The cystic fibrosis transmembrane conductance regulator interacts with and regulates the activity of the HCO3- salvage transporter human Na+-HCO3- cotransport isoform 3

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) regulates both HCOFormula secretion and HCOFormula salvage in secretory epithelia. At least two luminal transporters mediate HCOFormula salvage, the Na+/H+ exchanger (NHE3) and the Na+-HCOFormula cotransport (NBC3). In a previous work, we show that CFTR interacts with NHE3 to regulate its activity (Ahn, W., Kim, K. W., Lee, J. A., Kim, J. Y., Choi, J. Y., Moe, O. M., Milgram, S. L., Muallem, S., and Lee, M. G. (2001) J. Biol. Chem. 276, 17236–17243). In this work, we report that transient or stable expression of human NBC3 (hNBC3) in HEK cells resulted in a Na+-dependent, DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid)- and 5-ethylisopropylamiloride-insensitive HCOFormula transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. GlutathioneS-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCOFormula -transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCOFormula transport by regulating the activity of all luminal HCOFormula secretion and salvage mechanisms of secretory epithelial cells.