Dynamic ion selectivity of K2P channels and paradoxical depolarization

描述(由申请人提供)：在低钾血症或细胞外钾水平降低的情况下，人类心肌细胞可能会反常地去极化，这与K的能斯特方程不一致。这种矛盾的去极化现象已经被观察了30多年。它对低钾血症所致心律失常的病因也是至关重要的。然而，其分子机制还不是很清楚。钾通道的离子选择性通常被认为是静态的，不会随着生理刺激的变化而改变。K通道如何比其他一价阳离子选择K仍然是一个悬而未决的问题。特别是，与四聚体K通道相比，二聚体两孔结构域K通道(K2P)的离子选择性和选择性过滤机制还不是很清楚。我们的长期目标是了解钾通道的生理作用和钾通道功能的分子机制。本研究的目的是探讨哺乳动物K2P通道的第一个克隆成员twik-1在人低钾血症心肌细胞中的功能，以及K2P通道离子选择性和通透性的动态行为。基于过去30年的研究，这些研究的意义，以及我们对克隆的Twik-1 K通道的初步研究，我们假设：1)Twik-1 K通道通过改变离子选择性和传导内漏钠电流，响应细胞外K水平降低的挑战，并有助于低钾血症下人心肌细胞的反常去极化；2)K2P通道可以调节选择性过滤器的构象，并在单价阳离子的离子选择性和通透性方面表现出动态行为。通过使用离子通道和电生理学领域的标准方法和传统方法，我们将在两个特定的目标上验证这些假说：1)研究Twik-1 K通道如何在正常和低钾条件下调节人原代心肌细胞的静息电位和动作电位。2)研究了细胞内无钾时K2P通道对小碱金属离子和大有机一价阳离子的离子选择性和通透性。这项研究将证明Twik-1钾通道在人心肌细胞中的生理作用，描述一种新的调节心脏兴奋性的机制，为理解低钾血症时心脏的反常去极化提供新的见解，并阐明低钾致心律失常的病因。它还将引入K通道在生理条件下的动态离子选择性的概念，提供灵活的K选择性过滤器的证据，支持或补充关于K通道离子选择性的著名假说，并加深对K通道离子选择性和K_2P通道选择性过滤器的理解。 与公共卫生相关：低钾血症是指血钾水平低于正常水平，可导致人类心脏疾病。一种被称为“反常去极化”的现象在低钾血症所致心脏疾病的病理机制中是至关重要的。这个项目的目标是：1)通过研究人类心脏中功能未知的基因或遗传分子来了解这种现象的机制；2)描述同一家族中12个基因或分子的新行为。这项研究将为低钾引起的心脏疾病提供新的见解，可能为心脏保护和心脏疾病的治疗提供新的方法。

DESCRIPTION (provided by applicant): In hypokalemia or lowered extracellular K+ levels, human cardiac cells can become paradoxically depolarized inconsistent with the Nernst equation for K+. Such paradoxical depolarization has been observed for over thirty years. It is also crucial to the etiology of hypokalemia-induced cardiac arrhythmia. However, its molecular mechanism is not well understood. Ion selectivity of K+ channels is generally considered to be static and not changed in response to physiological stimuli. How K+ channels select K+ over other monovalent cations still remains an unsolved question. Particularly, ion selectivity and the selectivity filter of dimeric two-pore domain K+ channels (K2P) are less understood compared to those in tetrameric K+ channels. Our long-term goal is to understand physiological roles of K+ channels and molecular mechanisms of K+ channel function. The objective of this proposal is to explore functional roles of TWIK-1, the first cloned member of mammalian K2P channels in human cardiomyocytes in hypokalemia, and to characterize dynamic behaviors in ion selectivity and permeability of K2P channels. Based on studies in the past thirty years, the implications that are derived from these studies, and our preliminary studies on cloned TWIK-1 K+ channels, we hypothesize: 1) TWIK-1 K+ channels respond to challenges of lowered extracellular K+ levels and contribute to paradoxical depolarization in human cardiomyocytes in hypokalemia by changing ion selectivity and conducting inward leak Na+ currents; 2) K2P channels can adjust the conformations of the selectivity filter and exhibit dynamic behaviors in ion selectivity and permeability for monovalent cations. By employing standard methods and conventional approaches in the field of ion channels and electrophysiology, we will test these hypotheses in two specific aims: 1) Investigate how TWIK-1 K+ channels regulate the resting potential and action potential of human primary cardiomyocytes in both normal and hypokalemic conditions. 2) Study ion selectivity and permeability of K2P channels for small alkali metal ions and large organic monovalent cations in the absence of intracellular K+. The proposed research will demonstrate physiological roles of TWIK-1 K+ channels in human cardiomyocytes, describe a novel mechanism that regulates cardiac excitability, provide novel insights on the understanding of paradoxical depolarization in the heart in hypokalemia, and shed light on the etiology of hypokalemia-induced cardiac arrhythmias. It will also introduce the concept of dynamic ion selectivity of K+ channels under physiological conditions, provide evidence of a flexible K+ selectivity filter, which supports or supplements well-known hypotheses regarding ion selectivity of K+ channels, and improve the understanding of ion selectivity and the selectivity filter of K2P channels. PUBLIC HEALTH RELEVANCE: Hypokalemia refers to lower-than-normal blood potassium levels, which can cause human cardiac disorders. A phenomenon called "paradoxical depolarization" is crucial to the pathological mechanism of hypokalemia-induced cardiac disorders. The goal of this project is: 1) to understand the mechanism of such a phenomenon, by studying a gene or a genetic molecule in the human heart, which function is not known; 2) to characterize novel behaviors of twelve genes or molecules in the same family. This research will provide novel insights for hypokalemia-induced cardiac disorders, possibly suggesting new methods for cardio-protection and treatment of cardiac disorders.