Slc14a2 urea transporters UT‐A1 and UT‐A3 coexist in a high molecular weight complex in rat inner medullary collecting duct (IMCD)

UT‐A1 and UT‐A3 are vasopressin‐regulated urea channel proteins that mediate transepithelial urea transport across the IMCD. Previous studies have shown that UT‐A1 is present in a >200 kDa complex, but did not identify the other components in the complex. The present study sought to test the hypothesis suggested by a recent structural study of a bacterial urea transporter homolog that UT‐A1 and UT‐A3 may assemble to form a heteromeric complex (Levin et al. Nature 462:757–762, 2009). We solubilized the membrane fraction of inner medullary tissue using non‐ionic detergents. The stabilized complex can be recognized either by UT‐A1 or UT‐A3 antibody in immunoblotting. A similar complex was also identified in IMCD suspensions treated with homobifunctional cross‐linkers. To test whether UT‐A3 is present in the complex, we performed cross‐immunoprecipitation studies. Using the UT‐A1 antibody (L194), we pulled down UT‐A1 and UT‐A3. Using the UT‐A3 antibody (Q2695), we pulled down both UT‐A1 and UT‐A3. There was a virtual absence of the abundant water channel protein AQP2 in the immunoprecipitated material. Controls for the immunoprecipitation used a preimmune IgG. These results suggest that the high molecular weight urea transporter complex contains both UT‐A1 and UT‐A3, although the stoichiometry of the two proteins in the complex remains to be determined. (PA was a 2010 APS UGSRF intern from Dartmouth College)