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

 描述(申请人提供)：电压门控钾通道HERG(人醚-a-GO-GO相关基因)是心脏中快速激活的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。虽然已知某些HERG的基因突变(或后天条件，如低钾血症)可以影响IKR，但我们对HERG调控背后的特定蛋白质-蛋白质相互作用的了解还不是很清楚。HERG的细胞质C末端部分以前被用来对人类心脏文库进行酵母双杂交筛选，以寻找可能调节离子通道定位、稳定性和相对表面表达的蛋白质。该筛选鉴定了TRIOBP-1(也称为Tara)，一种与细胞骨架动力学相关的F-肌动蛋白结合蛋白，可能是一种Herg相互作用蛋白。我们假设TRIOBP-1与HERG相互作用，调节离子通道表面的表达、密度、定位和兴奋性。首先，我们将结合全细胞膜片钳电生理学、Förster共振能量转移(FRET)测量和Western blotting在HEK293细胞系以及hiPSC-CMS中验证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，我们将研究与长QT综合征II型(LQT2)相关的一系列TRIOBP-1点突变，以探索它们对HEK293细胞HERG通道特性的调制作用。总之，这些实验为HERG通道相关的LQT2提供了一个潜在的治疗靶点，并提供了对HERG通道调节及其与之相互作用的蛋白质的洞察。

 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.