Dominant-negative suppression of HCN channels markedly reduces the pacemaker current If and undermines spontaneous beating of neonatal cardiomyocytes
Dominant-negative suppression of HCN channels markedly reduces the pacemaker current If and undermines spontaneous beating of neonatal cardiomyocytes
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DOI：
10.1161/01.cir.0000045672.32920.cb
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发表时间：
2003-01-28
期刊：
影响因子：
37.8
通讯作者：
Hoppe, UC
中科院分区：
文献类型：
--
作者：
Er, F;Larbig, R;Hoppe, UC
起搏器电流I-f有助于心脏自主细胞的自发舒张去极化。在异源表达中，HCN通道表现出类似于I-f的向内超极化激活电流。然而，HCN基因与天然I-f之间的联系在很大程度上是推断性的，I-f是否对心脏起搏至关重要仍不清楚。方法和结果：为了澄清这一情况，我们在中国仓鼠卵巢细胞中产生了HCN2的GYG(402-404)AYA孔突变，使通道失去功能，并以显性阴性方式抑制野生型HCN2。此外，当HCN2- aya与野生型HCN4共表达时，以显性负向方式抑制了I-HCN4，这表明HCN2和HCN4这两种亚型能够共同组装形成异多聚体复合物。鉴于HCN2和HCN4是新生大鼠脑室中主要的HCN mRNA转录本，我们利用腺病毒基因转移在新生心肌细胞中表达HCN2- aya，以检测HCN抑制对天然I-f的影响。在-130 mV (P)下测量时，I-f密度确实显著降低，从对照细胞的7.8+/-1.6 pA/pF （n=13）降至hcn2 - aya感染细胞的0.3+/-0.2 pA/pF （n=11）
Background-The pacemaker current I-f contributes to spontaneous diastolic depolarization of cardiac autonomic cells. In heterologous expression, HCN channels exhibit a hyperpolarization-activated inward, current similar to I-f. However, the links between HCN genes and native I-f are largely inferential, and it remains unknown whether I-f is essential for cardiac pacing.Methods and Results-To clarify this situation, we generated a GYG(402-404)AYA pore mutation of HCN2, which rendered the channel nonfunctional and suppressed wild-type HCN2 in a dominant-negative manner in Chinese hamster ovary cells. In addition, HCN2-AYA suppressed I-HCN4 in a dominant-negative manner when coexpressed with wild-type HCN4, indicating that the 2 isoforms HCN2 and HCN4 are able to coassemble to form heteromultimeric complexes. Given that HCN2 and HCN4 are the dominant HCN mRNA transcripts in neonatal rat ventricle, we expressed HCN2-AYA in neonatal cardiocytes using adenoviral gene transfer to test the effect of HCN suppression on native I-f. I-f density was indeed reduced markedly, from 7.8+/-1.6 pA/pF (n=13) in control cells to 0.3+/-0.2 pA/pF (n=11) in HCN2-AYA-infected cells when measured at -130 mV (P