The Na,K-ATPase, or sodium pump, generates the ion gradients responsible for most fluid and electrolyte transport processes in the kidney. In keeping with this central role in homeostasis, the function of the renal sodium pump is regulated by signals that contribute to the control of body fluid volume and blood pressure. To drive vectorial trans-epithelial transport, the Na,K-ATPase must be restricted to the basolateral surfaces of renal tubule epithelial cells. While a great deal has been learned about the structure and catalytic cycle of the sodium pump, much less is known about the partner proteins and trafficking pathways that determine its subcellular distribution and modulate its activity. During the previous 4 year funding period of this award we have identified new sodium pump interacting proteins and explored their roles in controlling its properties. We have also adapted a novel labeling methodology that allows us to investigate the attributes of temporally defined cohorts of Na,K-ATPase. The SNAP tag technique endows a protein of interest with the ability to become covalently coupled to fluorophores or biochemically useful ligands. We have generated a SNAP-tagged Na,KATPase a-subunit fusion protein and expressed it in MDCK renal epithelial cells. The SNAP tagged a-subunit assembles with Na,K-ATPase ?-subunit to form functionally competent pumps that are sorted appropriately. We have developed pulse-chase protocols with which we can observe directly the trafficking itinerary pursued by newly synthesized Na,K-ATPase and isolate newly synthesized Na,K-ATPase in association with its collections of partner proteins. These efforts have already yielded surprising new insights into the mechanisms through which the pump is assembled and transported through epithelial cells. We will expand our analysis to: 1) define the pathways and parameters that govern the trafficking of newly synthesized Na,K-ATPase in polarized renal epithelial cells;2) identify and characterize the protein partners that interact with a temporally-defined cohort of Na,K-ATPase at each stage of its post-synthetic life span;and 3) establish the pathways and partners that regulate Na,K-ATPase sorting and function in the epithelial cells of each of the segments of the developing kidney and mature nephron in situ. This approach permits us to ask entirely new and previously experimentally inaccessible questions about the biology of the sodium pump, and will yield valuable insights into the cell biological and biochemical factors that govern this physiologically critical ion transport system.

Public Health Relevance

The kidney is responsible for determining the body's salt and water composition. The kidney depends upon an enzyme called the Na,K-ATPase to drive all of its salt and fluid transport processes. Changes in Na,K- ATPase function can produce profound changes in kidney salt and fluid transport, and hence in blood pressure. Our studies use a novel method to explore the mechanisms that kidney cells use to regulate the activity of this critical enzyme.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK072614-24
Application #
8665901
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Ketchum, Christian J
Project Start
1989-07-01
Project End
2015-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
24
Fiscal Year
2014
Total Cost
$452,526
Indirect Cost
$178,871
Name
Yale University
Department
Physiology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Stoops, Emily H; Hull, Michael; Caplan, Michael J (2016) Newly synthesized and recycling pools of the apical protein gp135 do not occupy the same compartments. Traffic 17:1272-1285
Farr, Glen A; Hull, Michael; Stoops, Emily H et al. (2015) Dual pulse-chase microscopy reveals early divergence in the biosynthetic trafficking of the Na,K-ATPase and E-cadherin. Mol Biol Cell 26:4401-11
Alves, Daiane S; Thulin, Gunilla; Loffing, Johannes et al. (2015) Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia. J Am Soc Nephrol 26:2765-76
Stoops, Emily H; Hull, Michael; Olesen, Christina et al. (2015) The periciliary ring in polarized epithelial cells is a hot spot for delivery of the apical protein gp135. J Cell Biol 211:287-94
Stoops, Emily H; Caplan, Michael J (2014) Trafficking to the apical and basolateral membranes in polarized epithelial cells. J Am Soc Nephrol 25:1375-86
Jouret, François; Wu, Jingshing; Hull, Michael et al. (2013) Activation of the Ca²+-sensing receptor induces deposition of tight junction components to the epithelial cell plasma membrane. J Cell Sci 126:5132-42
Seo-Mayer, Patricia W; Thulin, Gunilla; Zhang, Li et al. (2011) Preactivation of AMPK by metformin may ameliorate the epithelial cell damage caused by renal ischemia. Am J Physiol Renal Physiol 301:F1346-57
Zhang, Li; Jouret, Francois; Rinehart, Jesse et al. (2011) AMP-activated protein kinase (AMPK) activation and glycogen synthase kinase-3β (GSK-3β) inhibition induce Ca2+-independent deposition of tight junction components at the plasma membrane. J Biol Chem 286:16879-90
Kimura, Toru; Han, Wonsun; Pagel, Philipp et al. (2011) Protein phosphatase 2A interacts with the Na,K-ATPase and modulates its trafficking by inhibition of its association with arrestin. PLoS One 6:e29269
Alves, Daiane S; Farr, Glen A; Seo-Mayer, Patricia et al. (2010) AS160 associates with the Na+,K+-ATPase and mediates the adenosine monophosphate-stimulated protein kinase-dependent regulation of sodium pump surface expression. Mol Biol Cell 21:4400-8

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