, P<0.05) (Figure 6B and 6C).

Bottom Line: Furthermore, neferine (10 μmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation.Neferine-induced blocking of Kv4.3 currents was frequency-dependent.Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD10 at Epi sites of rabbit heart.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

ABSTRACT

Aim: Neferine is an isoquinoline alkaloid isolated from seed embryos of Nelumbo nucifera (Gaertn), which has a variety of biological activities. In this study we examined the effects of neferine on Kv4.3 channels, a major contributor to the transient outward current (I(to)) in rabbit heart, and on ex vivo electrophysiology of rabbit hearts.

Methods: Whole-cell Kv4.3 currents were recorded in HEK293 cells expressing human cardiac Kv4.3 channels using patch-clamp technique. Arterially perfused wedges of rabbit left ventricles (LV) were prepared, and transmembrane action potentials were simultaneously recorded from epicardial (Epi) and endocardial (Endo) sites with floating microelectrodes together with transmural electrocardiography (ECG).

Results: Neferine (0.1-100 μmol/L) dose-dependently and reversibly inhibited Kv4.3 currents (the IC50 value was 8.437 μmol/L, and the maximal inhibition at 100 μmol/L was 44.12%). Neferine (10 μmol/L) caused a positive shift of the steady-state activation curve of Kv4.3 currents, and a negative shift of the steady-state inactivation curve. Furthermore, neferine (10 μmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation. Neferine-induced blocking of Kv4.3 currents was frequency-dependent. In arterially perfused wedges of rabbit LV, neferine (1, 3, and 10 μmol/L) dose-dependently prolonged the QT intervals and action potential durations (APD) at both Epi and Endo sites, and caused dramatic increase of APD10 at Epi sites.

Conclusion: Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD10 at Epi sites of rabbit heart.

Show MeSH
Related in: MedlinePlus