Jow Flora, Tseng Eugene, Maddox Taneya, Shen Ru, Kowal Dianne, Dunlop John, Mekonnen Belew, Wang KeWei
Discovery Neuroscience, Wyeth Research, Princeton, NJ 08543, USA.
Assay Drug Dev Technol. 2006 Aug;4(4):443-50. doi: 10.1089/adt.2006.4.443.
The slow delayed rectifier K+ current, Iks, encoded by KCNQ1 (KvLQT1)/KCNE1 (mink) genes, contributes to cardiac action potential repolarization and determines the heartbeat rate. Mutations in either KCNQ1 or KCNE1 that reduce Iks cause long-QT syndrome (LQTS), a disorder of ventricular repolarization that results in cardiac arrhythmia and sudden death. A well-recognized potential treatment for LQTS caused by reduction of Iks is to enhance functional activation of cardiac KCNQ1/KCNE1 channels. In the present study, we generated a stable Chinese hamster ovary cell line that expresses KCNQ1/KCNE1 channels confirmed by electrophysiology. Using a pharmacological tool compound R-L3 (L-364,373 [(3-R)-1,3-dihydro-5-(2-fluorophenyl)-3-(1H-indol- 3-ylmethyl)-1-methyl-2H-1,4-benzodiazepin-2-one]), which activates KCNQ1/mink channels, we then developed and validated a non-radioactive rubidium (Rb+) efflux assay that directly measures the functional activity of KCNQ1/KCNE1 channels by atomic absorption spectroscopy. Our results show that the validated Rb+ efflux assay can be used for screening of KCNQ1/KCNE1 openers that potentially treat LQTS in both inherited and acquired forms. In addition, the assay also can be used for evaluation of possible long-QT liability during cardiac selectivity of new chemical entities.
由KCNQ1（KvLQT1）/KCNE1（mink）基因编码的缓慢延迟整流钾电流Iks，有助于心脏动作电位复极化并决定心率。KCNQ1或KCNE1中导致Iks降低的突变会引起长QT综合征（LQTS），这是一种心室复极化障碍，可导致心律失常和猝死。一种公认的针对因Iks降低引起的LQTS的潜在治疗方法是增强心脏KCNQ1/KCNE1通道的功能激活。在本研究中，我们构建了一个稳定的中国仓鼠卵巢细胞系，该细胞系表达经电生理学确认的KCNQ1/KCNE1通道。使用一种药理学工具化合物R-L3（L-364,373 [(3-R)-1,3-二氢-5-(2-氟苯基)-3-(1H-吲哚-3-基甲基)-1-甲基-2H-1,4-苯并二氮杂卓-2-酮]），它可激活KCNQ1/mink通道，然后我们开发并验证了一种非放射性铷（Rb+）外流测定法，该方法通过原子吸收光谱法直接测量KCNQ1/KCNE1通道的功能活性。我们的结果表明，经过验证的Rb+外流测定法可用于筛选可能治疗遗传性和获得性LQTS的KCNQ1/KCNE1开放剂。此外，该测定法还可用于评估新化学实体心脏选择性过程中可能的长QT风险。
