Abstract
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 ~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 ~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 (~135-kDa). The stably transfected cells exhibited a ~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:2CI. Preincubation in chloride-free media was necessary to activate fully the expressed cotransporter, suggesting a [CI]-dependent regulatory mechanism.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 17977-17985 |
| Number of pages | 9 |
| Journal | Journal of Biological Chemistry |
| Volume | 270 |
| Issue number | 30 |
| DOIs | |
| State | Published - 1995 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Primary structure, functional expression, and chromosomal localization of the bumetanide-sensitive Na-K-Cl cotransporter in human colon. / Payne, John A; Xu, J. C.; Haas, M.; Lytle, C. Y.; Ward, D.; Forbush, B.
In: Journal of Biological Chemistry, Vol. 270, No. 30, 1995, p. 17977-17985.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Primary structure, functional expression, and chromosomal localization of the bumetanide-sensitive Na-K-Cl cotransporter in human colon
AU - Payne, John A
AU - Xu, J. C.
AU - Haas, M.
AU - Lytle, C. Y.
AU - Ward, D.
AU - Forbush, B.
PY - 1995
Y1 - 1995
N2 - 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 ~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 ~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 (~135-kDa). The stably transfected cells exhibited a ~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:2CI. Preincubation in chloride-free media was necessary to activate fully the expressed cotransporter, suggesting a [CI]-dependent regulatory mechanism.
AB - 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 ~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 ~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 (~135-kDa). The stably transfected cells exhibited a ~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:2CI. Preincubation in chloride-free media was necessary to activate fully the expressed cotransporter, suggesting a [CI]-dependent regulatory mechanism.
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UR - http://www.scopus.com/inward/citedby.url?scp=0029074919&partnerID=8YFLogxK
U2 - 10.1074/jbc.270.30.17977
DO - 10.1074/jbc.270.30.17977
M3 - Article
C2 - 7629105
AN - SCOPUS:0029074919
VL - 270
SP - 17977
EP - 17985
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 30
ER -