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 (⌬N424) or the entire COOH (⌬C92) terminus and examined the effects of these truncations on targeting, cell membrane stability, and NBC1 activity. ⌬N424 and ⌬C92 targeted to the plasma membrane of HEK293 cells or to the basolateral

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... ne of opossum kidney (OK) cells at 24 h but did not display NBC1 activity. Unlike the NBC1 wild-type and the ⌬N424, ⌬C92 expression was significantly decreased in the basolateral membrane at 48 h and yet the total ⌬C92 expression in the cell was constant. We found that decreased ⌬C92 expression in the basolateral membrane was due to increased endocytosis and mistargeting to the apical membrane. Increased endocytosis was prevented when both ⌬N424 and ⌬C92 were cotransfected together and more stable expression of ⌬C92 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. endocytosis; basolateral membrane; apical membrane; protein-protein interaction THE SODIUM BICARBONATE COTRANSPORTER (NBC1) mediates bicarbonate reabsorption in the renal proximal tubule and is important for pH regulation in different cell types (2, 3, 21). Acute and chronic regulations of NBC1 have been studied extensively (7, 18, 22, 23) . However, the molecular determinants necessary for understanding NBC1 activity in normal and disease states are not known. NBC1 is localized in the basolateral membrane of the renal proximal tubule cells. NBC1 has 10 transmembrane-spanning domains with a long NH 2 and a short COOH terminus located intracellularly (31). Previous studies of other members of the bicarbonate superfamily, anion exchangers (AEs), have provided insights into the role of the NH 2 and the COOH termini on structure and function of the exchangers (14, 28, 33, 37, 39) . In these studies, the NH 2 and COOH termini play an important role in regulation, localization, dimerization, and function of AEs. The NH 2 terminus of NBC1 contains conserved amino acids found in anion exchanger 2 (AE2) necessary for pH sensing (28) and contains a stretch of conserved amino acids found in AE1 necessary for activity (14). The NH 2 terminus of AE1 is also necessary for interaction with other cytoplasmic loops of AE1 in erythrocytes (14) . Furthermore, crystallographic structure analysis of the NH 2 terminus of AE1 showed that its NH 2 terminus is important for cytoskeletal protein binding and dimerization (37) and is necessary for membrane insertion and transport activity (14) . The COOH terminus of NBC1 is very hydrophilic and is believed to be involved in protein-protein interaction and targeting to the plasma membrane (20). It contains 15 consecutive positively charged residues, 12 of which are lysine. It also contains an acidic motif, LDSDNDD, and a PKA phosphorylation site (8) . Toye et al. (33) reported that the COOH terminus of AE1 is involved in trafficking to the plasma membrane and further showed that the last 11 amino acids at the COOH terminus of the AE1 containing a tyrosine residue are responsible for basolateral targeting but are not sufficient for basolateral localization (32). Recently, Li et al. (17) showed that truncation of the last 23 amino acids of the COOH terminal caused mistargeting of NBC1 to the apical membrane at 48 h after transfection in Madin-Darby canine kidney (MDCK) cells. By contrast, Horita et al. (10) reported that deletion of 65 bp of SLC4A gene did not affect NBC1 function when expressed in ECV304 cells and in oocytes. This mutant, however, is retained in the intracellular compartment when expressed in MDCK cells. The reason for the apparent differences between the data of Li et al. (17) and Horita et al. (10) remained to be clarified. The role of the NH 2 terminus in targeting, cell membrane expression, and NBC1 activity is not known. Although there is no clear basolateral targeting motif, the NBC1 NH 2 terminus contains a stretch of acidic amino acids that could potentially contribute to basolateral targeting and activity. In addition, several mutations of NBC1 in the NH 2 terminus resulted in impaired bicarbonate reabsorption leading to proximal renal tubular acidosis (12, 13), suggesting an important role of the NH 2 terminus in NBC1 function. In the present study, we investigated whether the NH 2 and/or the COOH terminus plays a role in NBC1 activity, targeting, stability, and expression in the plasma membrane of a nonpolarized cell and in the basolateral membrane of polarized cells.