Submitted on May 16, 2007
Accepted on February 4, 2008
Functional characterization of mouse urea transporters UT-A2 and UT-A3 expressed in purified Xenopus laevis oocyte plasma membranes
Bryce MacIver1*, Craig P. Smith2, Warren G. Hill1, and Mark L. Zeidel3
1 Dept. of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States; Harvard Medical School, Boston, Massachusetts, United States
2 Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
3 Chair of the Dept of Medicine, Beth Israel Deaconess Medical Center, United States; Harvard Medical School, Boston, Massachusetts, United States
* To whom correspondence should be addressed. E-mail: imaciver{at}bidmc.harvard.edu.
Urea is a small solute synthesized by many terrestrial organisms as part of the catabolism of protein. In mammals it is transported across cellular membranes by specific urea transporter proteins that are the products of two separate, but closely related genes, referred to as UT-A and UT-B. Three major UT-A isoforms are found in the kidney; namely UT-A1, UT-A2 and UT-A3. UT-A2 is found in the thin descending limb of the loop of Henle whereas UT-A1 and UT-A3 are concentrated in the inner medullary collecting duct. UT-A2 and UT-A3 effectively represent two halves of the whole UT-A gene and when joined together by 73 hydrophilic amino acids constitute UT-A1. A biophysical characterization of mouse UT-A2 and UT-A3 was undertaken by expression in Xenopus laevis oocytes and subsequent preparation of highly enriched plasma membrane vesicles for use in stopped-flow fluorometry. Both isoforms were found to be highly specific for urea, and did not permeate water, ammonia or other molecules closely related to urea (formamide, acetamide, methylurea and dimethylurea). Single transporter flux rates of 46000 ± 10000 and 59000 ± 15000 (mean ± SEM) urea molecules per second per channel respectively for UT-A2 and UT-A3 were obtained. Overall, the UT-A2 and UT-A3 isoforms appear to have identical functional kinetics.
