Role of NH₂ and COOH termini in targeting, stability, and activity of sodium bicarbonate cotransporter 1

Abstract

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 membrane 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 insertio...

Cation‐Coupled Bicarbonate Transporters

“…Espiritu et al (97) demonstrated that deletion mutants lacking the cytoplasmic N- or C-terminal domains showed reduced basolateral membrane expression in opossum kidney OK cells. A recent report by Soleimani’s group shows that Asp 405 and Asp 416 in the N-terminal domain of NBCe1 are critical for protein expression in plasma membranes (190).…”

The role of aspartic acid residues 405 and 416 of the kidney isotype of sodium-bicarbonate cotransporter 1 in its targeting to the plasma membrane

“…Interestingly, mice with the disrupted NBCe1 gene showed metabolic acidosis (15), supporting both the clinical and in vitro observations mentioned above. The role of NH 2 -terminal of NBCe1A in its targeting, stability, and activity was previously examined in HEK293 cells and opossum kidney (OK) cells (12). In these studies, using an NH 2 terminus truncated mutant of NBCe1 (⌬N424), the importance of this region in NBCe1 targeting to the plasma membrane and functional activity was clearly demonstrated (12).…”

“…The role of NH 2 -terminal of NBCe1A in its targeting, stability, and activity was previously examined in HEK293 cells and opossum kidney (OK) cells (12). In these studies, using an NH 2 terminus truncated mutant of NBCe1 (⌬N424), the importance of this region in NBCe1 targeting to the plasma membrane and functional activity was clearly demonstrated (12). However, the scope of these data was limited in that it did not identify the specific motifs and amino acid residues that may play a role in the targeting of NBCe1.…”

“…Previous studies suggest that the NH 2 terminus of NBCe1A is important for its accurate targeting and function (12,24). In addition, the mutation R298S in the NH 2 -terminal cytoplasmic portion of human NBCe1A was reported to lead to its retention in the intracellular compartment and renal proximal tubular acidosis (17,20).…”

“…A broken salt bridge between E91 and R298 and a changed conformation of the protein may be responsible for mistargeting of NBCe1A (9,20). Using an NH 2 terminus truncated mutation of NBCe1 (⌬N424) recent studies have revealed the importance of this region in NBCe1 targeting to the plasma membrane and functional activity (12). In the present study, using PCR-mutagenesis approach, a series of green fluorescent protein (GFP)-tagged NBCe1 NH 2 -terminal deletion and amino acid-specific mutant proteins spanning the 424 amino acid NH 2 -terminal region of NBCe1A were generated.…”