This proposal describes a 5-year training program for the development of an academic research career in Gastroenterology and Medicine. The proposal builds upon the candidate's strengths and prior research skills, and takes advantage of a vibrant and stimulating scientific environment at The Johns Hopkins University. The principal investigator will be supported and mentored by Dr. Mark Donowitz, a leader in Na/H exchanger biology and diarrheal diseases. In addition, a panel of accomplished and well-established scientists will regularly provide the principal investigator with scientific and career advice. The goal is to foster the PI to develop a successful, independently funded research program and to gain the experience necessary to maintain a productive scientific career relevant to research on diarrheal disease. The objective of the proposed research is to identify and characterize the molecular and cellular mechanisms responsible for the dysfunction of sodium/hydrogen exchanger 3 (NHE3) we identified are present in NHE3 polymorphisms. Previous studies revealed that diarrhea is the major phenotype in NHE3 knockout mice, suggesting a potential involvement of NHE3 in diarrheal diseases. Single nucleotide polymorphisms (SNPs) account for many inherited diseases. Our preliminary studies 1) identified three missense SNPs in the NHE3 cytoplasmic tail, the regulatory domain for NHE3;2) demonstrated that these mutants, R474Q, V567M and R799C, caused reduced basal NHE3 activity;3) revealed the abnormal function of these SNPs in NHE3 appears to be due to either a change in intrinsic NHE3 function in R474Q, or abnormal trafficking in V567M and defect in both intrinsic NHE3 function and trafficking in R799C;4) demonstrated that R474Q and R799C mutants failed to respond to acute dexamethasone stimulation, suggesting a possible defect of NHE3 in response to stimulation postprandially;and 5) identified a mutation of NHE3 at a polymorphic site R474K in 2 of 3 patients with chronic diarrhea due to congenital sodium diarrhea (CSD) and this mutant failed to respond to dexamethasone. These findings suggest that poorly functioning NHE3 polymorphisms may act as genetic determinants that contribute to chronic diarrheal diseases. However, the molecular mechanisms by which the identified three NHE3 polymorphisms cause reduced sodium absorption remain ill defined. In this proposal, we will determine whether NHE3 protein complex size is altered in polymorphisms under basal and dexamethasone-stimulated conditions. We will also investigate whether the interactions of NHE3 mutants and NHE3 known regulatory proteins are changed under basal and dexamethesone- stimulated conditions. Finally, we will use proteomic approaches to identify novel proteins associated with NHE3 polymorphisms. Together, experiments proposed in these three specific aims will provide insight into the genetic basis of chronic diarrheal disease with the ultimate goal of establishing NHE3 and related proteins as novel therapeutic targets for treatment of these diarrheal diseases. This grant will also allow me to develop a solid foundation of knowledge in chronic diarrheal diseases and the qualifications of an independent investigator. To achieve this goal, formal education during the award period will be obtained through enrolling and auditing courses offered by the JHU Graduate School. Specific courses that are relevant to my training, but not limited to, are: BIophotonics (585.634) and Fundamentals of confocal Microscopy (110.807), Bioinformatics (410.639) and Role of chromatography and Mass Spectrometry in Biomedical research (330.804).
Sodium and hydrogen absorption that occurs in small intestine and colon is mainly by regulation of NHE3 activity. Inhibition of NHE3 activity is present in polymorphic NHE3. However, the mechanisms responsible for these poorly functioning NHE3 polymorphisms remain unknown. The current study will provide novel insight s into understanding genetic component accounts for the role of NHE3 in chronic diarrheal diseases.
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