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Acta Pharmacol Sin
2010 Mar 01;313:297-306. doi: 10.1038/aps.2010.8.
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Blocking effect of methylflavonolamine on human Na(V)1.5 channels expressed in Xenopus laevis oocytes and on sodium currents in rabbit ventricular myocytes.
Fan XR
,
Ma JH
,
Zhang PH
,
Xing JL
.
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To investigate the blocking effects of methylflavonolamine (MFA) on human Na(V)1.5 channels expressed in Xenopus laevis oocytes and on sodium currents (I(Na)) in rabbit ventricular myocytes. Human Na(V)1.5 channels were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. I(Na) and action potentials in rabbit ventricular myocytes were studied using the whole-cell recording. MFA and lidocaine inhibited human Na(V)1.5 channels expressed in Xenopus oocytes in a positive rate-dependent and concentration-dependent manner, with IC(50) values of 72.61 micromol/L and 145.62 micromol/L, respectively. Both of them markedly shifted the steady-state activation curve of I(Na) toward more positive potentials, shifted the steady-state inactivation curve of I(Na) toward more negative potentials and postponed the recovery of the I(Na) inactivation state. In rabbit ventricular myocytes, MFA inhibited I(Na) with a shift in the steady-state inactivation curve toward more negative potentials, thereby postponing the recovery of the I(Na) inactivation state. This shift was in a positive rate-dependent manner. Under current-clamp mode, MAF significantly decreased action potential amplitude (APA) and maximal depolarization velocity (V(max)) and shortened action potential duration (APD), but did not alter the resting membrane potential (RMP). The demonstrated that the kinetics of sodium channel blockage by MFA resemble those of class I antiarrhythmic agents such as lidocaine. MFA protects the heart against arrhythmias by its blocking effect on sodium channels.
Figure 1. MFA, plasmid and sodium current. (A) Diagram of the molecular structure of MFA. (B) Electrophoresis identification of the pcDNA 3.1 HA-SCN5A (NaV1.5) plasmid vector; Lane 1, the plasmid with Csp45 I restriction enzyme digestion; Lane 2, the pcDNA 3.1 HA-SCN5A (NaV1.5) plasmid vector. (C) Confirmation of INa. Depolarizing pulses from -120 mV to -40 mV for 100 ms were applied. Current tracings of human NaV1.5 channels expressed in Xenopus oocytes are superimposed before (control) and during superfusion of 20 μmol/L TTX.
Figure 2. Concentration-dependent blockage of INa by MFA or lidocaine. Depolarizing pulses from -120 mV to -40 mV for 100 ms were applied. (A) Current tracings of human NaV1.5 channels expressed in Xenopus oocytes are superimposed before (control) and during superfusion of MFA (5â250 μmol/L) or lidocaine (5â750 μmol/L). (B) The concentration-response curves are plotted based on data from panel A and fitted by the Hill equation. (C) Time-course of the effects of MFA on human NaV1.5 channels expressed in Xenopus oocytes. Oocytes were perfused with ND96 solution for 5 min before application of 100 μmol/L MFA or without drug and again with ND96 solution (washout). The normalized currents are plotted during the recording course. (D) The effects of MFA on the current-voltage relationship of human NaV1.5 channels expressed in Xenopus oocytes. Depolarizing pulses (100 ms duration) were applied from a holding potential of -120 mV to various potentials ranging from -100 mV to +40 mV with 5 mV increments.
Figure 3. Effects of MFA or lidocaine on steady-state activation and inactivation of INa in Xenopus oocytes. The activation protocol was obtained from the current-voltage relationship (100-ms depolarizing pulses to potentials ranging from -70 mV to -20 mV in 5 mV increments), and the inactivation curve was obtained using of a double-pulse protocol (100-ms conditioning prepulses from a holding potential of -100 mV to -40 mV in 5 mV increments), followed by a test pulse to -10 mV for 100 ms. (A and B) The activation and inactivation currents of human NaV1.5 channels expressed in Xenopus oocytes before and after superfusion of 100 μmol/L MFA or 250 μmol/L lidocaine, respectively. (C) The normalized steady-state activation and inactivation of human NaV1.5 channels expressed in Xenopus oocytes plotted in the absence and presence of 100 μmol/L MFA or 250 μmol/L lidocaine.
Figure 4. Effects of MFA or lidocaine on recovery from inactivation of INa in Xenopus oocytes. The recovery from inactivation was studied using a conventional double-pulse protocol (a 50-ms prepulse to -40 mV from a holding potential of -120 mV, followed by a variable recovery period from 2â80 ms and then by a test pulse to -40 mV for 50 ms). (A) Example tracing of the recovery from inactivation of human NaV1.5 channels expressed in Xenopus oocytes before and after superfusion of 100 μmol/L MFA or 250 μmol/L lidocaine. (B) The normalized recovery from inactivation of human NaV1.5 channels expressed in Xenopus oocytes plotted in the absence and presence of 100 μmol/L MFA or 250 μmol/L lidocaine.
Figure 5. Rate-dependent blockage of INa by MFA or lidocaine in Xenopus oocytes. A series of 30 depolarizing pulses with 30 ms duration from a holding potential of -120 mV to -40 mV at different stimulation frequencies was applied to rabbit ventricular myocytes. After stimulating at each frequency, cells were perfused with normal solution, and INa recovered substantially. (A and C) Superimposed tracings of INa obtained from different experiments: in control at 4 Hz, and in the presence of 75 μmol/L MFA or 250 μmol/L lidocaine at 1 Hz, 2 Hz and 4 Hz. (B and D) Relative INa was plotted against each pulse number. Relative INa was defined as the ratio of INa at each pulse number/INa at the first pulse.
Figure 6. Onset of INa blockage by MFA in Xenopus oocytes. Prepulses of various durations (1â100 ms) were applied to -30 mV from a holding potential of -120 mV, after a 250 ms recovery period at -120 mV and after a 20 ms test pulse was applied again to -30 mV. (A) Current tracings of human NaV1.5 channels expressed in Xenopus oocytes before and after superfusion of 100 μmol/L MFA. (B) The normalized onset blockage of INa in Xenopus oocytes plotted in the absence and presence of 100 μmol/L MFA.
Figure 7. Effects of MFA on current-voltage relationship, steady state activation and inactivation of INa in rabbit ventricular myocytes. The pulse protocols were as described in Figures 2 and 3. (A) Sodium current tracings of rabbit ventricular myocytes superimposed before (control), during superfusion of MFA 75 μmol/L and after washout. (B and C) Activation and inactivation currents of rabbit ventricular myocytes before and after superfusion of 100 μmol/L MFA, respectively. (DâF) The effects of 100 μmol/L MFA on the current-voltage relationship, steady-state activation and inactivation curves of INa in rabbit ventricular myocytes, respectively.
Figure 8. Effects of MFA on the recovery from inactivation and rate-dependent blockage of INa in rabbit ventricular myocytes. The clamp protocols were as described in Figures 4 and 5. (A and C) Current tracings of rabbit ventricular myocytes before and after superfusion of 100 or 75 μmol/L MFA, respectively. For reasons of clarity, only the first (P1) and the last (P30) currents are shown. (B and D) Normalized INa was plotted in the absence and presence of 100 or 75 μmol/L MFA and against the recovery time or each pulse number, respectively.
Figure 9. Effects of MFA on AP characteristics in rabbit ventricular myocytes. APs were elicited by depolarizing pulses delivered for 5 ms and at 1.5-fold above the threshold at a rate of 1 Hz. Representative recordings of APs are superimposed before (control), during superfusion of MFA 75 μmol/L and after washout.
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