Immunocytochemical studies of the Na-K-Cl cotransporter of shark kidney.

The Na-K-Cl cotransporter (NKCC or BSC) has been described in numerous secretory and reabsorptive epithelia and is an important part of the mechanism of NaCl reabsorption in both the mammalian and elasmobranch kidneys. We have recently developed a panel of four monoclonal antibodies (MAbs) raised to the 195-kDa Na-K-Cl cotransport protein of the shark rectal gland (sNKCC1), which is expressed along the basolateral plasma membrane of secretory cells in this tissue (29). Here, we report immunologic studies of the Na-K-Cl cotransporter in the kidney of the dogfish shark Squalus acanthias. Western blot analysis of shark renal microsomes using MAbs J3, J7, and J25 identified proteins of approximately 195 and 150 kDa, whereas MAb J4 was not reactive. To define the cellular and subcellular distribution of the cotransport protein, immunofluorescence and immunoelectron microscopy studies were performed on fixed kidneys. Immunofluorescence microscopy on semithin (0.5-micron) cryosections demonstrated that MAbs J3, J7, and J25 intensely stained the apical plasma membrane of all distal tubule segments. Weak staining was also seen along the basolateral membrane of most distal nephrons. Immunoelectron microscopy confirmed this observation and showed that some of these segments were morphologically similar to diluting segments from other species. MAbs also reacted with the brush border and, to a lesser extent, the basolateral membrane of proximal tubules. This study supports the hypothesis that the lateral bundle zone of the elasmobranch kidney functions as a countercurrent exchanger and is consistent with the presence of multiple isoforms of the Na-K-Cl cotransporter in the shark kidney.

Primary structure, functional expression, and chromosomal localization of the bumetanide-sensitive Na-K-Cl cotransporter in human colon.

By moving chloride into epithelial cells, the Na-K-Cl cotransporter aids transcellular movement of chloride across both secretory and absorptive epithelia. Using cDNA probes from the recently identified elasmobranch secretory Na-K-Cl cotransporter (sNKCC1) (Xu, J. C., Lytle, C. Zhu, T. T., Payne, J. A., Benz, E., and Forbush, B., III (1994) Proc. Natl. Acad. Sci. 91, 2201-2205), we have identified the human homologue. By screening cDNA libraries of a human colonic carcinoma line, T84 cell, we identified a sequence of 4115 bases from overlapping clones. The deduced protein is 1212 amino acids in length, and analysis of the primary structure indicates 12 transmembrane segments. The primary structure is 74% identical to sNKCC1, 91% identical to a mouse Na-K-Cl cotransporter (mNKCC1), 58% identical to rabbit and rat renal Na-K-Cl cotransporters (NKCC2), and 43% identical to the thiazide-sensitive Na-Cl cotransporters from flounder urinary bladder and rat kidney. Similar to sNKCC1 and mNKCC1, the 5'-end of the human colonic cotransporter is rich in G + C content. Interestingly, a triple repeat (GCG)7 occurs within the 5'-coding region and contributes to a large alanine repeat (Ala15). Two sites for N-linked glycosylation are predicted on an extracellular loop between putative transmembrane segments 7 and 8. A single potential site for phosphorylation by protein kinase A is present in the predicted cytoplasmic C-terminal domain. Northern blot analysis revealed a 7.4-7.5-kilobase transcript in T84 cells and shark rectal gland and a approximately 7.2-kilobase transcript in mammalian colon, kidney, lung, and stomach. Metaphase spreads from lymphocytes were probed with biotin-labeled cDNA and avidin fluorescein (the cotransporter gene was localized to human chromosome 5 at position 5q23.3). Human embryonic kidney cells stably transfected with the full-length cDNA expressed a approximately 170-kDa protein recognized by anti-cotransporter antibodies. Following treatment with N-glycosidase F, the molecular mass of the expressed protein was similar to that predicted for the core protein from the cDNA sequence (132-kDa) and identical to that of deglycosylated T84 cotransporter (approximately 135-kDa). The stably transfected cells exhibited a approximately 15-fold greater bumetanide-sensitive 86Rb influx than control cells, and this flux required external sodium and chloride. Flux kinetics were consistent with an electroneutral cotransport of 1Na:1K:2Cl. Preincubation in chloride-free media was necessary to activate fully the expressed cotransporter, suggesting a [Cl]-dependent regulatory mechanism.