Regulation of lysosomal potassium channels

项目负责人/主要研究者（末、首、中）：任德健 本研究的总体目标是了解钾离子的功能和调控 溶酶体功能的通道。钾是最丰富的细胞内离子，面对两个血浆 膜和细胞器膜。质膜对钾具有高度渗透性。80多 现已发现钾通道介导这种质膜钾渗透性。在 相比之下，钾如何通过细胞内细胞器如溶酶体的了解要少得多。 溶酶体在细胞清除、消化、再循环、胞吐和膜修复中起着重要作用。 由于腔中钙的高浓度，溶酶体也是钙的储存器， 被释放到胞质溶胶中以形成胞质溶胶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病理生理条件有关，如溶酶体储存 疾病、癌症和神经变性。因此，了解溶酶体膜如何介导离子 渗透性对我们理解细胞器的生理功能很重要。我们最近发现了一个 在溶酶体膜中形成钾选择性通道的新型蛋白质TMEM 175。遗传变异 在人类中，TMEM175也与帕金森病有关。我们提出了三个具体目标，以扩大我们的 初步调查结果。在目的1中，我们将使用膜片钳记录来比较野生型和TMEM 175 敲除动物，并将测试是否有其他主要的溶酶体钾通道，除了 TMEM175。与质膜通道不同，溶酶体钾通道的功能是 大部分未知。在目标2中，我们将使用TMEM175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、囊泡融合和自噬。在目标3中，我们将测试TMEM175，如 质膜上的许多其他钾通道，由蛋白激酶调节。使用蛋白 化学实验，我们也将确定这种调节的结构要求。因为 作为溶酶体的基本功能，这些研究将揭示溶酶体钾通道如何促进溶酶体的功能。 细胞器在生理和病理条件下的功能。 OMB编号0925 - 0001/0002（2018年1月修订版批准至2020年3月31日）页码

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