Parsing apical oxalate exchange in Caco-2BBe1 monolayers: siRNA knockdown of SLC26A6 reveals the role and properties of PAT-1.
The purpose of this investigation was to quantitate the contribution of the anion exchanger PAT-1 (putative anion transporter-1), encoded by SLC26A6, to oxalate transport in a model intestinal epithelium and to discern some characteristics of this exchanger expressed in its native environment. Control (Con) Caco-2 BBe1 monolayers, 6-8 days postseeding, were compared with those transfected with a small interfering RNA targeted to SLC26A6 (A6KD). Radiotracer and Ussing chamber techniques were used to determine the transepithelial unidirectional fluxes of Ox(2-), Cl(-), and SO(4)(2-) whereas fluorometric/BCECF measurements of intracellular pH were used to assess HCO(3)(-) exchange. PAT-1 was functionally targeted to the apical membrane, and SLC26A6 knockdown reduced PAT-1 protein (>60%) and mRNA (>75%) expression in A6KD. No net flux of Ox(2-), Cl(-), or SO(4)(2-) was detected in Con or A6KD monolayers, yet the unidirectional fluxes in A6KD were reduced 50, 35, and 15%, respectively. Cl(-)-dependent HCO(3)(-) efflux from A6KD was reduced 50% compared with Con. The difference between Con and A6KD properties represents that mediated solely by PAT-1, and by this approach we found that PAT-1-mediated oxalate influx and efflux are inhibited equally by mucosal DIDS (EC(50) approximately 5 microM) and that mucosal Cl(-) inhibits oxalate uptake with an EC(50) < 20 mM. Transepithelial Cl(-) gradients supported large, DIDS-sensitive net absorptive or secretory fluxes of oxalate in a direction opposite that of the imposed Cl(-) gradient. The overall symmetry of PAT-1-mediated oxalate exchange suggests that vectorial oxalate transport observed in vivo is principally dependent on the magnitude and direction of counterion gradients.
Freel
Robert W
RW
Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida 32610-00275, USA. rfreel@ufl.edu
Morozumi
Makoto
M
Hatch
Marguerite
M
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D018938:Caco-2 Cells; D027963:Chloride-Bicarbonate Antiporters / Q000235:genetics / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D055785:Gene Knockdown Techniques; D006801:Humans; D006863:Hydrogen-Ion Concentration; D026901:Membrane Transport Proteins / Q000172:deficiency* / Q000235:genetics / Q000502:physiology*; D019815:Oxalic Acid / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics*; D000076988:Sulfate Transporters; D013431:Sulfates / Q000378:metabolism
D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D026901:Membrane Transport Proteins; D034741:RNA, Small Interfering; C439529:SLC26A6 protein, human; D000076988:Sulfate Transporters; D013431:Sulfates; D019815:Oxalic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid