Mechanisms that alter Potassium channel trafficking in arrhythmias

项目摘要 促心律失常性长QT综合征（LQTS）通常由药物或突变引起， 心脏中快速激活延迟整流钾电流的幅度（IKr）。宏观IKr是一个直接的 细胞表面Kv11.1通道数量的函数。这反过来又取决于 信道插入、再循环和降级。KCNH 2中约90%的LQTS连锁错义突变 通过破坏通道运输来减少细胞表面的Kv11.1通道数量。对许多人来说， 通过在阻断Kv11.1电流（IKv11.1）的药物中培养细胞来增加这些突变体。我们建议 这些突变中的一些增加了早期内体中Kv11.1通道的活性 (EE)、再循环内体（RE）和/或反式高尔基体网络（TGN）。这些渠道开放的增加 细胞器改变细胞器的膜电位（pH）、pH和K+水平。细胞器的变化 电化学梯度改变Kv11.1通道的构象，阻止其向前运输和/或 促进降解。阻断IKv11.1的药物可防止突变通道开放，防止细胞器变化， pH值和K+水平，并改善向前运输/减少降解。 为了验证这一假设，我们将开发一种方法来测定Kv11.1通道开放的选择，相关的， 用于膜插入、回收和降解的细胞器。我们已经可以测量EE，RE和 TGN使用一种名为Voltair的基于DNA的记者，由Krishnan开发。现在我们将使用一个基于DNA的报告器 对于K+，我们最近开发了一种称为pHlicKer的方法，可以同时定量这些细胞中的内腔pH和[K+]。 细胞器我们将设计pHlicKer的变体，这些变体特异性地定位于活细胞中的EE、RE或TGN。 然后，我们将使用Voltair和pHlicKer明确确定突变Kv11.1通道如何增加通道 这些细胞器中的开放影响细胞膜、内腔pH和内腔[K+]水平。我们认为， 增加Kv11.1通道的开放，可降低K+浓度，增加[K+]浓度。的变化 EE、RE或TGN中的电化学梯度将阻止向前运输/促进降解 的Kv11.1通道到细胞表面。我们期望在药物中孵育表达突变Kv11.1通道的细胞， 阻断IKv11.1将阻止EE、RE和TGN的电化学梯度的变化， 突变Kv11.1通道运输增加功能半衰期。 第一代细胞器电化学梯度测量样机的研制 将使我们能够量化通道动力学如何变化，因为它们交通到质膜。这将是一 关键的一步，开发新的分子，可以选择性地靶向细胞内通道细胞内影响 其表达和/或降解。我们的研究也将导致第一个实用的方法来映射细胞器 K+和潜在的加速发现新的K+通道和转运蛋白的细胞器。

Project Summary The pro-arrhythmic long QT syndrome (LQTS) is commonly caused by drugs or mutations that decrease the amplitude of the rapidly activating delayed rectifier K+ current in the heart (IKr). Macroscopic IKr is a direct function of the number of Kv11.1 channels in the cell surface. This in turn, depends on the balance between channel insertion, recycling and degradation. About 90% of LQTS-linked missense mutations in KCNH2 decrease Kv11.1 channel number in the cell surface by disrupting channel trafficking. The trafficking for many of these mutants is increased by culturing cells in drugs that block Kv11.1 current (IKv11.1). We propose the innovative hypothesis that some of these mutations increase the activity of Kv11.1 channel in early endosomes (EE), recycling endosomes (RE) and/or the trans-Golgi network (TGN). The increase in channel opening in these organelles alters the organelle membrane potential (ψ), pH and K+ levels. The changes in the organelle electrochemical gradients alter the conformational of Kv11.1 channels that prevent their onward trafficking and/or promote degradation. Drugs that block IKv11.1 prevent mutant channel openings, prevent organelles changes in ψ, pH and K+ levels, and improve onward trafficking/decrease degradation. To test this hypothesis, we will develop a method to assay Kv11.1 channel opening in selected, relevant organelles for membrane insertion, recycling, and degradation. We can already measure ψ in EEs, REs and the TGN using a DNA-based reporter called Voltair, developed by Krishnan. Now, we will use a DNA-based reporter for K+ we have recently developed, called pHlicKer, to simultaneously quantitate lumenal pH and [K+] in these organelles. We will engineer variants of pHlicKer that localize specifically in EEs, REs, or the TGN in live cells. We will then use Voltair and pHlicKer to explicitly determine how mutant Kv11.1 channels that increase channel opening in these organelles impacts ψ, lumenal pH and lumenal [K+] levels. We expect that mutations that increase the opening of Kv11.1 channels will decrease ψ and increase lumenal [K+]. The changes in the electrochemical gradients in EEs, REs, or the TGN will prevent the onward trafficking/promote the degradation of Kv11.1 channels to the cell surface. We expect incubating cells expressing mutant Kv11.1 channels in drugs that block IKv11.1 will prevent the changes in the electrochemical gradients of EEs, REs, and TGN to improve mutant Kv11.1 channel trafficking increase functional half-life. The development of the first-generation prototypes to measure electrochemical gradients in organelles will allow us to quantify how channel dynamics change as they traffic to the plasma membrane. This will be a critical step to develop new molecules that can selectively target intracellular channels intracellular to impact their expression and/or degradation. Our research would also lead to the first practical method to map organellar K+ and potentially accelerate the discovery of new K+ channels and transporters in organelles.