In the gastrointestinal tract, the absorption of water and nutrients occurs as normal part of digestion. In the intestine, impaired sodium absorption results in decreased water absorption and diarrhea. In diarrheal diseases, elevation of intracellular second messengers, such as calcium (Ca2+), by neurohumoral substances and bacterial toxins results in decreased sodium absorption in the intestine and colon. The sodium/hydrogen exchanger (NHE) gene family plays an integral role in sodium absorption in the mammalian intestine. Regulation of brush border NHE3 accounts for most of the recognized digestive changes in sodium absorption which occur during digestion as well as the inhibition of sodium absorption that occurs during diarrhea. Inhibition of NHE3 activity by intracellular Ca2+ may involve the participation of phospholipase C gamma (PLCg), however the mechanism responsible for Ca2+ regulation of NHE3 activity is not well understood. Bioinformatics analysis has suggested PLCg may regulate NHE3 through a direct protein-protein interaction and results in the formation of an intermolecular PH domain. Therefore, the current study is designed to test the hypothesis that (1) NHE3 and PLCg directly interact;(2) PLCg contributes to basal activity and is necessary forCa2+ inhibition of NHE3 activity;(3) PLCg scaffolds NHE3 to signaling proteins, such as c-Src, which are required for proper function and regulation of NHE3 activity;(4) NHE3 and PLCg binding results in the formation of an intermolecular PH domain which regulates NHE3 activity by binding specific phospholipids in lipid raft microdomains of intestinal epithelial cells. To answer these questions, the principal investigator will utilize in vitro binding assays(e.g. pull-down, yeast two-hybrid) to determine the amino acids within NHE3, by mutagenesis, that are responsible for direct binding to PLCg. Investigator will determine the consequence of preventing PLCg binding to NHE3 on basal andCa2+ inhibition of NHE3 activity. Biochemical studies, including sucrose density gradient centrifugation, will be performed to analyze the contribution of PLCg on formation of NHE3 complexes. Finally, the investigator will perform lipid binding assays to uncover novel interactions between phospholipids and NHE3 as predicted by bioinformatics analysis. The results of this study will provide novel insights into the role of PLCg in regulation of NHE3 activity to further understand sodium absorption in digestion and diarrheal diseases.

Public Health Relevance

In the small intestine and colon, regulation of NHE3 activity accounts for most of the recognized changes in sodium absorption which occur during digestion as well as the inhibition of sodium absorption that occurs during diarrhea. Inhibition of NHE3 activity by intracellular calcium may involve the participation of PLCg, however, the mechansim [sic] responsible for the regulation is not well understood. The current study will provide novel insights into understanding the role of PLCg in regulating NHE3 activity in digestion and diarrhea.

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 #
5K01DK080930-02
Application #
7689177
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Podskalny, Judith M,
Project Start
2008-09-20
Project End
2012-08-31
Budget Start
2009-09-01
Budget End
2010-08-31
Support Year
2
Fiscal Year
2009
Total Cost
$144,914
Indirect Cost
Name
Johns Hopkins University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Grant, Christa N; Mojica, Salvador Garcia; Sala, Frederic G et al. (2015) Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function. Am J Physiol Gastrointest Liver Physiol 308:G664-77
Zachos, Nicholas C; Alamelumangpuram, Bharath; Lee, Luke J et al. (2014) Carbachol-mediated endocytosis of NHE3 involves a clathrin-independent mechanism requiring lipid rafts and Cdc42. Cell Physiol Biochem 33:869-81
Zachos, Nicholas C; Lee, Luke J; Kovbasnjuk, Olga et al. (2013) PLC-? directly binds activated c-Src, which is necessary for carbachol-mediated inhibition of NHE3 activity in Caco-2/BBe cells. Am J Physiol Cell Physiol 305:C266-75
Sarker, Rafiquel; Valkhoff, Vera E; Zachos, Nicholas C et al. (2011) NHERF1 and NHERF2 are necessary for multiple but usually separate aspects of basal and acute regulation of NHE3 activity. Am J Physiol Cell Physiol 300:C771-82
Donowitz, M; Singh, S; Singh, P et al. (2011) Alterations in the proteome of the NHERF2 knockout mouse jejunal brush border membrane vesicles. Physiol Genomics 43:674-84
Murtazina, Rakhilya; Kovbasnjuk, Olga; Chen, Tian-E et al. (2011) NHERF2 is necessary for basal activity, second messenger inhibition, and LPA stimulation of NHE3 in mouse distal ileum. Am J Physiol Cell Physiol 301:C126-36
Mohan, Sachin; Tse, Chung Ming; Gabelli, Sandra B et al. (2010) NHE3 activity is dependent on direct phosphoinositide binding at the N terminus of its intracellular cytosolic region. J Biol Chem 285:34566-78
Hartley, Jane Louise; Zachos, Nicholas C; Dawood, Ban et al. (2010) Mutations in TTC37 cause trichohepatoenteric syndrome (phenotypic diarrhea of infancy). Gastroenterology 138:2388-98, 2398.e1-2
Cha, Boyoung; Zhu, Xinjun Cindy; Chen, Weiping et al. (2010) NHE3 mobility in brush borders increases upon NHERF2-dependent stimulation by lyophosphatidic acid. J Cell Sci 123:2434-43
Donowitz, M; Singh, S; Singh, P et al. (2010) Alterations in the proteome of the NHERF1 knockout mouse jejunal brush border membrane vesicles. Physiol Genomics 42A:200-10

Showing the most recent 10 out of 13 publications