ABSTRACT

Coronary Artery Disease (CAD) typically kills more people globally each year than any other single cause of death. A better understanding of genetic predisposition to CAD and the underlying mechanisms will help to identify those most at risk and contribute to improved therapeutic approaches. KCNE2 is a functionally versatile, ubiquitously expressed potassium channel β subunit associated with CAD and cardiac arrhythmia susceptibility in humans and mice. Here, to identify novel KCNE2 interaction partners, we employed yeast two-hybrid screening of adult and fetal human heart libraries using the KCNE2 intracellular C-terminal domain as bait. Testin (encoded by TES), an endothelial cell-expressed, CAD-associated, focal adhesion protein, was identified as a high-confidence interaction partner for KCNE2. We confirmed physical association between KCNE2 and Testin in vitro by co-immunoprecipitation. Whole-cell patch clamp electrophysiology revealed that KCNE2 negative-shifts the voltage dependence and increases the rate of activation of the endothelial cell and cardiomyocyte-expressed Kv channel α subunit, Kv1.5 in CHO cells, whereas Testin did not alter Kv1.5 function. However, Testin nullified KCNE2 effects on Kv1.5 voltage dependence and gating kinetics. In contrast, Testin did not prevent KCNE2 regulation of KCNQ1 gating. The data identify a novel role for Testin as a tertiary ion channel regulatory protein. Future studies will address the potential role for KCNE2-Testin interactions in arterial and myocyte physiology and CAD.

 抽象的

冠状动脉疾病（CAD）每年在全球造成的死亡人数通常比任何其他单一死因都要多。更好地了解 CAD 的遗传易感性及其潜在机制将有助于识别高危人群，并有助于改进治疗方法。 KCNE2 是一种功能多样、普遍表达的钾通道 β 亚基，与人类和小鼠的 CAD 和心律失常易感性相关。在这里，为了识别新的 KCNE2 相互作用伙伴，我们使用 KCNE2 胞内 C 端结构域作为诱饵，对成人和胎儿人类心脏库进行酵母双杂交筛选。 Testin（由TES编码）是一种内皮细胞表达的 CAD 相关粘着斑蛋白，被确定为 KCNE2 的高可信度相互作用伴侣。我们通过免疫共沉淀在体外证实了 KCNE2 和 Testin 之间的物理关联。全细胞膜片钳电生理学显示，KCNE2 负移电压依赖性并增加 CHO 细胞中内皮细胞和心肌细胞表达的 Kv 通道 α 亚基 Kv1.5 的激活率，而 Testin 不会改变 Kv1.5 功能。然而，Testin 消除了 KCNE2 对 Kv1.5 电压依赖性和门控动力学的影响。相比之下，Testin 并没有阻止 KCNE2 对 KCNQ1 门控的调节。这些数据确定了 Testin 作为三级离子通道调节蛋白的新作用。未来的研究将探讨 KCNE2-Testin 相互作用在动脉和肌细胞生理学以及 CAD 中的潜在作用。