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Intracellular trafficking of the Na‐K‐Cl cotransporter protein

Although the Na‐K‐Cl cotransporter is functionally well‐characterized, virtually nothing is known about intracellular trafficking of this protein. We used biotinylation of intact cells, and pulse‐chase radiolabeling coupled with immunoprecipitation to characterize certain features of intra‐cellular trafficking of the NKCC protein in the human fetal lung fibroblast cell line, MRC‐5. We report the following. By immuno‐precipitating the NKCC protein (using T4 monoclonal Ab) from MRC‐5 cells pulsed for three hours with 35‐S labeled methionine and chased for various times in 35‐S‐free medium, we found that the half‐life of the NKCC protein ≈ 16 hours. Parallel experiments measuring the half‐life of the alpha‐subunit of the sodium pump (using the α‐6F Ab) gave a half‐life of ≈ 28 hours, which agrees well with other workers (Coupaye‐Gerard et al. AJP 1997 272: C1781). We measured the half‐life of the plasmalemmal fraction of the NKCC protein by biotinylating intact cells followed by western blotting at various post‐biotinylation times. This yielded a half‐life ≈ 12 hours for plasmalemmal NKCC. Using a combination of membrane protein biotinylation, western blotting and a selective proteasomal (MG132) or lysosomal (Leupeptin) inhibitor, we determined that the NKCC protein is subject to proteasomal degradation, while finding no evidence of lysosomal degradation. Finally, using a cleavable analog of biotin, and western blotting, we determined that internalization of the NKCC protein had a half‐time of about 5–10 minutes. Thus, although the NKCC protein has a relatively long plasmalemmal half‐life, it is rapidly internalized. This result suggests a robust trafficking mechanism for the protein into and out of the plasmalemma that may represent a potential regulatory component for the NKCC.