Wang W, Xia J, Kass R S
Department of Pharmacology, College of Physicians & Surgeons of Columbia University, New York, New York 10032, USA.
J Biol Chem. 1998 Dec 18;273(51):34069-74. doi: 10.1074/jbc.273.51.34069.
IsK, a slowly activating delayed rectifier K+ current through channels formed by the assembly of two channel proteins KvLQT1 and MinK, modulates the repolarization of cardiac action potentials. Mutations that map to the KvLQT1 and minK genes account for more than 50% of an inherited cardiac disorder, the Long QT syndrome (Splawski, I., Tristani-Firouzi, M., Lehmann, M. H., Sanguinetti, M. C., and Keating, M. T. (1997) Nat. Genet. 17, 338-340). Despite the importance of these channels to human cardiac function, the molecular basis of their uniquely slow gating properties as well as the stoichiometry and interaction sites of these two subunits are still unclear. We have constructed several fusion channel proteins to begin investigating the stoichiometry of these two subunits and the role of voltage-dependent subunit assembly in channel gating. Functional properties of these constructs were measured using whole cell patch clamp recordings of transiently transfected Chinese hamster ovary cells. The constructs we tested are as follows: MK24 (C terminus of MinK linked to N terminus of KvLQT1); KK40 (a tandem homodimer of KvLQT1); and MKK44 (C terminus of MinK linked to N terminus of KK40). In control experiments (no DNA, control DNA, or only MinK), no time-dependent K+ current was observed. Expression of KvLQT1 or KK40 produced currents that activate and inactivate in a voltage-dependent manner as reported by others for KvLQT1. In contrast, expression of MK24 and MKK44 elicited current with activation kinetics and voltage dependence very similar to native IsK and identical to currents expressed by cells co-transfected with independent MinK and KvLQT1 cDNA. Expression of MK24 plus additional MinK significantly slows current kinetics. Our data raise the possibility 1) of multiple MinK/KvLQT1 stoichiometries and 2) indicate that uniquely slow kinetics of IsK channels is due to voltage-dependent conformational changes of the channel protein and not to assembly of channel subunits.
IsK是一种通过由两个通道蛋白KvLQT1和MinK组装形成的通道的缓慢激活延迟整流钾电流，它调节心脏动作电位的复极化。定位到KvLQT1和minK基因的突变占遗传性心脏疾病长QT综合征的50%以上（斯普拉夫斯基，I.，特里斯坦尼 - 菲鲁齐，M.，莱曼，M. H.，桑吉内蒂，M. C.，和基廷，M. T.（1997年）《自然遗传学》17卷，338 - 340页）。尽管这些通道对人类心脏功能很重要，但其独特的缓慢门控特性的分子基础以及这两个亚基的化学计量和相互作用位点仍不清楚。我们构建了几种融合通道蛋白，以开始研究这两个亚基的化学计量以及电压依赖性亚基组装在通道门控中的作用。使用瞬时转染的中国仓鼠卵巢细胞的全细胞膜片钳记录来测量这些构建体的功能特性。我们测试的构建体如下：MK24（MinK的C末端与KvLQT1的N末端相连）；KK40（KvLQT1的串联同型二聚体）；和MKK44（MinK的C末端与KK40的N末端相连）。在对照实验（无DNA、对照DNA或仅MinK）中，未观察到时间依赖性钾电流。KvLQT1或KK40的表达产生的电流如其他人报道的KvLQT1那样以电压依赖性方式激活和失活。相比之下，MK24和MKK44的表达引发的电流具有与天然IsK非常相似的激活动力学和电压依赖性，并且与用独立的MinK和KvLQT1 cDNA共转染的细胞表达的电流相同。MK24加上额外的MinK的表达显著减慢电流动力学。我们的数据提出了以下可能性：1）多种MinK/KvLQT1化学计量；2）表明IsK通道独特的缓慢动力学是由于通道蛋白的电压依赖性构象变化，而不是通道亚基的组装。
