Dr. Kashlan is currently conducting his research in the laboratory of Dr. Kleyman at the University of Pittsburgh. His career goals for the award period are to further develop his critical thinking skills, new approaches towards laboratory research, and technical expertise in electrophysiological, biochemical, and molecular biological methods. After 1-2 years of mentored research training in Dr. Kleyman's laboratory, Dr. Kashlan plans to make the transition to a tenure-track faculty position. His long-term career goals are to become a fully independent academic investigator in the broad fields of biophysics and molecular physiology, performing research that gives insight into important biological problems that impact clinical issues, with a particular focus on the structure and function of proteins. The basis for the first specific aim of the proposed research is the discovery in our laboratory of an interaction surface on the first transmembrane segment of the alpha subunit (alphaM1) within the epithelial Na(+) channel (ENaC). We propose experiments to identify the transmembrane segment(s) that interact with alphaM1, and then to characterize the interaction between these two segments. To that end, we will develop a novel reporter assay to identify interactions between different transmembrane segments and perform complementary mutagenesis paired with functional assays to characterize the interaction between the two segments. The second specific aim will characterize the structure of the ENaC inner pore by utilizing engineered histidines and Ni(2+), which may give insights into the mechanisms of ENaC gating, conductance and ion selectivity. We will perform scanning histidine mutagenesis throughout pore lining ENaC structural elements and measure the effects of adding Ni(2+) to the intracellular side of the mutant channels using the cut open oocyte and excised inside-out macro patch techniques. This research proposal should result in a greater understanding of the structure and function of the ENaC pore, and may give insights into channel selectivity, conductance and gating. The research proposed here may inform mechanisms underlying Na(+) homeostasis, whose failure leads to alterations in blood pressure and abnormal mucociliary clearance.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
5K01DK078734-05
Application #
8245814
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Rankin, Tracy L
Project Start
2008-04-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2014-03-31
Support Year
5
Fiscal Year
2012
Total Cost
$131,266
Indirect Cost
$9,723
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Mukherjee, Anindit; Mueller, Gunhild M; Kinlough, Carol L et al. (2014) Cysteine palmitoylation of the ? subunit has a dominant role in modulating activity of the epithelial sodium channel. J Biol Chem 289:14351-9
Youker, Robert T; Bruns, Jennifer R; Costa, Simone A et al. (2013) Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization. Mol Biol Cell 24:1996-2007
Kashlan, Ossama B; Blobner, Brandon M; Zuzek, Zachary et al. (2012) Inhibitory tract traps the epithelial Na+ channel in a low activity conformation. J Biol Chem 287:20720-6
Kashlan, Ossama B; Kleyman, Thomas R (2012) Epithelial Na(+) channel regulation by cytoplasmic and extracellular factors. Exp Cell Res 318:1011-9
Pan, Jianjun; Chen, Qiang; Willenbring, Dan et al. (2012) Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist acetylcholine. Nat Commun 3:714
Shi, Shujie; Blobner, Brandon M; Kashlan, Ossama B et al. (2012) Extracellular finger domain modulates the response of the epithelial sodium channel to shear stress. J Biol Chem 287:15439-44
Kashlan, Ossama B; Adelman, Joshua L; Okumura, Sora et al. (2011) Constraint-based, homology model of the extracellular domain of the epithelial Na+ channel ? subunit reveals a mechanism of channel activation by proteases. J Biol Chem 286:649-60
Tolino, Lindsey A; Okumura, Sora; Kashlan, Ossama B et al. (2011) Insights into the mechanism of pore opening of acid-sensing ion channel 1a. J Biol Chem 286:16297-307
Kashlan, Ossama B; Kleyman, Thomas R (2011) ENaC structure and function in the wake of a resolved structure of a family member. Am J Physiol Renal Physiol 301:F684-96
Shi, Shujie; Ghosh, D Dipon; Okumura, Sora et al. (2011) Base of the thumb domain modulates epithelial sodium channel gating. J Biol Chem 286:14753-61

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