Substitution of transmembrane domain Cys residues alters pH^sub o^-sensitive anion transport by AE2/SLC4A2 anion exchanger

AE2/SLC4A2 is the most widely expressed of the Na^sup +^-independent SLC4 Cl^sup -^/HCO3 ^sup -^ exchangers and is essential for postnatal survival, but its structure remains unknown. We have generated and expressed a mouse AE2 construct devoid of transmembrane domain cysteine (Cys) residues, mAE2^sub Cys-less^, to enhance the utility of Cys-substitution mutagenesis for structural and structure-function studies of mAE2. mAE2^sub Cys-less^ expressed in Xenopus oocytes exhibited partial reduction of stilbene disulfonate-sensitive anion exchange activity. This activity was independent of the mAE2 N-terminal cytosolic domain and was accompanied by near-normal surface expression, without change in K ^sub 1/2^ for extracellular Cl^sup -^. mAE2^sub Cys-less^ exhibited wildtype activation of anion exchange by hypertonicity and by NH^sub 4^Cl, and wildtype inhibition of anion exchange by acidic intracellular pH (pH^sub i^) in the absence of NH^sub 4^ ^sup +^. However, inhibition of anion exchange by extracellular pH (pH^sub o^) exhibited an alkaline shifted pH^sub o(50)^ value of at least 0.6-0.7 pH units. Although SO^sub 4^ ^sup 2-^ transport by mAE2^sub Cys-less^ resembled wildtype mAE2 in its stimulation by acidic pH^sub o^, the absence of transmembrane domain Cys residues abrogated activation of oxalate transport by acidic pH^sub o^. The contrasting enhancement of SO^sub 4^ ^sup 2-^ transport by alkaline pH^sub o^ in the mAE1 anion translocation pathway mutant E699Q (Am J Physiol Cell Physiol 295: C302) was phenocopied by the corresponding mutant E1007Q in both AE2 and AE2^sub Cys-less^. However, the absence of transmembrane domain Cys residues exacerbated the reduced basal anion transport function exhibited by this and other missense substitutions at AE2 residue E1007. AE2^sub Cys-less^ will be a valuable experimental tool for structure-function studies of the SLC4 gene family, but its utility for studies of AE2 regulation by extracellular pH must be evaluated in the context of its alkaline-shifted pH^sub o^ sensitivity, resembling that of AE2 gastric parietal cell variant AE2c1.[PUBLICATION ABSTRACT]