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Core A: Cellular Physiology
The Cellular Physiology Core provides investigators with in vitro systems for the study of transport processes at the molecular, cell, and epithelial levels. These systems facilitate basic studies of structure, function and regulation not only of membrane transport proteins, but also of membrane target proteins of epithelia identified in other Cores, thereby supporting many nationally funded research projects focusing on epithelial cell biology and transport protein regulation, promoting investigation within this area. The objectives of this core are:

• To provide a center for the study of transport proteins and other membrane resident proteins in isolated in vitro systems. Single cell expression systems such as oocytes and naïve mammalian cells (e.g., HEK-293 cells) are used to describe and elucidate structure-function relationships, cellular processing steps, membrane interactions, molecular dynamics and turnover of surface resident proteins. Such systems may be employed to examine directly the interaction of wild-type and mutated proteins with respect to both trafficking patterns and function, and provide the basis for studies which may then be carried to organized epithelia or tissues. These systems are ideal for electrophysiological characterization of ion channel activity, both at the single-channel and whole-cell levels. This Core provides technology for measurements of surface expression, whole-cell channel activity, single-channel characteristics and conformational changes of wild-type and mutated channels expressed with or without putative interacting or regulatory partners. The core also provides voltage clamp fluorometry, a technique that assesses conformational rearrangements that occur in restricted areas of ion channels and transporters during gating.

• To establish systems to expand the study of transport proteins and other regulatory proteins in organized epithelia, either natively expressing the proteins of interest or in model epithelia. To examine membrane protein regulation in the setting of organized epithelia, we will make available electrophysiologic techniques for examining transport function, expression and turnover. Such techniques complement studies that may be performed in single cell systems and in the Kidney Imaging Core. These techniques include standard voltage clamp analysis as well as direct measures of tissue capacitance and impedance. Studies may be carried out either in epithelia that natively express channels of interest or in model epithelia (e.g., MDCK or FRT cells), where mutated forms of the channel are expressed. These systems lend themselves to modulation of gene expression by either overexpression or silencing techniques.

• To provide methods for modulating gene expression in organized epithelia. To take advantage of the model systems provided in this Core and in the Kidney Imaging Core, a viral vector Subcore has been established to enable investigators to over-express genes of interest in cells and epithelia using recombinant adenoviruses and lentiviruses. To silence gene expression, short hairpin and small interfering RNAs are expressed using these viruses or by nucleofection and lipid-mediated transfer methods.

• To provide technical resources for the analysis of post-translational modifications of transport and other associated proteins. Post-translational modifications, including phosphorylation, ubiquitination and palmitoylation, represent key mechanisms for the regulation of epithelial transport. This Subcore enables investigators to examine post-translational modifications in proteins to gain mechanistic insights into the regulation of membrane transport and associated proteins at the molecular level. Biochemical assays to identify and examine these post-translational modifications will be supplemented by mass spectrometry analysis.