Grant: Discovery Grants Program - Individual

Background: This basic research program is set up to discover novel nutrient membrane transporters in order to understand the biology of transport pathways. Our research group has a track record of identifying novel transporters and we have developed a “Research Pipeline” for membrane transporters discovery. We have four current research leads on “missing links” in ascorbic acid and organic cation pathways, which we propose to explore using a novel methodology developed by us. Due to a key role of intestinal absorption as a determinant of systemic homoeostasis the long term focus of this research program lays on transport pathways in the absorbing epithelial cell, the enterocyte. Since we do not have a complete understanding of all the transporters expressed in the enterocyte, we propose two experimental approaches to identify future targets. Objectives: Four existing candidate genes in pathways of vitamin C and organic cation transport will be explored. Two subprojects will inform on future research targets. The individual objectives are: 1. Ascorbic Acid Uptake: Identification of additional candidates through differential gene expression in CaCo-2 cells exposed to varying concentration of ascorbic acid. 2. Ascorbic Acid Release: Characterization of slc23a3 and slc22a23 as “releaser”. 3. Dehydroascorbic Acid Uptake in the Small Intestine: Characterization of GLUT14's role in the pathway. 4. Organic Cations Uptake: Characterization of slc22a23 as multispecific “uptaker”.5. Organic Cations Release: Characterization of mfsd14b as “releaser” of primary amines. 6. The Transcriptome of the Enterocyte to generate future research targets. Design: Our molecular “Research Pipeline” utilizes common methodology to characterize individual membrane transporters in objectives 2-5. This will result in genes (a) annotation, (b) Subcloning of major protein isoforms, (c) determination of subcellular localization, (d) substrate studies. Two transcriptomics studies aid the identification of another elusive ascorbic acid uptake transporter and the transcriptome of the enterocyte and will inform on future targets. Significance: The program will discover novel membrane transporters. Its methodology will be a template for further studies. The basic discoveries will prime translation to enhance physiologic performance or environmental challenges.