Cellular Physiology

项目SUMIWARY（见说明书）：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，用于研究转运和其他膜蛋白在分离表达系统和组织上皮中的功能和调控。核心将以这样一种方式与其他核心相互作用，即在模型系统或分离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其在体外系统中的功能来利用，这些系统也可以通过成像进一步分析。核心技术也可直接应用于新模型系统鉴定的质膜蛋白的研究。为了实现这些目标，Core将：a)提供一个转运蛋白和调控/相互作用蛋白在分离体外系统中的表达中心，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。这些系统包括爪蟾卵母细胞和原始细胞表达系统（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特征；b)建立系统，以扩大有组织上皮中转运蛋白和相互作用蛋白/途径的研究，无论是在天然表达转运蛋白还是在模型上皮（例如MDCK和FRT细胞）中，运输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上皮电阻的标准电压钳和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。将产生重组病毒，以允许重组野生型或突变的通道亚基和表达其他感兴趣的基因。沉默rna将使用重组病毒和脂质介导的转移方法表达，以允许基因表达下调；d)使用生化和质谱方法分析转运蛋白和调节蛋白的翻译后修饰，包括磷酸化、泛素化、糖基化和棕榈酰化。

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.