This proposal will increase understanding of acute regulation of the small intestinal brush border Na+/H+ exchanger NHE3, which accounts for the majority of Na+ absorption in the intestine and contributes to abnormal water and Na+ absorption in most diarrheal diseases. NHE3 transports Na+ and H+ under basal conditions, and in the post-prandial state is first inhibited and then stimulated later in digestion. Studies ae proposed based on our findings during the previous funding period that A) A large, multiprotein complex forms on the NHE3 C-terminus acting as a scaffold between aa 586-605 which is necessary for the post-prandial inhibition of NHE3 activity as is mimicked by elevated Ca2+ and cAMP. Proteins shown to be part of this complex include NHERF 1-4, CaMKII, CK2, PLC?, and probably CaM and the complex is dynamic, changing in composition with conditions which mimick the post-prandial state. B) NHE3 is fixed to the microvillar cytoskeleton under basal conditions, but reversibly dissociates with signaling that mimics the post-prandial state. This proposal will increase understanding of the role of this signaling complex in NHE3 regulation and diarrheal diseases by: A) Testing the hypothesis that the NHE3 C-terminal signaling complex is a physiologic and pathophysiologic regulator of NHE3, which can be affected to stimulate NHE3 activity. The role of CaM KII will be studied, which we have shown is part of the signaling complex and which appears to inhibit basal NHE3 activity and to phosphorylate NHE3. Also, a peptide that mimics the part of the NHE3 C-terminus to which the signaling complex attaches can be loaded into cells, and stimulates basal NHE3 activity and reduces Ca2+ and cAMP-induced NHE3 inhibition, including in an in vivo model of cholera. This peptide will be examined for effects on NHE3 regulation in polarized intestinal cells and intact rodent intestine as well as models of acute diarrhea and used to probe biochemically the nature of the NHE3 signaling complex. This peptide may be useful to develop as a drug to treat diarrhea. B) Testing the hypothesis that acute regulation of NHE3 activity involves two separate events: changes in NHE3 trafficking, which has been studied in the past, and the newly recognized dynamic aspects in the NHE3/microvillar cytoskeletal-NHERF2 association. Advanced imaging/confocal microscopy with polarized epithelial cells will determine which NHE3 regulatory processes are associated with freeing up NHE3 from NHERF2 and the microvillar cytoskeleton and determine which steps in signaling are responsible for this dynamic interaction of NHE3 with the microvillar actin cytoskeleton as well as the consequences on regulation of NHE3 activity of disrupting the dissociation, including in in vivo conditions. These studies will provide insights into how this NHE3 signaling complex helps set NHE3 activity under basal and regulated conditions, including in two in vivo diarrhea models;and also how information about this complex can be used translationally to develop drug therapy of diarrhea by stimulating NHE3.

Public Health Relevance

This proposal will increase understanding of acute regulation of the small intestinal brush border Na/H exchanger NHE3, which is the major way Na is absorbed in the intestine and which contributes to abnormal water and Na absorption in most diarrheal diseases. NHE3 is regulated by a large multiprotein complex which forms on its C-terminus. This proposal will examine how this complex accomplishes the regulation, including: A) Determining how calmodulin kinase II, which is part of the complex, inhibits NHE3 activity under basal conditions;B) Defining the effects of a peptide that mimics the part of NHE3 on which the complex forms and which can be loaded into intact cells and small intestine, including in an in vivo mouse jejunal model and models of acute diarrhea;and B) Examining a newly recognized aspect of signal transduction that regulates NHE3, which is dynamic control of the association of NHE3 with NHERF2 and the microvillar cytoskeleton to allow newly trafficked NHE3 to move up and down the microvillus.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK026523-34
Application #
8724471
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Grey, Michael J
Project Start
1988-06-01
Project End
2017-08-31
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
34
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Baltimore
State
MD
Country
United States
Zip Code
21218
In, Julie; Foulke-Abel, Jennifer; Zachos, Nicholas C et al. (2016) Enterohemorrhagic Escherichia coli reduce mucus and intermicrovillar bridges in human stem cell-derived colonoids. Cell Mol Gastroenterol Hepatol 2:48-62.e3
Zachos, Nicholas C; Kovbasnjuk, Olga; Foulke-Abel, Jennifer et al. (2016) Human Enteroids/Colonoids and Intestinal Organoids Functionally Recapitulate Normal Intestinal Physiology and Pathophysiology. J Biol Chem 291:3759-66
Foulke-Abel, Jennifer; In, Julie; Yin, Jianyi et al. (2016) Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology. Gastroenterology 150:638-649.e8
In, Julie G; Foulke-Abel, Jennifer; Estes, Mary K et al. (2016) Human mini-guts: new insights into intestinal physiology and host-pathogen interactions. Nat Rev Gastroenterol Hepatol 13:633-642
Janecke, Andreas R; Heinz-Erian, Peter; Yin, Jianyi et al. (2015) Reduced sodium/proton exchanger NHE3 activity causes congenital sodium diarrhea. Hum Mol Genet 24:6614-23
Chen, Tiane; Kocinsky, Hetal S; Cha, Boyoung et al. (2015) Cyclic GMP kinase II (cGKII) inhibits NHE3 by altering its trafficking and phosphorylating NHE3 at three required sites: identification of a multifunctional phosphorylation site. J Biol Chem 290:1952-65
Saxena, Kapil; Blutt, Sarah E; Ettayebi, Khalil et al. (2015) Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology. J Virol 90:43-56
Zhu, Xinjun Cindy; Sarker, Rafiquel; Horton, John R et al. (2015) Nonsynonymous single nucleotide polymorphisms of NHE3 differentially decrease NHE3 transporter activity. Am J Physiol Cell Physiol 308:C758-66
Yang, Jianbo; Sarker, Rafiquel; Singh, Varsha et al. (2015) The NHERF2 sequence adjacent and upstream of the ERM-binding domain affects NHERF2-ezrin binding and dexamethasone stimulated NHE3 activity. Biochem J 470:77-90
Singh, Varsha; Yang, Jianbo; Cha, Boyoung et al. (2015) Sorting nexin 27 regulates basal and stimulated brush border trafficking of NHE3. Mol Biol Cell 26:2030-43

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