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K+水平降低时，人类心脏细胞可能与K+的能斯特方程不一致，矛盾地去极化。这种矛盾的去极化现象已经被观察了30多年。这对于低钾血症引起的心律失常的病因学也是至关重要的。然而，其分子机制尚不清楚。K+通道的离子选择性通常被认为是静态的，不会随着生理刺激而改变。K+通道如何选择K+而不是其他单价阳离子仍然是一个未解决的问题。特别是，与四聚体K+通道相比，二聚体两孔域K+通道（K2P）的离子选择性和选择性过滤器的了解较少。我们的长期目标是了解K+通道的生理作用和K+通道功能的分子机制。本研究的目的是探索哺乳动物K2P通道的首个克隆成员TWIK-1在低钾血症时在人心肌细胞中的功能作用，并表征K2P通道的离子选择性和通透性的动态行为。基于过去三十年的研究成果，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向内泄漏的Na+电流来响应低钾血症时细胞外K+水平降低的挑战，并促进人心肌细胞的矛盾去极化；2) K2P通道可以调节选择性过滤器的构象，对一价阳离子表现出离子选择性和渗透性的动态行为。通过采用离子通道和电生理学领域的标准方法和传统方法，我们将在两个具体目标中验证这些假设：1)研究TWIK-1 K+通道如何调节正常和低钾血症条件下人类原代心肌细胞的静息电位和动作电位。2)研究在细胞内缺乏K+的情况下，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。该研究将证明TWIK-1 K+通道在人类心肌细胞中的生理作用，描述一种调节心脏兴奋性的新机制，为理解低钾血症时心脏的矛盾去极化提供新的见解，并阐明低钾血症诱发心律失常的病因。本文还将介绍生理条件下K+通道动态离子选择性的概念，提供柔性K+选择性过滤器的证据，支持或补充关于K+通道离子选择性的已知假设，并提高对离子选择性和K2P通道选择性过滤器的理解。

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.