The oligomeric state of the secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) in rat parotid plasma membranes was studied using the reversible chemical cross-linker DTSSP [3, 3'-dithiobis(sulfosuccinimidyl propionate)]. The monomeric apparent molecular mass of NKCC1 is approximately 170 kDa. However, we show here that this protein migrates as a approximately 355 kDa complex on SDS-PAGE gels after membrane treatment with DTSSP, indicating that NKCC1 exists as an oligomer in the plasma membrane. The stability of this oligomer is such that it is not disrupted by solubilization of the membrane by low concentrations of the nonionic detergent Triton X-100 (0.3%) or the mild ionic detergent deoxycholate (20 mM); however, higher concentrations of Triton X-100 or treatment with the denaturing detergent SDS do result in destabilization of the NKCC1 complex. In additional experiments, we immunoprecipitated the 355 kDa cross-linked complex from biotinylated membranes, then cleaved the cross-linking bonds and analyzed the resulting components of the NKCC1 oligomer by avidin blotting, silver staining, and 2D electrophoresis. In these studies, we were unable to detect the presence of any proteins other than NKCC1 itself in the 355 kDa oligomer, suggesting that this complex is an NKCC1 dimer. Strong evidence for this conclusion was provided by a quantitative analysis of the molecular sizes of oligomers formed by full-length NKCC1 and an N-terminally truncated version of NKCC1 expressed in HEK293 cells. Taken together, our data provide convincing evidence that the dominant structural unit of NKCC1 in the plasma membrane is a homodimer.
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