Barry D M, Xu H, Schuessler R B, Nerbonne J M
Department of Molecular Biology, Washington University Medical School, St Louis, MO 63110, USA.
Circ Res. 1998 Sep 7;83(5):560-7. doi: 10.1161/01.res.83.5.560.
A novel in vivo experimental strategy, involving cell type-specific expression of a dominant-negative K+ channel pore-forming alpha subunit, was developed and exploited to probe the molecular identity of the cardiac transient outward K+ current (I(to)). A point mutation (W to F) was introduced at position 362 in the pore region of Kv4.2 to produce a nonconducting mutant (Kv4.2W362F) subunit. Coexpression of Kv4.2W362F with Kv4.2 (or Kv4.3) attenuates the wild-type currents, and the effect is subfamily specific; ie, Kv4.2W362F does not affect heterologously expressed Kv1.4 currents. With the use of the alpha-myosin heavy chain promoter to direct cardiac-specific expression, several lines of Kv4.2W362F transgenic mice were generated. Electrophysiological recordings reveal that I(to) is selectively eliminated in ventricular myocytes isolated from transgenic mice expressing Kv4.2W362F, thereby demonstrating directly that the Kv 4 subfamily underlies I(to) in the mammalian heart. Functional knockout of I(to) leads to marked increases in action potential durations in ventricular myocytes and to prolongation of the QT interval in surface ECG recordings. In addition, a novel rapidly activating and inactivating K+ current, which is not detectable in myocytes from nontransgenic littermates, is evident in Kv4.2W362F-expressing ventricular cells. Importantly, these results demonstrate that electrical remodeling occurs in the heart when the expression of endogenous K- channels is altered.
一种新的体内实验策略被开发并应用,该策略涉及细胞类型特异性表达显性负性钾通道孔形成α亚基,以探究心脏瞬时外向钾电流(I(to))的分子特性。在Kv4.2的孔区域第362位引入点突变(W到F)以产生非传导性突变体(Kv4.2W362F)亚基。Kv4.2W362F与Kv4.2(或Kv4.3)共表达会减弱野生型电流,且这种作用具有亚家族特异性;即,Kv4.2W362F不影响异源表达的Kv1.4电流。利用α-肌球蛋白重链启动子指导心脏特异性表达,产生了几株Kv4.2W362F转基因小鼠。电生理记录显示,在从表达Kv4.2W362F的转基因小鼠分离的心室肌细胞中,I(to)被选择性消除,从而直接证明Kv4亚家族是哺乳动物心脏中I(to)的基础。I(to)的功能性敲除导致心室肌细胞动作电位持续时间显著增加,并使体表心电图记录中的QT间期延长。此外,一种新的快速激活和失活的钾电流在表达Kv4.2W362F的心室细胞中明显可见,而在非转基因同窝小鼠的心肌细胞中则检测不到。重要的是,这些结果表明,当内源性钾通道的表达改变时,心脏会发生电重构。