Electrogenic sulfate/chloride exchange in Xenopus oocytes mediated by murine AE1 E699Q.
Functional evaluation of chemically modified human erythrocytes has led to the proposal that amino acid residue E681 of the band 3 anion exchanger AE1 lies on the anion translocation pathway and is a proton carrier required for H+/SO4(2-) cotransport. We have tested in Xenopus oocytes the functional consequences of mutations in the corresponding residue E699 of mouse AE1. Most mutations tested abolished AE1-mediated Cl- influx and efflux. Only the E699Q mutation increased stilbene disulfonate-sensitive efflux and influx of SO4(2-). E699Q-mediated Cl- influx was activated by elevation of intracellular SO4(2-), but E699Q-mediated Cl- efflux was undetectable. The DNDS (4,4'-dinitrostilbene-2,2'-disulfonic acid) sensitivity of E699Q-mediated SO4(2-) efflux was indistinguishable from that of wt AE1-mediated Cl- efflux. The extracellular anion selectivity of E699Q-mediated SO4(2-) efflux was similar to that of wt AE1-mediated Cl- efflux. The stoichiometry of E699Q-mediated exchange of extracellular Cl- with intracellular SO4(2-) was 1:1. Whereas SO4(2-) injection into oocytes expressing wt AE1 produced little change in membrane potential or resistance, injection of SO4(2-), but not of Cl- or gluconate, into oocytes expression E699Q depolarized the membrane by 17 mV and decreased membrane resistance by 66%. Replacement of bath Cl- with isethionate caused a 28-mV hyperpolarization in SO4(2-)-loaded oocytes expressing E699Q, but had no effect on oocytes expressing wt AE1. Extracellular Cl(-)-dependent depolarization of SO4(2-)-preloaded oocytes was blocked by DNDS. AE1 E699Q-mediated inward current measured in the presence of extracellular Cl- was of magnitude sufficient to account for measured 35SO4(2-) efflux. Thus, AE1 E699Q-mediated SO4(2-)/Cl- exchange operated largely, if not exclusively, as an electrogenic, asymmetric, 1:1 anion exchange. The data confirm the proposal that E699 resides on or contributes to the integrity of the anion translocation pathway of AE1. A single amino acid change in the sequence of AE1 converted electroneutral to electrogenic anion exchange without alteration of SO4(2-)/Cl- exchange stoichiometry.
Chernova
M N
MN
Molecular Medicine Unit, Beth Israel Hospital, Boston, Massachusetts 02215, USA.
Jiang
L
L
Crest
M
M
Hand
M
M
Vandorpe
D H
DH
Strange
K
K
Alper
S L
SL
D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000378:metabolism*; D017920:Antiporters / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism; D002460:Cell Line; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides / Q000378:metabolism; D005260:Female; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D009154:Mutation; D009865:Oocytes / Q000378:metabolism*; D011233:Precipitin Tests; D018075:RNA, Complementary / Q000378:metabolism; D012996:Solutions; D013267:Stilbenes / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014982:Xenopus laevis
D001457:Anion Exchange Protein 1, Erythrocyte; D017920:Antiporters; D001639:Bicarbonates; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D018075:RNA, Complementary; D012996:Solutions; D013267:Stilbenes; D013431:Sulfates; D013462:Sulfur Radioisotopes