Regulation of lysosomal potassium channels

项目总监/首席调查员(最后、第一、中间)：任德建 拟议研究的总体目标是了解钾离子的功能和调节。 通向溶酶体功能的通道。钾是面对两种血浆的最丰富的细胞内离子 细胞膜和细胞器膜。质膜对钾具有高度的渗透性。超过80人 现在已经发现钾通道可以调节这种质膜的钾通透性。在……里面 相比之下，钾是如何通过溶酶体等细胞内细胞器的，人们却知之甚少。 溶酶体在细胞清除、消化、循环、胞吐和膜修复等过程中起着重要作用。 由于管腔中的钙浓度很高，溶酶体也是钙的储存库，其中的钙 释放到胞浆中，以形成胞浆钙动力学，并调节肌肉收缩和激素 分泌物。溶酶体功能障碍与诸如溶酶体储存等病理生理状况有关 疾病、癌症和神经退化。因此了解溶酶体膜是如何介导离子的 通透性对于我们理解细胞器的生理功能很重要。我们最近发现了一种 在溶酶体膜上形成钾选择性通道的新蛋白TMEM175。遗传变异 在人类中，TMEM175也与帕金森氏症有关。我们提出了三个具体目标来扩大我们的 初步调查结果。在目标1中，我们将使用膜片钳记录来比较野生型和TMEM175 并将测试除了基因敲除外是否还有其他主要的溶酶体钾通道 TMEM175。与质膜通道不同，溶酶体钾通道的功能是 很大程度上是未知的。在目标2中，我们将使用TMEM175基因敲除小鼠模型来检验以下假设 钾通道调节溶酶体的正常工作，包括细胞器膜电位 调节、管腔酸化、小泡融合和自噬。在目标3中，我们将测试TMEM175，如 质膜上的许多其他钾通道都是由蛋白激酶调节的。使用蛋白质 在化学实验中，我们还将确定这样的结构要求。因为 溶酶体的基本功能，这些研究将揭示溶酶体钾通道如何在 细胞器在生理和病理条件下的功能。 OMB编号0925-0001/0002(01/18修订版批准至2020年3月31日)页面

Program Director/Principal Investigator (Last, First, Middle): Ren, Dejian The overall goal of the proposed research is to understand the function and the regulation of potassium ion channels to the function of lysosomes. Potassium is the most abundant intracellular ion that faces both plasma membrane and organelle membrane. Plasma membranes are highly permeable to potassium. More than 80 potassium channels have now been discovered to mediate such plasma membrane potassium permeability. In contrast, how potassium passes intracellular organelles such as lysosomes is much less understood. Lysosomes play fundamental roles in cellular clearance, digestion, recycling, exocytosis and membrane repair. Because of high concentration of calcium in the lumen, lysosomes are also calcium stores from which calcium is released into cytosol to shape cytosolic calcium kinetics and to regulate muscle contraction and hormone secretion. Lysosomal dysfunction has been linked to pathophysiological conditions such as lysosomal storage diseases, cancer and neurodegeneration. Therefore understanding how lysosomal membrane mediates ionic permeability is important to our understanding of the organelle’s physiology function. We recently discovered a novel protein TMEM175 that forms a potassium-selective channel in lysosomal membrane. Genetic variation in human TMEM175 is also implicated in Parkinson’s disease. We propose three specific aims to expand our preliminary findings. In Aim 1, we will use patch clamp recordings to compare wild-type and TMEM175 knockout animals and will test whether there are other major lysosomal potassium channels in addition to TMEM175. Unlike those of plasma membrane channels, the functions of lysosomal potassium channels are largely unknown. In Aim 2, we will use the TMEM175 knockout mouse model to test the hypothesis that potassium channels regulate the normal working of lysosomes including organelle membrane potential regulation, luminal acidification, vesicle fusion and autophagy. In Aim 3, we will test whether TMEM175, like many other potassium channels on plasma membrane, is regulated by protein kinases. Using protein chemistry experiments, we will also determine the structural requirements for such regulation. Because of the fundamental function of lysosomes, the studies will reveal how lysosomal potassium channels contribute to the organelle’s function under physiological and pathological conditions. OMB No. 0925-0001/0002 (Rev. 01/18 Approved Through 03/31/2020) Page