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.

摘要

缓慢激活的延迟整流器K ⁺电流（I _Ks）在人心室动作电位的复极化中起关键作用，并且由KCNQ1（Kv7.1）和β-亚基编码的成孔性α-亚基形成由KCNE1编码。证据表明，我 _Ks的是通过蛋白激酶C（PKC）途径调节，但其作用机理是有争议的。这项研究的目的是找出参与的长期调控的具体PKC亚型我 _Ks的电流。该我 _了Ks使用全细胞膜片钳技术记录了与人KCNQ1 / KCNE1基因共转染的人胚胎肾（HEK）293B细胞中的电流。结果表明，Ang II和PMA的慢性激活均会降低长期调节（约24小时）中的I _Ks电流。进一步的证据表明，siRNA敲低PKCε拮抗了AngII诱导的对I _Ks电流的慢性抑制，而敲低cPKC（PKCα和PKCβ）则减弱了PMA对电流的抑制作用。而且，随着布雷菲德菌素A的信道的前向输送抑制减轻对血管紧张素Ⅱ诱导的慢性抑制我 _Ks的电流，而信道的内吞作用与dynasore抑制缓和二者血管紧张素Ⅱ和PMA诱导的慢性抑制上我 _Ks的电流。以上结果表明，PKCε的激活促进了通道的内吞作用，并抑制了通道向质膜的正向转运，而cPKC的激活仅促进了通道的内吞作用，两者均下调了通道电流。