Role of NH(2) and COOH termini in targeting, stability, and activity of sodium bicarbonate cotransporter 1.
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 NH(2) (DeltaN424) or the entire COOH (DeltaC92) terminus and examined the effects of these truncations on targeting, cell membrane stability, and NBC1 activity. DeltaN424 and DeltaC92 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 DeltaN424, DeltaC92 expression was significantly decreased in the basolateral membrane at 48 h and yet the total DeltaC92 expression in the cell was constant. We found that decreased DeltaC92 expression in the basolateral membrane was due to increased endocytosis and mistargeting to the apical membrane. Increased endocytosis was prevented when both DeltaN424 and DeltaC92 were cotransfected together and more stable expression of DeltaC92 was observed. Immunoprecipitation studies using NBC1 antibody specific for the COOH epitope were able to detect the COOH truncated NBC1 when probed with NH(2) 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.
Espiritu
Doris Joy D
DJ
Section of Nephrology, Department of Medicine, University of Illinois at Chicago, Chicago, Chicago, IL 60612-7378, USA.
Bernardo
Angelito A
AA
Arruda
Jose A L
JA
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D004705:Endocytosis / Q000502:physiology; D006801:Humans; D007668:Kidney / Q000378:metabolism; D009893:Opossums; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000502:physiology*
C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters