Activation of phospholipase C beta enzymes by G beta-gamma and corresponding regulation of downstream ion channels

G beta-gamma 激活磷脂酶 C beta 酶以及下游离子通道的相应调节

• 批准号: 10392874
• 负责人: maria Falzone
• 金额: $ 6.76万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392874
• 关键词: Agonist; Binding; Binding Sites; Biological; Bioluminescence; Cell membrane; Cells; Complex; Conflict (Psychology); Coupled; Cryoelectron Microscopy; Electron Microscopy; Electrophysiology (science); Energy Transfer; Environment; Enzymes; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Funding; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Grant; Heart Rate; Hormones; Image; In Vitro; Inflammation; Intracellular Membranes; Investigation; Ion Channel; Knowledge; Label; Link; Lipid Bilayers; Liposomes; Location; Luciferases; Measurable; Measurement; Measures; Membrane; Molecular Conformation; Mutagenesis; Neurotransmitters; Nociception; Nucleotides; Pathway interactions; Phosphatidylinositol 4,5-Diphosphate; Phospholipase C; Phospholipases A; Physiological Processes; Physiology; Play; Potassium Channel; Proteins; Regulation; Reporter; Research; Research Personnel; Resources; Role; Second Messenger Systems; Side; Signal Pathway; Signal Transduction; Signaling Molecule; Stimulus; Structure; Supervision; System; Time; TimeLine; Training; Universities; Writing; career development; cryogenics; design; experience; experimental study; extracellular; fluorescence microscope; fluorophore; improved; insight; meetings; molecular modeling; nanomolar; phospholipase C beta; post-doctoral training; programs; receptor; reconstitution; response; single molecule; student mentoring; symposium; voltage

Project Summary Cells respond to many extracellular stimuli via G protein coupled receptors (GPCR). Extracellular signaling molecules bind GPCR’s and catalyze the release of intracellular membrane anchored G proteins, Ga and Gbg, which act on downstream targets. Ion channels are a downstream target of numerous GPCR signaling cascades, connecting the cellular response to these stimuli to membrane excitability. GPCR-dependent regulation of ion channels plays an essential role in many physiological processes including nociception, regulation of heart rate, and inflammation; therefore, it is essential to understand this regulation. Ion channels can be regulated by GPCR signaling directly by G proteins or by G protein-regulated second messengers, including phosphatidylinositol- 4,5-bisphosphate (PIP2). PIP2 is degraded by the G protein-dependent b family of phospholipase C (PLC) enzymes, which cleave PIP2 to produce IP3 and DAG. Numerous families of ion channels are regulated by PIP2 in a PLCb-dependent manner, including inwardly rectifying K+ (Kir) channels and voltage-dependent K+ channels. PLCb enzymes are activated by both Gaq and Gbg, linking their function to both Gaq and Gai-coupled receptors. While the regulation by Gaq is well-understood, much is unknown regarding the Gbg-dependent activation. In order to understand the GPCR-dependent regulation of downstream ion channels and the associated physiological processes, it is necessary to understand the G protein regulation of PLCb enzymes, which are the key signaling intermediate. To this end, I propose to study the Gbg-dependent activation of PLCb enzymes via the following two aims: (1) Investigate the minimal requirements for Gbg-dependent activation of PLCb enzymes and the regulation of downstream ion channels using a cell-free reconstituted system, (2) Characterize the interaction between Gbg and PLCb including localization of the binding site and characterization of the Gbg- dependent conformational changes using cryogenic electron microscopy and bioluminescence resonance energy transfer. Successful completion of these aims will directly connect PLCb regulation to its effect on ion channels, expanding the knowledge of these signaling cascades. This project will be conducted under the supervision of Dr. Roderick MacKinnon at Rockefeller University. Dr. MacKinnon has extensive experience studying the function and regulation of ion channels as well as training postdoctoral researchers to succeed as independent researchers. Together, the lab and Rockefeller University generate an environment with the resources and intellectual input necessary for completing the proposed project. The accompanying training plan includes a timeline describing the completion of the proposed experiments and allocates time for personal and career development including frequent meetings with Dr. MacKinnon, attending conferences to present the findings, mentoring students, and grant writing to acquire funding for an independent research program.

项目概要 细胞通过 G 蛋白偶联受体 (GPCR) 对许多细胞外刺激做出反应。细胞外信号传导 分子结合 GPCR 并催化细胞内膜锚定 G 蛋白、Ga 和 Gbg 的释放， 作用于下游目标。离子通道是众多 GPCR 信号级联的下游目标， 将细胞对这些刺激的反应与膜兴奋性联系起来。 GPCR 依赖性离子调节 通道在许多生理过程中发挥着重要作用，包括伤害感受、心率调节、 和炎症；因此，了解该规定非常重要。 GPCR 可以调节离子通道 直接通过 G 蛋白或 G 蛋白调节的第二信使（包括磷脂酰肌醇）发出信号 4,5-二磷酸（PIP2）。 PIP2 被磷脂酶 C (PLC) 的 G 蛋白依赖性 b 家族降解 酶，裂解 PIP2 产生 IP3 和 DAG。许多离子通道家族均受 PIP2 调节 以 PLCb 相关的方式，包括内向整流 K+ (Kir) 通道和电压相关的 K+ 通道。 PLCb 酶由 Gaq 和 Gbg 激活，将其功能与 Gaq 和 Gai 偶联受体联系起来。 虽然 Gaq 的调节已被充分理解，但有关 Gbg 依赖性激活的情况尚不清楚。在 为了了解下游离子通道的 GPCR 依赖性调节以及相关的 生理过程中，有必要了解PLCb酶的G蛋白调节，它们是 关键信号中间体。为此，我建议通过以下方式研究 PLCb 酶的 Gbg 依赖性激活： 以下两个目标：（1）研究 PLCb 酶的 Gbg 依赖性激活的最低要求 以及使用无细胞重组系统调节下游离子通道，（2）表征 Gbg 和 PLCb 之间的相互作用，包括结合位点的定位和 Gbg- 的表征 使用低温电子显微镜和生物发光共振进行依赖性构象变化 能量转移。成功完成这些目标将直接将 PLCb 调节与其对离子的影响联系起来 通道，扩展了这些信号级联的知识。该项目将在 洛克菲勒大学罗德里克·麦金农 (Roderick MacKinnon) 博士的指导。 MacKinnon博士拥有丰富的经验 研究离子通道的功能和调节，并培养博士后研究人员取得成功 独立研究人员。该实验室和洛克菲勒大学共同创造了一个环境 完成拟议项目所需的资源和智力投入。随附的培训计划 包括描述拟议实验的完成情况的时间表，并为个人和个人分配时间 职业发展，包括与麦金农博士经常会面，参加会议以介绍 调查结果、指导学生以及撰写赠款以获得独立研究项目的资金。