Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative Kv2 alpha subunit.
Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative Kv2 alpha subunit.
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DOI：
10.1161/01.res.85.7.623
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发表时间：
1999-10
影响因子：
20.1
通讯作者：
Haodong Xu;D. Barry;Huilin Li;Sylvain Brunet;Weinong Guo;J. Nerbonne
中科院分区：
文献类型：
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作者：
Haodong Xu;D. Barry;Huilin Li;Sylvain Brunet;Weinong Guo;J. Nerbonne
体内实验策略涉及作为显性失活功能的突变型 Kv 2.1 亚基的心脏特异性表达，该策略被用于专注于探索成孔 (α) 亚基 Kv2 亚家族成员在哺乳动物心脏中功能性电压门控 K(+) 通道生成中的作用的研究。突变的 Kv2.1 α 亚基 (Kv2.1N216) 被设计为产生包含细胞内 N 末端、S1 跨膜结构域和部分 S1/S2 环的截短蛋白。截短的 Kv2.1N216 在 C 末端用 8 个氨基酸的 FLAG 肽标记表位，以生成 Kv2。 1N216 标志。 Kv2 的表达没有检测到离子电流。 HEK-293 细胞中的 1N216FLAG，尽管该构建体与野生型 Kv2.1 共表达显着降低了 Kv2 的幅度。 1-感应电流。使用α-肌球蛋白重链启动子指导转基因的心脏特异性表达，2个Kv2系。产生表达1N216FLAG的转基因小鼠。从这些动物中分离出的心室肌细胞的电生理记录显示，I（K，慢）选择性减少。 I（K，慢）的衰减伴随着明显的动作电位延长，并且偶尔观察到自发触发的活动（显然是由早期后除极引起的）。 Kv2 中 I(K, Slow) 失活的时间常数。与非转基因细胞中I(K，慢速)失活的时间常数(平均值+/-SEM=1147+/-57 ms；n=25)相比，表达1N216FLAG的细胞(平均值+/-SEM=830+/-103ms；n=17)加速。此外，与野生型细胞中的I(K，slow)不同，Kv2.1N216FLAG表达细胞中残留的I(K，slow)成分对25mmol/L四乙铵不敏感。总而言之，这些观察结果表明，小鼠心室肌细胞中有 2 个不同的 I(K，慢) 成分，并且 Kv2 α 亚基是 I(K，慢) 失活速度较慢、四乙铵敏感成分的基础。体内遥测记录还揭示了表达 Kv2.1N216FLAG 的转基因小鼠中明显的 QT 延长，与心室复极缺陷一致。
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.