Role of NH2 and COOH termini in targeting, stability, and activity of sodium bicarbonate cotransporter 1
| Autor: | Doris Joy Espiritu, A. A. Bernardo, Jose A.L. Arruda |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Kidney
Physiology Chemistry Sodium-Bicarbonate Symporters Cell Membrane Opossums Bicarbonate reabsorption Apical membrane Endocytosis Sodium-Bicarbonate Cotransporter Protein–protein interaction Cell membrane medicine.anatomical_structure Biochemistry medicine Animals Humans Amino Acid Sequence Cells Cultured Epithelial polarity |
| Zdroj: | American Journal of Physiology-Renal Physiology. 291:F588-F596 |
| ISSN: | 1522-1466 1931-857X |
| Popis: | Sodium bicarbonate cotransporter 1 (NBC1) mediates 80% of bicarbonate reabsorption by the kidney, but the molecular determinants for activity, targeting, and cell membrane stability are poorly understood. We generated truncation mutants involving the entire NH2 (ΔN424) or the entire COOH (ΔC92) terminus and examined the effects of these truncations on targeting, cell membrane stability, and NBC1 activity. ΔN424 and ΔC92 targeted to the plasma membrane of HEK293 cells or to the basolateral membrane of opossum kidney (OK) cells at 24 h but did not display NBC1 activity. Unlike the NBC1 wild-type and the ΔN424, ΔC92 expression was significantly decreased in the basolateral membrane at 48 h and yet the total ΔC92 expression in the cell was constant. We found that decreased ΔC92 expression in the basolateral membrane was due to increased endocytosis and mistargeting to the apical membrane. Increased endocytosis was prevented when both ΔN424 and ΔC92 were cotransfected together and more stable expression of ΔC92 was observed. Immunoprecipitation studies using NBC1 antibody specific for the COOH epitope were able to detect the COOH truncated NBC1 when probed with NH2 epitope-specific antibody or vice versa. Similar findings were observed with Ni-NTA pull-down assay. Cotransfection of both mutants partially restored NBC1 activity. In summary, NBC1 targets to the basolateral membrane of OK cells by a default mechanism and the COOH terminus plays a role on NBC1 stability in the basolateral membrane. |
| Databáze: | OpenAIRE |
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