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'-disulfonate (DNDS) 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 K(i) values of the stilbene disulfonate compound DNDS were 2.5 and 4 μM 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.