title: 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 HCO(3)(-) secretion and HCO(3)(-) salvage in secretory epithelia. At least two luminal transporters mediate HCO(3)(-) salvage, the Na(+)/H(+) exchanger (NHE3) and the Na(+)-HCO(3)(-) 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 HCO(3)(-) 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. Glutathione S-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 HCO(3)(-)-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 HCO(3)(-) transport by regulating the activity of all luminal HCO(3)(-) secretion and salvage mechanisms of secretory epithelial cells.
authors.affiliation: Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Meeyoung
authors.initials: M
authors.lastname: Park
authors.forename: Shigeru B H
authors.initials: SB
authors.lastname: Ko
authors.forename: Joo Young
authors.initials: JY
authors.lastname: Choi
authors.forename: Gaia
authors.initials: G
authors.lastname: Muallem
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Thomas
authors.forename: Alexander
authors.initials: A
authors.lastname: Pushkin
authors.forename: Myeong-Sok
authors.initials: MS
authors.lastname: Lee
authors.forename: Joo Young
authors.initials: JY
authors.lastname: Kim
authors.forename: Min Goo
authors.initials: MG
authors.lastname: Lee
authors.forename: Shmuel
authors.initials: S
authors.lastname: Muallem
authors.forename: Ira
authors.initials: I
authors.lastname: Kurtz
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D001483:Base Sequence; D001639:Bicarbonates / Q000378:metabolism; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism / Q000502:physiology*; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007668:Kidney; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D014162:Transfection
chemical_list: D001639:Bicarbonates; C505032:CFTR protein, human; D017931:DNA Primers; D004791:Enzyme Inhibitors; D020033:Protein Isoforms; D011994:Recombinant Proteins; C422022:SLC4A7 protein, human; C000613385:SLC9A3 protein, human; D027982:Sodium-Bicarbonate Symporters; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid