SULFATE TRANSPORT MEDIATED BY THE MAMMALIAN ANION-EXCHANGERS IN RECONSTITUTED PROTEOLIPOSOMES

The kinetic properties of sulfate transport mediated by the anion exch angers AE1 and AE2 have been examined, Microsomes isolated from HEK ce lls transiently overexpressing either protein were reconstituted in un ilamellar, 200-600-nm diameter proteoliposomes. Transport mediated by the exchangers was monitored by loading the reconstituted proteoliposo mes with the slowly transportable anion SO42- using [S-35]SO42- as a t racer and performing [S-35]SO42-/SO42- exchange. The following data su ggest that AE1 and AE2 have been functionally reconstituted: (i) the r ate of SO42- transport in AE1 and AE2 containing proteoliposomes was 1 0-20 times higher than in proteoliposomes derived from control microso mes; (ii) the transport of SO42- was strongly dependent on the presenc e of a trans anion; and (iii) the anion exchanger inhibitors, 4,4'-dii sothiocyanostilbene-2,2'-disulfonate (DIDS) and 4,4'-dinitrostilbene-2 ,2'-disulfonate (DNDS) totally abolished SO42- transport, Furthermore, DIDS inhibits SO42- transport only when occluded inside the vesicles, indicating a uniform, asymmetrical, inside out orientation of the rec onstituted exchangers, The K-i values of the stilbene disulfonate comp ound DNDS were 2.5 and 4 mu M for AE1 and AE2, respectively, suggestin g that the two exchangers pos sess similar high affinity sites for sti lbene 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 su lfate transport.