Xu H, Barry D M, Li H, Brunet S, Guo W, Nerbonne J M
Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, MO 63110, USA.
Circ Res. 1999 Oct 1;85(7):623-33. doi: 10.1161/01.res.85.7.623.
An in vivo experimental strategy, involving cardiac-specific expression of a mutant Kv 2.1 subunit that functions as a dominant negative, was exploited in studies focused on exploring the role of members of the Kv2 subfamily of pore-forming (alpha) subunits in the generation of functional voltage-gated K(+) channels in the mammalian heart. A mutant Kv2.1 alpha subunit (Kv2.1N216) was designed to produce a truncated protein containing the intracellular N terminus, the S1 membrane-spanning domain, and a portion of the S1/S2 loop. The truncated Kv2.1N216 was epitope tagged at the C terminus with the 8-amino acid FLAG peptide to generate Kv2. 1N216FLAG. No ionic currents are detected on expression of Kv2. 1N216FLAG in HEK-293 cells, although coexpression of this construct with wild-type Kv2.1 markedly reduced the amplitudes of Kv2. 1-induced currents. Using the alpha-myosin heavy chain promoter to direct cardiac specific expression of the transgene, 2 lines of Kv2. 1N216FLAG-expressing transgenic mice were generated. Electrophysiological recordings from ventricular myocytes isolated from these animals revealed that I(K, slow) is selectively reduced. The attenuation of I(K, slow) is accompanied by marked action potential prolongation, and, occasionally, spontaneous triggered activity (apparently induced by early afterdepolarizations) is observed. The time constant of inactivation of I(K, slow) in Kv2. 1N216FLAG-expressing cells (mean+/-SEM=830+/-103 ms; n=17) is accelerated compared with the time constant of I(K, slow) inactivation (mean+/-SEM=1147+/-57 ms; n=25) in nontransgenic cells. In addition, unlike I(K, slow) in wild-type cells, the component of I(K, slow) remaining in the Kv2.1N216FLAG-expressing cells is insensitive to 25 mmol/L tetraethylammonium. Taken together, these observations suggest that there are 2 distinct components of I(K, slow) in mouse ventricular myocytes and that Kv2 alpha subunits underlie the more slowly inactivating, tetraethylammonium-sensitive component of I(K, slow). In vivo telemetric recordings also reveal marked QT prolongation, consistent with a defect in ventricular repolarization, in Kv2.1N216FLAG-expressing transgenic mice.
一种体内实验策略被用于专注探索成孔（α）亚基的Kv2亚家族成员在哺乳动物心脏中功能性电压门控K⁺通道生成过程中的作用，该策略涉及心脏特异性表达一种作为显性负性起作用的突变型Kv 2.1亚基。设计了一种突变型Kv2.1 α亚基（Kv2.1N216），以产生一种截短的蛋白质，其包含细胞内N端、S1跨膜结构域以及S1/S2环的一部分。截短的Kv2.1N216在C端用8个氨基酸的FLAG肽进行表位标记，以产生Kv2. 1N216FLAG。在HEK - 293细胞中表达Kv2. 1N216FLAG时未检测到离子电流，尽管将该构建体与野生型Kv2.1共表达会显著降低Kv2. 1诱导电流的幅度。利用α - 肌球蛋白重链启动子指导转基因的心脏特异性表达，产生了2株表达Kv2. 1N216FLAG的转基因小鼠品系。对从这些动物分离的心室肌细胞进行的电生理记录显示，I(K, slow)被选择性降低。I(K, slow)的衰减伴随着动作电位的显著延长，并且偶尔会观察到自发触发活动（显然由早期后去极化诱导）。与非转基因细胞中I(K, slow)失活的时间常数（平均值±标准误 = 1147±57 ms；n = 25）相比，表达Kv2. 1N216FLAG的细胞中I(K, slow)失活的时间常数（平均值±标准误 = 830±103 ms；n = 17）加快。此外，与野生型细胞中的I(K, slow)不同，表达Kv2.1N216FLAG的细胞中剩余的I(K, slow)成分对25 mmol/L四乙铵不敏感。综上所述，这些观察结果表明，小鼠心室肌细胞中的I(K, slow)有2个不同的成分，并且Kv2 α亚基是I(K, slow)中失活更慢、对四乙铵敏感的成分的基础。体内遥测记录还显示，表达Kv2.1N216FLAG的转基因小鼠出现明显的QT延长，这与心室复极化缺陷一致。
