ABSTRACT

The slowly activating delayed rectifier K⁺ current (I _Ks) plays a key role in the repolarization of ventricular action potential in the human heart and is formed by the pore-forming α-subunit encoded by KCNQ1 (Kv7.1) and β-subunit encoded by KCNE1. Evidence suggested that I _Ks was regulated through protein kinase C (PKC) pathway, but the mechanism is controversial. This study was designed to identify the specific PKC isoform involved in the long-term regulation of I _Ks current. The I _Ks current was recorded using whole-cell patch-clamp technique in human embryonic kidney (HEK) 293B cell co-transfected with human KCNQ1/KCNE1 genes. The results revealed that both chronic activation of Ang II and PMA reduced the I _Ks current in a long-term regulation (about 24 hours). Further evidence showed that PKCε knockdown by siRNA antagonized the AngII-induced chronic inhibition on the I _Ks current, whereas knockdown of cPKC (PKCα and PKCβ) attenuated the inhibition effect of PMA on the current. Moreover, the forward transport inhibition of the channel with brefeldin A alleviated the Ang II-induced chronic inhibition on I _Ks current, while the channel endocytosis inhibition with dynasore alleviated both Ang II and PMA-induced chronic inhibition on I _Ks current. The above results showed that PKCε activation promoted the channel endocytosis and inhibited the channel forward transport to the plasma membrane, while cPKC activation only promoted the channel endocytosis, which both down regulated the channel current.

 抽象的

缓慢激活的延迟整流K ⁺电流（ I _Ks ）在人体心脏心室动作电位的复极中起着关键作用，由KCNQ1（Kv7.1）编码的成孔α亚基和β亚基形成由 KCNE1 编码。有证据表明IKs通过蛋白激酶 C (PKC) 途径进行调节，但_其机制存在争议。本研究旨在确定参与I _Ks电流长期调节的特定 PKC 亚型。使用全细胞膜片钳技术在与人 KCNQ1/KCNE1 基因共转染的人胚胎肾 (HEK) 293B 细胞中记录I _Ks电流。结果显示，Ang II和PMA的慢性激活均在长期调节（约24小时）中降低了I _Ks电流。进一步的证据表明，siRNA敲低PKCε可拮抗AngII诱导的I _Ks电流慢性抑制，而cPKC（PKCα和PKCβ）敲低则减弱PMA对电流的抑制作用。此外，布雷菲德菌素 A 对通道的正向转运抑制减轻了 Ang II 诱导的对I _Ks电流的慢性抑制，而 dynasore 的通道内吞作用抑制则减轻了 Ang II 和 PMA 诱导的对I _Ks电流的慢性抑制。上述结果表明，PKCε激活促进通道内吞作用并抑制通道向前转运至质膜，而cPKC激活仅促进通道内吞作用，两者均下调通道电流。