Regulation of Kv1.3 Channels and T Cell Activity by Phospholipases

磷脂酶对 Kv1.3 通道和 T 细胞活性的调节

• 批准号: 8050301
• 负责人: David John Combs
• 金额: $ 4.31万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8050301
• 关键词: Action Potentials; Affect; Antigens; Autoimmune Diseases; B-Lymphocytes; Bacterial Infections; Blood Circulation; Cell membrane; Cells; Ceramides; Charge; Choline; Cytosol; Electrophysiology (science); Endocrine; Endoplasmic Reticulum; Enzymes; Eukaryotic Cell; Event; Excision; Flow Cytometry; Foundations; Gated Ion Channel; Head; Human; Immune response; In Situ; Interleukin-2; Left; Lipase; Lipids; Lymphocyte; Lymphocyte Activation; Mammals; Mediating; Membrane; Membrane Proteins; Mitogens; Modification; Molecular Biology; Molecular Conformation; Multiple Sclerosis; Muscle; Nerve; Outcome Study; Phospholipase; Phospholipids; Phosphorylcholine; Production; Prokaryotic Cells; Reaction; Regulation; Signal Transduction; Solutions; Sphingomyelinase; Sphingomyelins; Spiders; System; T cell differentiation; T-Cell Activation; T-Lymphocyte; Testing; Therapeutic Intervention; Work; Xenopus; acidic sphingomyelinase; autocrine; ceramide 1-phosphate; ceramide kinase; cytokine; expectation; extracellular; inorganic phosphate; novel; paracrine; prevent; protein function; receptor; sensor; sphingomyelin phosphodiesterase D; therapeutic target; transcription factor; voltage; voltage gated channel

DESCRIPTION (provided by applicant): Voltage-gated ion channels produce electrical signals in the so-called excitable cells including nerve, muscle, and endocrine cells. These channels are, however, also present in non-excitable eukaryotic cells (e.g. lymphocytes) and prokaryotes. This phenomenon raises the fundamental question of whether voltage-gated channels may be regulated by a mechanism other than changing the transmembrane voltage. The answers to this question will point to new strategies for achieving therapeutic interventions via the control of voltage-gated ion channels. Previous work from our lab showed that in a heterologous expression system, certain bacterial sphingomyelinases can activate voltage-gated (Kv) Kv1.3 channels whereas others suppress their activity. In T lymphocytes, the activity of Kv1.3 provides crucial support for signaling events that mediate T cell immune responses. Therefore, I will test the hypothesis that sphingomyelinases can regulate T cell status by modulating Kv1.3 activity, activity known to be essential for T cell activation. I will first test, in T lymphocytes, the hypothesis that enzymatic removal of positively charged choline from the membrane phospholipid sphingomyelin activates Kv1.3 channels whereas removal of the negatively charged phosphate group, along with choline, inhibits the channels (Aim#1; spider, bacterial and human enzymes will be used). Furthermore, I will test the hypothesis that removal of choline from sphingomyelin promotes T cell activation whereas removal of phosphate, along with choline, inhibits T cell activation (Aim#2). These studies will be carried out with a combination of electrophysiology, flow cytometry and molecular biology. The studies outlined above represent initial efforts to test an important novel hypothesis that specific lipases regulate T cell activity via modifying membrane phospholipids that interact with voltage sensors of Kv1.3 channels. Through these studies, we will obtain important information leading to new strategies for treating autoimmune diseases, including multiple sclerosis, and bacterial infection. The outcome of the studies will help establish a new conceptual framework for understanding the regulation of voltage-gated ion channel activity in non-excitable cells, laying the foundation for uncovering a potentially general mechanism underlying the regulation of membrane protein function. PUBLIC HEALTH RELEVANCE: Project Narrative The proposed project is designed to uncover an important, novel type of regulation of immune cell activity and investigate its underlying mechanism. The information yielded by this study will lead to new ways to treat multiple sclerosis (MS), other autoimmune diseases, and notorious methicillin-resistant Staphylococcus aureus (MRSA) or Bacillus anthracis infection.

描述（由申请人提供）： 电压门控离子通道在所谓的可兴奋细胞中产生电信号，包括神经、肌肉和内分泌细胞。然而，这些通道也存在于不可兴奋的真核细胞（例如淋巴细胞）和原核生物中。这一现象提出了一个根本性的问题，即电压门控通道是否可以通过改变跨膜电压以外的机制来调节。这个问题的答案将指向通过控制电压门控离子通道实现治疗干预的新策略。我们实验室以前的工作表明，在异源表达系统中，某些细菌鞘磷脂酶可以激活电压门控（Kv）Kv1.3通道，而其他人则抑制其活性。在T淋巴细胞中，Kv1.3的活性为介导T细胞免疫应答的信号传导事件提供了关键支持。因此，我将测试的假设，鞘磷脂酶可以通过调节Kv1.3的活性，已知是T细胞活化所必需的调节T细胞的状态。我将首先在T淋巴细胞中测试以下假设：从膜磷脂鞘磷脂酶中酶促去除带正电荷的胆碱激活Kv 1.3通道，而去除带负电荷的磷酸基团，沿着胆碱，抑制通道（目的#1;将使用蜘蛛、细菌和人类酶）。此外，我将检验从鞘磷脂中去除胆碱促进T细胞活化，而去除磷酸盐（沿着胆碱）抑制T细胞活化的假设（目标#2）。这些研究将结合电生理学、流式细胞术和分子生物学进行。 以上概述的研究代表了测试一个重要的新假设的初步努力，即特定的脂肪酶通过修饰与Kv1.3通道的电压传感器相互作用的膜磷脂来调节T细胞活性。通过这些研究，我们将获得重要的信息，导致治疗自身免疫性疾病，包括多发性硬化症和细菌感染的新策略。这些研究的结果将有助于建立一个新的概念框架，用于理解非兴奋细胞中电压门控离子通道活性的调节，为揭示膜蛋白功能调节的潜在一般机制奠定基础。 公共卫生相关性：该项目旨在揭示一种重要的新型免疫细胞活性调节并研究其潜在机制。这项研究产生的信息将导致治疗多发性硬化症（MS），其他自身免疫性疾病和臭名昭著的耐甲氧西林金黄色葡萄球菌（MRSA）或炭疽杆菌感染的新方法。