Point mutations involved in red cell stomatocytosis convert the electroneutral anion exchanger 1 to a nonselective cation conductance

Abstract

The anion exchanger 1 (AE1) is encoded by the SLC4A1 gene and catalyzes the electroneutral anion exchange across cell plasma membrane. It is the most abundant transmembrane protein expressed in red cell where it is involved in CO 2 transport. Recently, 4 new point mutations of SLC4A1 gene have been described leading to missense mutations in the protein sequence (L687P, D705Y, S731P, or H734R). These point mutations were associated with hemolytic anemia, and it was shown that they confer a cation transport feature to the human AE1. Facing this unexpected property for an electroneutral anion exchanger, we have studied the transport features of mutated hAE1 by expression in xenopus oocytes. Our results show that the point mutations of hAE1 convert the electroneutral anion exchanger to a cation conductance: the exchangers are no longer able to exchange Cl ؊ and HCO 3 ؊ , whereas they transport Na ؉ and K ؉ through a conductive mechanism. These data shed new light on transport mechanisms showing the tiny difference, in terms of primary sequence, between an electroneutral exchange and a conductive pathway. (Blood.

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

“…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).…”

“…For Na ϩ and K ϩ content measurements, oocytes were incubated in MBS with 0. 5 (13), oocytes incubated in MBS with ouabain and bumetanide were quickly washed two times in 7.5 ml of Milli-Q water (Millipore) and dried on aluminum foil overnight at 80°C after removing excess extracellular fluid. Dried oocytes were weighted to determine dry cell solids.…”

“…Cl Ϫ /HCO 3 Ϫ Exchange Activity Measurements-Oocyte intracellular pH was measured using selective microelectrodes as published previously (5).…”

“…It was already known that either AE1 could be converted into a cation conductance or that a cation conductance could be observed in a still functional anion exchanger. These peculiar transport features result from specific point mutations in the transmembrane domain (such as H734R in TM10), and some of these point mutations are associated with hereditary red cell or renal diseases (5,(7)(8)(9)(10)46). Our present results extend to TM5 the list of amino acids that, when substituted in AE1, induce a Na ϩ and K ϩ leak in Xenopus oocytes.…”

“…In physiological conditions, AE1 exchanges one chloride for one bicarbonate by an electroneutral transport mechanism with the driving force for ion movement provided by their electrochemical gradient. A few years ago, some specific point mutations were characterized and proposed to convert the electroneutral anion exchanger into a cation-conductive pathway (5,6). These mutations are associated with human pathologies (hereditary hemolytic anemia and distal renal tubular acidosis) (7)(8)(9)(10).…”