Five aquaporins (AQPs) have been cloned from mammalian cells. Only the AQP1 protein, found in red blood cells, is available in quantities sufficient for detailed biophysical and structural analyses. AQPs will be expressed in yeast mutants which accumulate plasma membrane-targeted secretory vesicles. These vesicles can be assayed directly for water and solute transport using stopped-flow fluorescence quenching or used as an abundant source of protein for reconstitution and structural studies. Therefore, activation energies for water transport, solute permeabilities, and unitary conductances will be determined. High levels of protein will allow reconstitution and crystallization of planar bilayers for structural studies as previously described for AQP1. Mutant forms of AQP2 responsible for nephrogenic diabetes insipids (NDI) will be quantitatively analyzed. Mathematical models of channel pores based on solute permeabilities will be tested. Aquaporin homologues which do not function as water channels will be used to make chimeras to determine the structural determinants of water permeability. overall, quantitative functional studies and structural studies of wild-type and mutant AQPs that were not previously possible will now be possible.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32DK009485-02
Application #
2591199
Study Section
General Medicine B Study Section (GMB)
Program Officer
Rankin, Tracy L
Project Start
1997-11-01
Project End
Budget Start
1997-11-01
Budget End
1998-08-31
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Tuot, Delphine S; Plantinga, Laura C; Judd, Suzanne E et al. (2013) Healthy behaviors, risk factor control and awareness of chronic kidney disease. Am J Nephrol 37:135-43
Coury, L A; Zeidel, M L; Brodsky, J L (1999) Use of yeast sec6 mutant for purification of vesicles containing recombinant membrane proteins. Methods Enzymol 306:169-86