The F-actin binding protein TRIOBP-1 regulates hERG K+ channels

 描述（由申请人提供）：电压门控钾通道hERG（人ether-a-go-go-related基因）是心脏中快速激活延迟整流钾通道电流（IKr）的主要成孔亚基。心脏IKr的生理作用是心室动作电位的复极化。虽然已知hERG的某些基因突变（或获得性疾病，如低钾血症）可影响IKr，但我们对hERG调控的特定蛋白质-蛋白质相互作用的了解尚不清楚。hERG的细胞质C-末端部分先前用于对人心脏文库进行酵母双杂交筛选，以筛选可能调节离子通道定位、稳定性和相对表面表达的蛋白质。该筛选将TRIOBP-1（也称为塔拉）（一种与细胞骨架动力学相关的F-肌动蛋白结合蛋白）鉴定为推定的hERG相互作用蛋白。我们假设TRIOBP-1与hERG相互作用以调节离子通道表面表达、密度、定位和兴奋性。首先，我们将在HEK 293细胞系以及hiPSC-CM中使用全细胞膜片钳电生理学、Förster共振能量转移（FRET）测量和Western印迹相结合的方法来测试TRIOBP- 1结构域与hERG蛋白功能相关以调节hERG通道电流和表面表达的预测。其次，我们将研究一系列先前与长QT综合征II型（LQT 2）相关的TRIOBP-1点突变，以探索其对HEK 293细胞中hERG通道特性的调节作用。这些实验一起为hERG通道相关LQT 2提供了潜在的治疗靶点，并提供了对hERG通道调节及其相互作用蛋白的深入了解。

 DESCRIPTION (provided by applicant): The voltage-gated potassium channel hERG (human ether-a-go-go-related gene) is the primary pore-forming subunit of the rapidly activating delayed rectifier potassium channel current (IKr) in the heart. The physiological role of cardiac IKr is th repolarization of the ventricular action potential. Although it is known that certain genetic mutations of hERG (or acquired conditions such as hypokalemia) can affect IKr, our understanding of the specific protein-protein interactions underlying hERG regulation are not well known. The cytoplasmic C- terminal portion of hERG was previously used to conduct a yeast two hybrid screen of a human cardiac library for proteins that might regulate ion channel localization, stability, and relative surface expression. This screen identified TRIOBP-1 (also known as Tara), an F-actin binding protein associated with cytoskeletal dynamics, as a putative hERG interacting protein. We hypothesize that TRIOBP-1 interacts with hERG to regulate ion channel surface expression, density, localization, and excitability. First, we will test the prediction that TRIOBP- 1 domains functionally associate with hERG protein to modulate hERG channel currents and surface expression using a combination of whole-cell patch clamp electrophysiology, Förster resonance energy transfer (FRET) measurements, and Western blotting in a HEK293 cell line as well as in hiPSC-CMs. Second, we will investigate a series of TRIOBP-1 point mutations previously associated with Long QT Syndrome Type II (LQT2) to explore their modulatory effects on hERG channel properties in HEK293 cells. Together these experiments provide a potential therapeutic target for hERG channel-related LQT2 and provide insight into hERG channel regulation and the proteins with which it interacts.