Department of Physiology, School of Medicine, Zhengzhou University, Zhengzhou, China.
Faculty of Medicine, KU Leuven, Leuven, Belgium.
Acta Physiol (Oxf). 2018 Mar;222(3). doi: 10.1111/apha.12986. Epub 2017 Dec 7.
Junctophilins (JPs), a protein family of the junctional membrane complex, maintain the close conjunction between cell surface and intracellular membranes in striate muscle cells mediating the crosstalk between extracellular Ca entry and intracellular Ca release. The small-conductance Ca -activated K channels are activated by the intracellular calcium and play an essential role in the cardiac action potential profile. Molecular mechanisms of regulation of the SK channels are still uncertain. Here, we sought to determine whether there is a functional interaction of junctophilin type 2 (JP2) with the SK channels and whether JP2 gene silencing might modulate the function of SK channels in cardiac myocytes.
Association of JP2 with SK2 channel in mouse heart tissue as well as HEK293 cells was studied using in vivo and in vitro approaches. siRNA knockdown of JP2 gene was assessed by real-time PCR. The expression of proteins was analysed by Western blotting. Ca -activated K current (I ) in infected adult mouse cardiac myocytes was recorded using whole-cell voltage-clamp technique. The intracellular Ca transient was measured using an IonOptix photometry system.
We showed for the first time that JP2 associates with the SK2 channel in native cardiac tissue. JP2, via the membrane occupation and recognition nexus (MORN motifs) in its N-terminus, directly interacted with SK2 channels. A colocalization of the SK2 channel with its interaction protein of JP2 was found in the cardiac myocytes. Moreover, we demonstrated that JP2 is necessary for the proper cell surface expression of the SK2 channel in HEK293. Functional experiments indicated that knockdown of JP2 caused a significant decrease in the density of I and reduced the amplitude of the Ca transient in infected cardiomyocytes.
The present data provide evidence that the functional interaction between JP2 and SK2 channels is present in the native mouse heart tissue. Junctophilin 2, as junctional membrane complex (JMC) protein, is an important regulator of the cardiac SK channels.
连接蛋白（JPs）是连接膜复合蛋白家族的成员，维持纹状肌细胞中细胞表面和细胞内膜的紧密连接，介导细胞外 Ca 内流与细胞内 Ca 释放之间的串扰。小电导钙激活钾通道（SK 通道）被细胞内 Ca 激活，在心肌动作电位形态中发挥重要作用。SK 通道的调节分子机制尚不确定。本研究旨在确定连接蛋白 2（JP2）与 SK 通道是否存在功能相互作用，以及 JP2 基因沉默是否可能调节心肌细胞中 SK 通道的功能。
采用体内和体外方法研究了 JP2 与 SK2 通道在小鼠心脏组织和 HEK293 细胞中的结合情况。通过实时 PCR 评估 JP2 基因的沉默。通过 Western 印迹分析蛋白表达。使用全细胞膜片钳技术记录感染成年小鼠心肌细胞中的 Ca 激活的 K 电流（I ）。使用 IonOptix 光度测定系统测量细胞内 Ca 瞬变。
我们首次表明，JP2 在天然心脏组织中与 SK2 通道相互作用。JP2 通过其 N 端的膜占领和识别结构域（MORN 基序）与 SK2 通道直接相互作用。在心肌细胞中发现 SK2 通道与其相互作用蛋白 JP2 的共定位。此外，我们证明 JP2 是 SK2 通道在 HEK293 细胞中正确细胞表面表达所必需的。功能实验表明，JP2 敲低导致 I 的密度显著降低，并降低感染心肌细胞中的 Ca 瞬变幅度。
本研究数据提供了证据，表明 JP2 与 SK2 通道之间的功能相互作用存在于天然小鼠心脏组织中。连接蛋白 2 作为连接膜复合蛋白（JMC）蛋白，是心脏 SK 通道的重要调节因子。
