Cellular Physiology

SUMIWARY项目(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有组织的上皮细胞中转运和其他膜蛋白的功能和调节。核心将以这样的方式与其他核心相互作用，无论是在模型系统中开发的运输蛋白和相互作用蛋白的研究，还是在分离的小管中，都可以通过在体外系统中直接测量它们的功能来利用，这也有助于通过成像进行进一步的分析。这些核心技术也直接适用于由新的模型系统识别的质膜蛋白的研究。为了实现这些目标，核心将：a)在分离的体外系统中为运输蛋白和调节/相互作用蛋白的表达提供一个中心，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性的影响。这些系统包括非洲爪哇卵母细胞和幼稚细胞表达系统(例如HEK-293细胞)，并将能够直接测量通道的质膜表达及其电生理特性；b)建立系统来扩大对运输蛋白的研究以及在有组织的上皮细胞(无论是天然表达运输蛋白的还是模型上皮细胞)(如MDCK和FRT细胞)中，可以比单细胞表达系统更全面地评估运输蛋白突变的情况。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以及组织电容的直接测量；c)提供调节上皮细胞基因表达的方法。重组病毒将被产生，以允许重组野生型或突变的通道亚基，并表达其他感兴趣的基因。沉默RNA将使用重组病毒和脂类介导的转移方法表达，以允许下调基因表达；以及d)使用生化和质谱学方法分析运输蛋白和调节蛋白的翻译后修饰，包括磷酸化、泛素化、糖基化和棕榈酸化。

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.