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.

项目概要 促心律失常性长 QT 综合征 (LQTS) 通常是由药物或突变导致 心脏中快速激活的延迟整流 K+ 电流 (IKr) 的幅度是直接的宏观 IKr。 细胞表面 Kv11.1 通道数量的功能取决于两者之间的平衡。 KCNH2 中大约 90% 的 LQTS 相关错义突变。 通过破坏通道运输来减少细胞表面的 Kv11.1 通道数量。 这些突变体通过在阻断 Kv11.1 电流 (IKv11.1) 的药物中培养细胞而增加。 创新假设，其中一些突变增加了早期内体中 Kv11.1 通道的活性 (EE)、再循环内体 (RE) 和/或跨高尔基体网络 (TGN) 这些通道开放的增加。 细胞器改变细胞器膜电位 (ψ)、pH 和 K+ 水平。 电化学梯度改变 Kv11.1 通道的构象，阻止其继续运输和/或 促进降解，阻断 IKv11.1 的药物可防止突变通道开放，防止细胞器发生变化。 ψ、pH 和 K+ 水平，并改善后续运输/减少降解。 为了检验这一假设，我们将开发一种方法来检测选定的相关 Kv11.1 通道的开放情况。 我们已经可以测量 EE、RE 和膜插入、回收和降解的细胞器。 TGN 使用基于 DNA 的报告器 Voltair，由 Krishnan 开发。现在，我们将使用基于 DNA 的报告器。 对于 K+，我们最近开发了一种名为 pHlicKer 的试剂盒，可同时定量这些物质中的管腔 pH 值和 [K+] 我们将设计专门定位于活细胞中的 EE、RE 或 TGN 的 pHlicKer 变体。 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 这些细胞器的开口会影响 ψ、腔内 pH 值和腔内 [K+] 水平。 增加 Kv11.1 通道的开放度会降低 ψ 并增加腔内 [K+] 的变化。 EE、RE 或 TGN 中的电化学梯度将阻止继续运输/促进降解 我们期望在药物中培养表达突变型 Kv11.1 通道的细胞。 阻断 IKv11.1 将阻止 EE、RE 和 TGN 电化学梯度的变化，从而改善 突变体 Kv11.1 通道运输增加功能半衰期。 开发第一代测量细胞器电化学梯度的原型 将使我们能够量化通道动态在进入质膜时如何变化。 开发新分子的关键一步，可以选择性地靶向细胞内通道以影响 它们的表达和/或降解也将导致第一个绘制细胞器图谱的实用方法。 K+ 并有可能加速细胞器中新 K+ 通道和转运蛋白的发现。