Co-transporters are an important class of membrane proteins responsible for the accumulation of sugars, amino acids, peptides, neurotransmitters and ions in cells. These proteins are now known to also co-transport water (200-500 molecules per cycle) and small polar molecules such as urea. The energy for the substrate transport comes from the Na+ (or H=) electrochemical potential gradient across the plasma membrane. Our goal is to understand how co-transporters use the free energy released from downhill Na+ transport to drive uphill substrate and water transport. Using the intestinal Na+/glucose co-transporter (SGLT1), and a bacterial homolog (Vibrio SGLT), we plan to identify and determine the architecture of the Na+ and substrate transport pathways, and to determine how Na+ changes the conformation of SGLTs to couple Na+ transport to sugar and water transport. The experimental strategy is to express truncated parts of the co-transporters in Xenopus laevis oocytes and bacteria, and to determine which parts of the protein retain partial reactions, e.g. Na+ uniport, sugar uniport, Na+/sugar co-transport, and water or urea transport. Once parts of the protein are found that exhibit the partial transport reactions, we will use a combination of molecular, biophysical and biochemical techniques to determine their structure and how they interact. Preliminary studies indicate that the C-terminal part of SGLT1 can function as a glucose uniporter, and we have identified several residues in this domain that interact with sugar during Na+/glucose co- transport. The plan is to locate the other residues in the sugar transport pathway, and carry out similar studies on the Na+ transport pathway. These studies will provide a low resolution topological map of the co- transporter and structural information about Na+/sugar/water coupling, and lay the ground work for obtaining higher resolution structures.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK044602-08
Application #
6380710
Study Section
Physiology Study Section (PHY)
Program Officer
Haft, Carol R
Project Start
1992-09-30
Project End
2002-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
8
Fiscal Year
2001
Total Cost
$217,846
Indirect Cost
Name
University of California Los Angeles
Department
Physiology
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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