A conductive pathway generated from fragments of the human red cell anion exchanger AE1.
Human red cell anion exchanger AE1 (band 3) is an electroneutral Cl-HCO3- exchanger with 12-14 transmembrane spans (TMs). Previous work using Xenopus oocytes has shown that two co-expressed fragments of AE1 lacking TMs 6 and 7 are capable of forming a stilbene disulphonate-sensitive (36)Cl-influx pathway, reminiscent of intact AE1. In the present study, we create a single construct, AE1Delta(6: 7), representing the intact protein lacking TMs 6 and 7. We expressed this construct in Xenopus oocytes and evaluated it employing a combination of two-electrode voltage clamp and pH-sensitive microelectrodes. We found that, whereas AE1Delta(6: 7) has some electroneutral Cl-base exchange activity, the protein also forms a novel anion-conductive pathway that is blocked by DIDS. The mutation Lys(539)Ala at the covalent DIDS-reaction site of AE1 reduced the DIDS sensitivity, demonstrating that (1) the conductive pathway is intrinsic to AE1Delta(6: 7) and (2) the conductive pathway has some commonality with the electroneutral anion-exchange pathway. The conductance has an anion-permeability sequence: NO3- approximately I- > NO2- > Br- > Cl- > SO4(2-) approximately HCO3- approximately gluconate- approximately aspartate- approximately cyclamate-. It may also have a limited permeability to Na+ and the zwitterion taurine. Although this conductive pathway is not a usual feature of intact mammalian AE1, it shares many properties with the anion-conductive pathways intrinsic to two other Cl-HCO3- exchangers, trout AE1 and mammalian SLC26A7.
Parker
Mark D
MD
Department of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK. mark.parker@yale.edu
Young
Mark T
MT
Daly
Christopher M
CM
Meech
Robert W
RW
Boron
Walter F
WF
Tanner
Michael J A
MJ
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D026921:Amino Acid Transport Systems, Neutral / Q000502:physiology; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000502:physiology*; D027963:Chloride-Bicarbonate Antiporters / Q000502:physiology*; D004594:Electrophysiology; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013654:Taurine / Q000502:physiology; D014982:Xenopus laevis
D026921:Amino Acid Transport Systems, Neutral; D001457:Anion Exchange Protein 1, Erythrocyte; D027963:Chloride-Bicarbonate Antiporters; D010446:Peptide Fragments; D013654:Taurine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid