The Na-K-Cl cotransporter NKCC1 is activated by phosphorylation of a regulatory domain in its N terminus. In the accompanying paper (Darman, R. B., and Forbush, B. (2002) J. Biol. Chem. 277, 37542-37550), we identify three phosphothreonines important in this process. Using a phospho-specific antibody (anti-phospho-NKCC1 antibody R5) raised against a diphosphopeptide containing Thr 212 and Thr 217 of human NKCC, we were readily able to monitor the cotransporter activation state. In 32 P

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... lation experiments with rectal gland tubules, we show that the R5 antibody signal is proportional to the amount of 32 P incorporated into NKCC1; and in experiments with NKCC1-transfected HEK-293 cells, we demonstrate that R5-detected phosphorylation directly mirrors functional activation. Immunofluorescence analysis of shark rectal gland shows activation-dependent R5 antibody staining along the basolateral membrane. In perfused rat parotid glands, isoproterenol induced staining of both acinar and ductal cells along the basolateral membrane. Isoproterenol also induced basolateral staining of the epithelial cells in rat trachea, whereas basal cells in the subepithelial tissue displayed heavy, non-polarized staining of the cell membrane. In rat colon, agonist stimulation induced staining along the basolateral membrane of crypt cells. These data provide direct evidence of NKCC1 regulation in these tissues, and they further link phosphorylation of NKCC1 with its activation in transfected cells and native tissue. The high conservation of the regulatory threonine residues among NKCC1, NKCC2, and NCC family members, together with the fact that tissues from divergent vertebrate species exhibit similar R5-binding profiles, lends further support to the role of this regulatory locus in vivo. NKCC1, the secretory or housekeeping isoform of the Na-K-Cl cotransporter, is expressed in most cell types, aiding in the regulation of cell volume. In polarized cells of secretory epithelia, NKCC1 is heavily expressed along the basolateral 1 The abbreviations used are: sNKCC1, shark NKCC1; hNKCC1, human NKCC1; ELISA, enzyme-linked immunosorbent assay; PBS, phosphatebuffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.