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: 10228308
• 负责人: maria Falzone
• 金额: $ 6.6万
• 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/10228308
• 关键词: Agonist; Binding; Binding Sites; Biological; Bioluminescence; Cell membrane; Cells; Cleaved cell; 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.

项目摘要 细胞通过G蛋白偶联受体（GPCR）对许多细胞外刺激反应。细胞外信号传导 分子结合GPCR并催化细胞内膜锚定G蛋白的释放，GA和GBG， 在下游目标上起作用。离子通道是许多GPCR信号级联的下游目标， 将细胞反应与这些刺激的响应连接到令人兴奋的膜。离子的GPCR依赖性调节 通道在许多物理过程中起着至关重要的作用，包括伤害感受，心率调节， 和炎症；因此，必须了解这一法规至关重要。离子通道可以通过GPCR调节 直接通过G蛋白或G蛋白调节的第二个使者信号传导，包括磷脂酰肌醇 - 4,5-三磷酸（PIP2）。 PIP2被G蛋白依赖性B家族降解的磷脂酶C（PLC）降解 酶，清除PIP2生产IP3和DAG。许多离子通道家庭受PIP2的调节 以PLCB依赖性方式，包括向内整流K+（KIR）通道和电压依赖性K+通道。 PLCB酶都被GAQ和GBG激活，将其功能链接到GAQ和GAI耦合接收机。 尽管GAQ的调节是充分理解的，但在依赖GBG依赖性激活方面却尚不清楚。在 为了了解下游离子通道的GPCR依赖性调节和相关的 生理过程，有必要了解PLCB酶的G蛋白调节，这是 关键信号中间体。为此，我建议通过研究PLCB酶的GBG依赖性激活 以下两个目的：（1）研究PLCB酶的GBG依赖性激活的最小要求 以及使用无单元重构系统对下游离子通道进行调节，（2）表征 GBG和PLCB之间的相互作用，包括结合位点的定位以及GBG-的表征 使用低温电子显微镜和生物发光共振的依赖通信 能量转移。这些目标的成功完成将直接将PLCB调节与对离子的影响联系起来 渠道，扩展了这些信号级联的知识。该项目将在 洛克菲勒大学Roderick Mackinnon博士的监督。麦金农博士有丰富的经验 研究离子渠道的功能和调节以及培训博士后研究人员成功 独立研究人员。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。参与培训计划 包括一个时间表，描述了拟议实验的完成，并为个人和个人分配时间 职业发展包括与Mackinnon博士的经常会面，参加会议，展示 调查结果，心理学生和授予写作，以获得独立研究计划的资金。