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Am J Physiol Renal Physiol 291: F588-F596, 2006. First published April 18, 2006; doi:10.1152/ajprenal.00361.2005
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Role of NH2 and COOH termini in targeting, stability, and activity of sodium bicarbonate cotransporter 1

Doris Joy D. Espiritu,1,2 Angelito A. Bernardo,1,3 and Jose A. L. Arruda1,2,3

1Section of Nephrology, Department of Medicine, and 2Department of Physiology and Biophysics (M/C 901), University of Illinois at Chicago, Chicago; and 3Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois

Submitted 1 September 2005 ; accepted in final form 24 March 2006

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 ({Delta}N424) or the entire COOH ({Delta}C92) terminus and examined the effects of these truncations on targeting, cell membrane stability, and NBC1 activity. {Delta}N424 and {Delta}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 {Delta}N424, {Delta}C92 expression was significantly decreased in the basolateral membrane at 48 h and yet the total {Delta}C92 expression in the cell was constant. We found that decreased {Delta}C92 expression in the basolateral membrane was due to increased endocytosis and mistargeting to the apical membrane. Increased endocytosis was prevented when both {Delta}N424 and {Delta}C92 were cotransfected together and more stable expression of {Delta}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


Address for reprint requests and other correspondence: J. A. L. Arruda, UIC Section of Nephrology (M/C 793), 820 S. Wood St., Rm. 418 W CSN, Chicago, IL 60612-7378 (e-mail: JAArruda{at}uic.edu)






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