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Stoichiometry determination of UT‐A1 and UT‐A3 within collecting duct urea transporter complex

The collecting duct urea channel proteins UT‐A1 and UT‐A3 play vital roles in the urine concentrating mechanism. Both proteins are splice variants of the same gene Slc14a2, with the entire UT‐A3 identical to N‐terminus of UT‐A1 except for the last amino acid. A previous study showed that UT‐A1 is present in a >200 kDa complex in rat inner medullary collecting duct (IMCD). Here, we tested the hypothesis raised by a recent structural study of bacterial urea transport homologue that UT‐A1 and UT‐A3 may assemble to form a dimeric complex. IMCD membrane samples were immunoprecipitated using antibodies against UT‐A1 or UT‐A3 and resolved by SDS‐PAGE. Gel bands corresponding to the UT‐A complex band and UT‐A1 monomer were excised for in‐gel digestion and LC/MS‐MS analysis. The stoichiometry of UT‐A1 versus UT‐A3 in the complex was determined by the ratio of intensities of peptides identified from the region shared by the two proteins compared to those unique to UT‐A1. The calculated ratio from all trypsinized peptides showed no significant difference between the UT‐A complex versus UT‐A1 monomer bands (1.18±0.12, n=6). Independently, using stable‐isotope labeled peptides for absolute quantification of peptides unique to UT‐A1 and UT‐A3, we found that UT‐A3 was present only in very small amounts in the UT‐A complex relative to UT‐A1. Together, these results argue against that the UT‐A complex contains both UT‐A1 and UT‐A3.