Hong-xue ZHANG,Hua ZHANG,Yu-hong WANG,Yan-fang XU

(1.Department of Pharmacology,Hebei Medical University;the Key Laboratory of New Drug Pharmacology and Toxicolog,Hebei Province;the Key Laboratory of Neural and Vascular Biology,Ministry of Education,Shijiazhuang 050017,China;2.Institute of Masteria Medica,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100050,China)

T3-19

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

Hong-xue ZHANG1,Hua ZHANG1,Yu-hong WANG2,Yan-fang XU1

(1.Department of Pharmacology,Hebei Medical University;the Key Laboratory of New Drug Pharmacology and Toxicolog,Hebei Province;the Key Laboratory of Neural and Vascular Biology,Ministry of Education,Shijiazhuang 050017,China;2.Institute of Masteria Medica,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100050,China)

OBJECTIVETransient outward potassium current(Ito)plays a crucial role in cardiac phase 1 repolarization and the channels are assembled by pore-forming α-subunits(Kv4.2 or Kv4.3)and auxiliary subunits(KChIP2 and DPP6).Previous studies have found that the compound NS5806 increases Itoin canine ventricular cardiomyocytes through slowing current decay.Here,we reported that NS5806 produced an acute inhibitory action on Itoin mouse ventricular cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes(hiPS-CM).METHODSWhole-cell patch-clamp was used to record Itoin native myocytes and in HEK cells expressing cloned Kv4.x/KChIP2/DPP6 channels;Western-blot detected the channel protein expression.RESULTSIn isolated mouse ventricular cardiomyocytes,NS5806(0.1-30 μmol·L-1)inhibited Itoin a concentration-dependent manner with IC50of 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 mV(P＜0.01).Similarly,NS5806 concentration-dependently reduced the Itopeak current amplitude with an acceleration of current decay.In addition,NS5806 increased IKv4.2/KChIP2and delayed current decay,but decreased IKv4.2/KChIP2/DPP6with the acceleration of current decay.The inhibitory action on the current was more potent if DPP6 expression level was increased from Kv4.2/KChIP2/DPP6 1∶1∶1 to 1∶1∶3.Western-blot showed a higher expression of DPP6 protein in mouse heart and in hiPS-CM compared to canine heart.Moreover,specific knock-down DPP6 expression by siRNA antagonized the inhibitory action of NS5806 in hiPS-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 KChIP2 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.CONCLUSIONNS5806 significantly inhibits Itoby accelerating current decay in mouse cardiomyocytes and hiPS-CM.The effect depends on the interaction between DPP6 and KChIP2 subunits.

NS5806;Ito;hiPS-CM;molecular docking

The project supported by University Innovation Team Leading Talent of Hebei Province(LJRC019)

Yan-fang XU,Tel:(0311)86266431,E-mail：yanfangxu@hotmail.com