Suppression of neuronal and cardiac transient outward currents by viral gene transfer of dominant-negative Kv4.2 constructs
Suppression of neuronal and cardiac transient outward currents by viral gene transfer of dominant-negative Kv4.2 constructs
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DOI：
10.1074/jbc.272.50.31598
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发表时间：
1997-12-12
影响因子：
4.8
通讯作者：
Marban, E
中科院分区：
文献类型：
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作者：
Johns, DC;Nuss, HB;Marban, E
为了探索瞬时外向（a型）钾电流的分子特性，我们在心脏细胞和神经元中表达了Kv4.2的截断版本。大鼠Kv4.2编码序列在第一个跨膜段的位置被截断，并被亚克隆到巨细胞病毒启动子（pE1Kv4.2ST）下游的腺病毒穿梭载体中。我们假设这种结构可以作为Kv4家族编码电流的显性负抑制因子，类似于Kv1通道。将野生型Kv4.2与β -半乳糖苷酶表达载体共转染中国仓鼠卵巢(CHO)-K1细胞，两天后产生强大的瞬时外向电流（I-to） （14.0 pA/pF, 50 mV, n = 5）。pE1Kv4.2ST共转染可显著抑制Kv4.2电流（0.8 pA/pF, n = 6, p < 0.02； cDNA比值为2:1的Kv4.2 st：野生型），但平行实验中，其未改变共表达Kv1.4和Kv1.5通道的电流密度，Kv4.2 st在co - k1细胞中共表达时也能有效抑制大鼠Kv4.3电流。然后，我们设计了一种重组腺病毒（AdKv4.2ST），在感染细胞中过表达Kv4.2ST。与β -半乳糖苷酶报告病毒感染相比，AdKv4.2ST感染2天后大鼠小脑颗粒细胞的a型电流降低（Ad β -半乳糖苷酶细胞的a型电流为116.0 pA/pF，组为281.4 pA/pF, n = 8, p < 0.001）。同样，成年大鼠心室肌细胞的I-to被AdKv4.2ST抑制，而被Ad β -半乳糖苷酶抑制不了（β -半乳糖苷酶细胞的I-to为8.8 pA/pF比21.4 pA/pF，每组n = 6, p < 0.05）。GFP-Kv4.2ST融合构建体的表达使得共聚焦显微镜能够对亚细胞蛋白定位进行成像。我们认为Kv4家族基因是大鼠小脑颗粒细胞a型电流和脑室I-to电流的主要贡献者。显性负构念的过表达可能在解剖各种离子通道基因对兴奋性的贡献方面具有普遍的实用价值。
To probe the molecular identity of transient outward (A-type) potassium currents, we expressed a truncated version of Kv4.2 in heart cells and neurons. The rat Kv4.2 coding sequence was truncated at a position just past the first transmembrane segment and subcloned into an adenoviral shuttle vector downstream of a cytomegalovirus promoter (pE1Kv4.2ST). We hypothesized that this construct would act as a dominant-negative suppressor of currents encoded by the Kv4 family by analogy to Kv1 channels. Cotransfection of wild-type Kv4.2 with a beta-galactosidase expression vector in Chinese hamster ovary (CHO)-K1 cells produced robust transient outward currents (I-to) after two days (14.0 pA/pF at 50 mV, n = 5). Cotransfection with pE1Kv4.2ST markedly suppressed the Kv4.2 currents (0.8 pA/pF, n = 6, p < 0.02; cDNA ratio of 2:1 Kv4.2ST:wild type), but in parallel experiments, it did not alter the current density of coexpressed Kv1.4 or Kv1.5 channels, Kv4.2ST also effectively suppressed rat Kv4.3 current when coexpressed in CHO-K1 cells. We then engineered a recombinant adenovirus (AdKv4.2ST) designed to overexpress Kv4.2ST in infected cells. A-type currents in rat cerebellar granule cells were decreased two days after AdKv4.2ST infection as compared with those infected by a beta-galactosidase reporter virus (116.0 pA/pF versus 281.4 pA/pF in Ad beta-galactosidase cells, n = 8 each group, p < 0.001). Likewise, I-to in adult rat ventricular myocytes was suppressed by AdKv4.2ST but not by Ad beta-galactosidase (8.8 pA/pF versus 21.4 pA/pF in beta-galactosidase cells, n = 6 each group, p < 0.05). Expression of a GFP-Kv4.2ST fusion construct enabled imaging of subcellular protein localization by confocal microscopy. The protein was distributed throughout the surface membrane and intracellular membrane systems, We conclude that genes from the Kv4 family are the predominant contributors to the A-type currents in cerebellar granule cells and I-to in rat ventricle. Overexpression of dominant-negative constructs may be of general utility in dissecting the contributions of various ion channel genes to excitability.