Inhibitory effect of compound NS5806 on cardiac transient outward potassium channel dependents on interaction between auxiliary subunits

OBJECTIVE Transient outward potassium current(I_(to)) plays a crucial role in cardiac phase 1 repolarization and the channels are assembled by pore-forming α-subunits(K_v4.2 or K_v4.3) and auxiliary subunits(KCh IP2 and DPP6).Previous studies have found that the compound NS5806 increases I_(to) in canine ventricular cardiomyocytes through slowing current decay.Here,we reported that NS5806 produced an acute inhibitory action on I_(to) in mouse ventricular cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes(hi PS-CM).METHODS Whole-cell patch-clamp was used to record I_(to) in native myocytes and in HEK cells expressing cloned K_v4.x/KCh IP2/DPP6 channels;Western-blot detected the channel protein expression.RESULTS In isolated mouse ventricular cardiomyocytes,NS5806(0.1-30 μmol·L^(-1)) inhibited I_(to) in a concentration-dependent manner with IC50 of 6.6±1.9 μmol·L^(-1).The current decay was significantly accelerated with a time constant from 53.8±5.5 to 41.8±3.0 ms at +60 m V(P<0.01).Similarly,NS5806 concentration-dependently reduced the I_(to) peak current amplitude with an acceleration of current decay.In addition,NS5806 increased I Kv4.2/KCh IP2 and delayed current decay,but decreased I Kv4.2/KCh IP2/DPP6 with the acceleration of current decay.The inhibitory action on the current was more potent if DPP6 expression level was increased from K_v4.2/KCh IP2/DPP6 1∶1∶1 to 1∶1∶3.Western-blot showed a higher expression of DPP6 protein in mouse heart and in hi PS-CM compared to canine heart.Moreover,specific knock-down DPP6 expression by si RNA antagonized the inhibitory action of NS5806 in hi PS-CM.Our results pointed to an important role of DPP6 subunit in the regulation of NS5806 on the channel.By using molecular docking simulation,five interaction sites with high possibility between KCh IP2 and DPP6 were identified.Mutations of those sites changed the inhibitory action of NS58056 into excitatory effect on the current with the delay of current decay.CONCLUSION NS5806 significantly inhibits I_(to) by accelerating current decay in mouse cardiomyocytes and hi PS-CM.The effect depends on the interaction between DPP6 and KCh IP2 subunits.