title: Substitution of transmembrane domain Cys residues alters pH(o)-sensitive anion transport by AE2/SLC4A2 anion exchanger.
abstract: AE2/SLC4A2 is the most widely expressed of the Na(+)-independent SLC4 Cl(-)/HCO3 (-) 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, mAE2Cys-less, to enhance the utility of Cys-substitution mutagenesis for structural and structure-function studies of mAE2. mAE2Cys-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 1/2 for extracellular Cl(-). mAE2Cys-less exhibited wildtype activation of anion exchange by hypertonicity and by NH4Cl, and wildtype inhibition of anion exchange by acidic intracellular pH (pHi) in the absence of NH4 (+). However, inhibition of anion exchange by extracellular pH (pHo) exhibited an alkaline shifted pHo(50) value of at least 0.6-0.7 pH units. Although SO4 (2-) transport by mAE2Cys-less resembled wildtype mAE2 in its stimulation by acidic pHo, the absence of transmembrane domain Cys residues abrogated activation of oxalate transport by acidic pHo. The contrasting enhancement of SO4 (2-) transport by alkaline pHo 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 AE2Cys-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. AE2Cys-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 pHo sensitivity, resembling that of AE2 gastric parietal cell variant AE2c1.
authors.affiliation: Renal Division and Molecular and Vascular Medicine Division, Beth Israel Deaconess Medical Center, 99 Brookline Avenue, RN-380F, Boston, MA 02215, USA.
authors.forename: Fabian R
authors.initials: FR
authors.lastname: Reimold
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Stewart
authors.forename: Kathleen
authors.initials: K
authors.lastname: Stolpe
authors.forename: John F
authors.initials: JF
authors.lastname: Heneghan
authors.forename: Boris E
authors.initials: BE
authors.lastname: Shmukler
authors.forename: Seth L
authors.initials: SL
authors.lastname: Alper
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D027963:Chloride-Bicarbonate Antiporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D051379:Mice; D018345:Mice, Knockout; D010070:Oxalates / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013431:Sulfates / Q000378:metabolism*; D014981:Xenopus
chemical_list: D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D010070:Oxalates; C579495:Slc4a2 protein, mouse; D013431:Sulfates; D003545:Cysteine