Sulfate transport mediated by the mammalian anion exchangers in reconstituted proteoliposomes.
The kinetic properties of sulfate transport mediated by the anion exchangers AE1 and AE2 have been examined. Microsomes isolated from HEK cells transiently overexpressing either protein were reconstituted in unilamellar, 200-600-nm diameter proteoliposomes. Transport mediated by the exchangers was monitored by loading the reconstituted proteoliposomes with the slowly transportable anion SO4(2-) using [35S]SO4(2-) as a tracer and performing [35S]SO4(2-)/SO4(2-) exchange. The following data suggest that AE1 and AE2 have been functionally reconstituted: (i) the rate of SO4(2-) transport in AE1 and AE2 containing proteoliposomes was 10-20 times higher than in proteoliposomes derived from control microsomes; (ii) the transport of SO4(2-) was strongly dependent on the presence of a trans anion; and (iii) the anion exchanger inhibitors, 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 4,4'-dinitrostilbene-2,2'-di-sulfonate (DIDS) totally abolished SO4(2-) transport. furthermore, DIDS inhibits SO4(2-) transport only when occluded inside the vesicles, indicating a uniform, asymmetrical, inside-out orientation of the reconstituted exchangers. The Ki values of the stilbene disulfonate compound DNDS were 2.5 and 4 microM for AE1 and AE2, respectively, suggesting that the two exchangers possess similar high affinity sites for stilbene compounds. Both AE1 and AE2 showed the same steep pH dependence of sulfate transport, which was maximal at pH 5.5 and reduced to less than 10% (of the value at pH 5.5) at pH 8.5, suggesting that an acidic residue shared by AE1 and AE2 participates in the pH regulation of sulfate transport.
Sekler
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Department of Biological Sciences, Stanford University, California 94305-5020, USA.
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Mastrocola
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Kopito
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D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D017920:Antiporters / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney; D007700:Kinetics; D008081:Liposomes; D051379:Mice; D008854:Microscopy, Electron; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D011994:Recombinant Proteins / Q000378:metabolism; D013267:Stilbenes / Q000494:pharmacology; D013431:Sulfates / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014162:Transfection
D017920:Antiporters; D008081:Liposomes; D011510:Proteolipids; D011994:Recombinant Proteins; D013267:Stilbenes; D013431:Sulfates; D013462:Sulfur Radioisotopes; C020485:proteoliposomes; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid