Cellular Physiology

细胞生理学

• 批准号: 8734387
• 负责人: KENNETH R HALLOWS
• 金额: $ 21.87万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8734387
• 关键词: Adenoviruses; Area; Biochemical; Biological Assay; Biological Models; Carrier Proteins; Cell membrane; Cell physiology; Cells; Cellular biology; Characteristics; Complement; Down-Regulation; Electric Capacitance; Epithelial; Epithelial Cells; Epithelium; Funding; Gene Expression; Genes; Goals; Image; Image Analysis; In Vitro; Instruction; International; Investigation; Ion Transport; Kidney; Kidney Diseases; Lipids; Mammalian Cell; Mass Spectrum Analysis; Measurement; Measures; Mediating; Membrane; Membrane Proteins; Membrane Transport Proteins; Methods; Modeling; Molecular; Molecular Conformation; Mutate; Mutation; Oocytes; Organ; Pathway interactions; Pattern; Phosphorylation; Post Translational Modification Analysis; Post-Translational Protein Processing; Proteins; RNA Interference; Recombinants; Regulation; Research; Research Personnel; Research Project Grants; Resistance; Resources; Role; Series; Small Interfering RNA; Structure; Structure-Activity Relationship; Subfamily lentivirinae; Surface; System; Techniques; Technology; Tissues; Transmembrane Transport; Ubiquitination; Viral Vector; Virus; Xenopus oocyte; base; design; electric impedance; genetic regulatory protein; glycosylation; in vitro Model; insight; interest; kidney imaging; knock-down; novel; overexpression; palmitoylation; recombinant virus; reconstitution; trafficking; voltage clamp

PROJECT SUMIWARY (See instructions): The Cellular Physiology Core provides a series of graded in vitro model systems and technologies for studying the function and regulation of transport and other membrane proteins in isolated expression systems and organized epithelia. The Core will interact with the other Cores in such a way that studies of transport proteins and interacting proteins developed either in model systems or isolated tubules may be exploited by direct measures of their function in in vitro systems that also lend themselves to further analysis by imaging. The Core technologies also are directly applicable to the study of plasma membrane proteins identified by novel model systems. To accomplish these goals, the Core will: a) provide a center for expression of transport proteins and regulatory/interacting proteins in isolated in vitro systems, which will permit direct assessment of the influence of these interactions on electrophysiologic characteristics both at the macroscopic and single channel level. These systems include Xenopus oocytes and naive cell expression systems (e.g., HEK-293 cells) and will permit direct measure of plasma membrane expression of channels and their electrophysiologic features; b) establish systems to expand the study of transport proteins and interacting proteins/pathways in organized epithelia either natively expressing the transport proteins or in model epithelia (e.g., MDCK and FRT cells) where transport protein mutations may be evaluated more fully than in single cell expression systems. These techniques will include standard voltage clamp for assessment of short-circuit current and transepithelial resistance and direct measures of tissue capacitance; c) provide methods for modulating gene expression in epithelia. Recombinant viruses will be generated to allow reconstitution of wild-type or mutated channel subunits and expression of other genes of interest. Silencing RNAs will be expressed using recombinant viruses and lipid-mediated transfer methods to permit downregulation of gene expression; and d) provide analysis of post-translational modifications of transport proteins and regulatory proteins, including phosphorylation, ubiquitination, glycosylation and palmitoylation using both biochemical and mass spectrometry approaches.

项目摘要（见说明）：细胞生理学核心提供了一系列分级的体外模型系统和技术，用于研究分离表达系统和组织上皮中转运和其他膜蛋白的功能和调节。核心将与其他核心以这样的方式相互作用，即在模型系统或分离的小管中开发的转运蛋白和相互作用蛋白的研究可以通过直接测量它们在体外系统中的功能来利用，这些系统也有助于通过成像进行进一步分析。核心技术也可直接应用于通过新型模型系统鉴定的质膜蛋白的研究。为了实现这些目标，核心将：a）提供在分离的体外系统中表达转运蛋白和调节/相互作用蛋白的中心，这将允许在宏观和单通道水平上直接评估这些相互作用对电生理特征的影响。这些系统包括非洲爪蟾卵母细胞和幼稚细胞表达系统（例如，HEK-293细胞），并将允许直接测量通道的质膜表达及其电生理学特征; B）建立系统以扩大天然表达转运蛋白的有组织上皮或模型上皮（例如，MDCK和FRT细胞），其中转运蛋白突变可以比在单细胞表达系统中更充分地评估。这些技术将包括用于评估短路电流和跨上皮电阻的标准电压钳和组织电容的直接测量; c）提供用于调节上皮中基因表达的方法。将产生重组病毒以允许野生型或突变的通道亚基的重建和其他感兴趣的基因的表达。沉默RNA将使用重组病毒和脂质介导的转移方法表达，以允许下调基因表达;和d）使用生物化学和质谱方法提供转运蛋白和调节蛋白的翻译后修饰的分析，包括磷酸化、泛素化、糖基化和棕榈酰化。