Aconitine Blocks Human Kv1.5 And HERG Potassium Channels Expressed In Xenopus Laevis Oocytes

Objective:Aconitine(ACO) is a classic drug of arrhythmogenic agents for establishment of arrhythmias in animal models. However its mechanisms of inducing arrhythmias are not clear at present. To elutriate the mechanisms of arrhythmias induced by aconitine, the effects of ACO on HERG and hKvl.5 channels were investigated.Materials and methods:HERG and Kvl.5 channels were expressed in Xenopus laevis oocytes and the resulting currents were recorded using a two-microelectrode voltage-clamp technique. In this study, we used Ala-scanning mutagenesis and voltage clamp analysis of wild type and mutant Kvl.5 and HERG channels expressed in Xenopus laevis oocytes to characterize the blocking activity and molecular basis of aconitine interaction with the pore domain of Kvl.5 and HERG.Results:(1)HERG and HKv1.5 channels could be stably expressed in Xenopus laevis oocytes (2) In HERG channels, ACO exhibited a blockade of voltage and concentration dependent with IC50 1.8±0.33μM (n=6). Meanwhile, the inhibition on HERG channels was time-dependent; the blockade was enhanced by further activation of currents, which were consistent with the open channel blockers.(3)In hKvl.5 channels, ACO produced a voltage-and time-dependent inhibition; which was consistent with open-state blocker. Meanwhile, ACO blocked hKvl.5 channels with a concentration-dependent manner and was partly reversible; the IC50 value for ACO's blockade in Kv1.5 channels was acquired to be 0.8±0.12μM with a Hill coefficient 0.796±0.123μM.(4)The mutation of Y652A significantly suppressed the potency of aconitine to block HERG channel and the IC50 was increased to 381-folded compared with the wild type of HERG channel (The IC50 was 685.545±213.629μM for Y652A, andl.8±0.33μM for WT HERG channel).(5) V505A mutation caused a 21-fold increase in the IC50 (IC50=16.828±2.203μM)for ACO, while I508A,and V512A mutation increased the ICso 1199-fold (IC50=954.671±55.135μM), and 1150-fold (IC50=915.840±213.953μM) respectively..Conclusions:In this study, we first find that aconitine blocks HERG and hKv1.5 channel expressed in Xenopus laevis oocytes in a voltage-and time-and concentrate-dependent manner.In addition, the feature of its blockage is consistent with the open-state channel blocker. Furthermore, aconitine blocks HERG channels as potent as E-4031 which is a typical and selective blocker of HERG channels, on the other hand, it blocks hKvl.5 channel as potent as the specific blockers of hKv1.5 channels such as DPO-1,S0100176 and more potent than AVE0118, the other selective blocker of hKv1.5 channels. These results suggest that blockage of potassium channels, particular HERG channel may be one of important mechanisms of arrhythmias indced by ACO. The mutations of HERG and hKvl.5 channel can result in decrease of block potency for aconitine. Y-652 may be the key binding site of ACO interacting with HERG channel, on the other hand,V505,1508 and V512 may be the key binding site for Kv1.5 channel.