Mechanisms that alter Potassium channel trafficking in arrhythmias

改变心律失常中钾通道运输的机制

• 批准号: 10676958
• 负责人: Yamuna Krishnan
• 金额: $ 19.91万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10676958
• 关键词: Acceleration; Arrhythmia; Binding; Biological Assay; Cell membrane; Cell surface; Cells; Cultured Cells; DNA; Development; Early Endosome; Endosomes; Engineering; Environment; Equilibrium; Generations; Golgi Apparatus; Half-Life; Heart; Heterogeneity; Incubated; Induced Mutation; Ion Channel; Ions; Killer Cells; Knowledge; Link; Long QT Syndrome; Maps; Measures; Membrane; Membrane Potentials; Methods; Missense Mutation; Molecular Conformation; Mutation; Organelles; Pathway interactions; Pharmaceutical Preparations; Potassium Channel; Probability; Recycling; Reporter; Research; Technology; Testing; Time; Variant; Visualization; Work; endosome membrane; experience; extracellular; high throughput screening; improved; innovation; mutant; pH gradient; preservation; prevent; prototype; tool; trafficking; trans-Golgi Network; voltage clamp

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.

项目摘要 促心律失常的长QT综合征(LQTS)通常是由药物或基因突变减少引起的 心脏快速激活延迟整流钾电流的幅度(Ikr)。宏观的IKR是一个直接的 细胞表面Kv11.1通道数的函数。这又取决于两者之间的平衡 渠道的插入、回收和降解。KCNH2中约90%的LQTS连锁错义突变 通过中断通道传输，减少细胞表面的Kv11.1通道数量。对许多人的贩卖 这些突变体通过在阻断Kv11.1电流的药物(IKv11.1)中培养细胞而增加。我们建议 创新假设，其中一些突变增加了早期内小体中Kv11.1通道的活性 (EE)、循环内体(RE)和/或反式高尔基体网络(TGN)。这些地区的航道开口量增加 细胞器改变细胞器膜电位(ψ)、pH和K+水平。细胞器的变化 电化学梯度改变了Kv11.1通道的构象，从而阻止了它们的继续运输和/或 促进降级。阻断IKv11.1的药物可以阻止突变的通道开放，防止细胞器的变化 ψ、pH和K+水平，并改善向前迁移/减少降解。 为了验证这一假设，我们将开发一种方法来测试Kv11.1通道在选定的相关 用于膜插入、循环和降解的细胞器。我们已经可以在EES、RES和 TGN使用的是由Krishnan开发的名为Voltair的DNA记者。现在，我们将使用基于DNA的记者 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管腔pH和[K+] 细胞器。我们将设计PHlicKer的变种，专门定位于EES、RES或活细胞中的TGN。 然后我们将使用Voltair和PHlicKer来明确确定突变的Kv11.1通道如何增加通道 这些细胞器的开放影响ψ、管腔pH和管腔[K+]水平。我们预计这种突变 增加Kv11.1通道的开放将降低ψ，增加管腔[K+]。中的变化 EES、RES或TGN中的电化学梯度将防止向前传输/促进降解 Kv11.1通道连接到细胞表面。我们期待在药物中培养表达突变Kv11.1通道的细胞 阻断IKv11.1将阻止EES、RES和TGN的电化学梯度的变化以改善 突变的Kv11.1通道转运增加了功能半衰期。 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在它们流向质膜时发生变化。这将是一个 开发新分子的关键一步，这种分子可以选择性地靶向细胞内通道以影响 它们的表达和/或降解。我们的研究还将带来绘制细胞器图的第一种实用方法 K+并有可能加速细胞器中新的K+通道和转运蛋白的发现。