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 亲心律失常长QT综合征（LQT）通常是由降低的药物或突变引起的 心脏（IKR）中快速激活的延迟整流器K+电流的放大器。宏观IKR是直接的 细胞表面中KV11.1通道数的功能。反过来，这取决于 通道插入，回收和降解。 KCNH2中约有90％的LQTS连接错义突变 通过破坏通道运输来减少细胞表面中的KV11.1通道数。许多人的贩运 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 创新的假设是，其中一些突变增加了早期内体中KV11.1通道的活性 （EE），回收内体（RE）和/或反式高尔基网络（TGN）。这些频道开放的增加 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渠道的构象，以防止其贩运和/或 促进退化。阻止IKV11.1阻止突变通道开口的药物，防止细胞器发生变化 ψ，pH和k+水平，并改善向前运输/减少降解。 为了检验这一假设，我们将开发一种方法来测定KV11.1频道在所选的，相关的 膜插入，回收和降解的细胞器。我们已经可以测量EE，RES和 使用基于DNA的记者的TGN，称为Krishnan开发的名为Voltair。现在，我们将使用基于DNA的记者 对于K+，我们最近开发了称为Phlicker，以同时定量lumenal pH和[k+] 细胞器。我们将设计专门在活细胞中的EE，RES或TGN中定位的Phlicker的变体。 然后，我们将使用Voltair和Phlicker明确确定突变体KV11.1如何增加通道 这些细胞器中的开口会影响ψ，Lumenal pH和Lumenal [K+]水平。我们期望这种突变 增加KV11.1通道的开放将减少ψ并增加流体[K+]。变化 EES，RES或TGN中的电化学梯度将防止贩运/促进降解 kv11.1通道到细胞表面。我们期望在药物中孵化表达突变体KV11.1通道 该块IKV11.1将防止EES，RES和TGN的电化学梯度的变化以改进 突变KV11.1通道运输增加了功能性半衰期。 第一代原型的开发以测量细胞器中的电化学梯度 将允许我们量化通道动力学在流动到质膜时的变化。这将是一个 开发新分子的关键步骤，这些分子可以选择性地靶向细胞内通道以撞击 它们的表达和/或退化。我们的研究还将导致第一种实用方法来映射有机机 K+，并可能加速细胞器中新的K+通道和转运蛋白的发现。