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.

Public Health Relevance

The Pittsburgh Center for Kidney Research Cellular Physiology Core provides mechanistic analyses of the functions of membrane transport and other associated proteins through a series of graded in vitro model systems. This Core interfaces with and complements the other Cores and has the overall goal of elucidating at a molecular and cellular level the function and regulation of key proteins involved in kidney diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Center Core Grants (P30)
Project #
5P30DK079307-07
Application #
8734387
Study Section
Special Emphasis Panel (ZDK1-GRB-6)
Project Start
Project End
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
7
Fiscal Year
2014
Total Cost
$218,680
Indirect Cost
$76,680
Name
University of Pittsburgh
Department
Type
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Sheng, Shaohu; Chen, Jingxin; Mukherjee, Anindit et al. (2018) Thumb domains of the three epithelial Na+ channel subunits have distinct functions. J Biol Chem 293:17582-17592
Hughes, Andrew D; Lakkis, Fadi G; Oberbarnscheidt, Martin H (2018) Four-Dimensional Imaging of T Cells in Kidney Transplant Rejection. J Am Soc Nephrol 29:1596-1600
Theodoraki, M-N; Hoffmann, T K; Jackson, E K et al. (2018) Exosomes in HNSCC plasma as surrogate markers of tumour progression and immune competence. Clin Exp Immunol 194:67-78
Apodaca, Gerard (2018) Role of Polarity Proteins in the Generation and Organization of Apical Surface Protrusions. Cold Spring Harb Perspect Biol 10:
Jackson, Travis C; Kotermanski, Shawn E; Kochanek, Patrick M et al. (2018) Oxidative Stress Induces Release of 2'-AMP from Microglia. Brain Res :
Balchak, Deidra M; Thompson, Rebecca N; Kashlan, Ossama B (2018) The epithelial Na+ channel ? subunit autoinhibitory tract suppresses channel activity by binding the ? subunit's finger-thumb domain interface. J Biol Chem 293:16217-16225
Sun, Zhihao; Brodsky, Jeffrey L (2018) The degradation pathway of a model misfolded protein is determined by aggregation propensity. Mol Biol Cell 29:1422-1434
Boyd-Shiwarski, Cary R; Shiwarski, Daniel J; Roy, Ankita et al. (2018) Potassium-regulated distal tubule WNK bodies are kidney-specific WNK1 dependent. Mol Biol Cell 29:499-509
Kashlan, Ossama B; Kinlough, Carol L; Myerburg, Michael M et al. (2018) N-linked glycans are required on epithelial Na+ channel subunits for maturation and surface expression. Am J Physiol Renal Physiol 314:F483-F492
Jackson, Edwin K; Mi, Eric; Ritov, Vladimir B et al. (2018) Extracellular Ubiquitin(1-76) and Ubiquitin(1-74) Regulate Cardiac Fibroblast Proliferation. Hypertension 72:909-917

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