A conductive pathway generated from fragments of the human red cell anion exchanger AE1

Abstract

Human red cell anion exchanger AE1 (band 3) is an electroneutral Cl-HCO 3− 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, AE1Δ(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 AE1Δ(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 AE1Δ(6 : 7) and (2) the conductive pathway has some commonality with the electroneutral anion-exchange pathway. The conductance has an anion-permeability sequence: NO 3It 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-HCO 3 − exchangers, trout AE1 and mammalian SLC26A7.

Structural Model of the Anion Exchanger 1 (SLC4A1) and Identification of Transmembrane Segments Forming the Transport Site

“…The region containing TM spans 6 -7 and 13-14 were proposed to be at the periphery of the core structure. In addition, it has been shown that deletion of TM6 and TM7 from full-length AE1 does not prevent anion exchange activity (12). However, this deletion changes AE1 transport features because the protein develops an anion conductance in place of an anion exchange.…”

“…This kind of structure, observed in Na ϩ symporters for example, allows transport of many different solutes: amino acids, sugar, nucleobase, glycerol, and water with H ϩ , Na ϩ , K ϩ , and/or Cl Ϫ ions (28 -31). As mentioned previously, AE1 is able to transport amino acids, urea, Na ϩ , and K ϩ and form an anion conductive pathway in trout erythrocytes (5,12,13,15,16). Point mutations convert the electroneutral human AE1 into a Na ϩ and K ϩ conductance (5).…”

“…In addition to naturally occurring mutations identified in patients with hereditary stomatocytosis or distal renal tubular acidosis, a sequence-function analysis of AE1 revealed other amino acids crucial for transport properties of the protein (11). Unexpected transport activity of AE1 had already been suggested in the past: Tanner and co-workers (12) had shown that human AE1 could behave as a conductive pathway when two putative membranespanning helices in the membrane domain of the protein were removed. These data supported the work of Motais and coworkers (13)(14)(15)(16) who showed that trout AE1 forms an anion conductive pathway permeable to organic (taurine and choline) and inorganic (Na ϩ and K ϩ ) ions in response to decreased ionic strength in red cells.…”

Leaky Cl − –HCO 3 − exchangers: cation fluxes via modified AE1

“…There have been truncation studies with AE1 fragments (Parker et al 2007), and functional expression studies of chimeras between conductive and non-conductive AE1 (Borgese et al 2004) as well as cysteine mutation studies , which have yielded some structural information. However, at the present time, we can only point to residues on domains 7, 8, 9 and 10 as being critical to gating properties of an inner pore so that channel-like properties emerge when point mutations are carried out.…”

Reduced DIDS-sensitive chloride conductance in Ae1−/− mouse erythrocytes

“…Engineered removal of transmembrane spans 6 and 7 from human AE1 elicited DIDS-sensitive anion conductance in parallel with a decrease in anion exchange activity [52]. In contrast, several individual missense mutations in AE1 associated with spherocytic or ovalocytic variants of stomatocytosis elicited apparent cation conductance of variable stilbene sensitivity in parallel with decreased anion exchange activity [53;54].…”