Alkaline-shifted pHo sensitivity of AE2c1-mediated anion exchange reveals novel regulatory determinants in the AE2 N-terminal cytoplasmic domain.
The mouse anion exchanger AE2/SLC4A2 Cl(-)/HCO(-)(3) exchanger is essential to post-weaning life. AE2 polypeptides regulate pH(i), chloride concentration, cell volume, and transepithelial ion transport in many tissues. Although the AE2a isoform has been extensively studied, the function and regulation of the other AE2 N-terminal variant mRNAs of mouse (AE2b1, AE2b2, AE2c1, and AE2c2) have not been examined. We now present an extended analysis of AE2 variant mRNA tissue distribution and function. We show in Xenopus oocytes that all AE2 variant polypeptides except AE2c2 mediated Cl(-) transport are subject to inhibition by acidic pH(i) and to activation by hypertonicity and NH(+)(4). However, AE2c1 differs from AE2a, AE2b1, and AE2b2 in its alkaline-shifted pH(o)((50)) (7.70 +/- 0.11 versus 6.80 +/- 0.05), suggesting the presence of a novel AE2a pH-sensitive regulatory site between amino acids 99 and 198. Initial N-terminal deletion mutagenesis restricted this site to the region between amino acids 120 and 150. Further analysis identified AE2a residues 127-129, 130-134, and 145-149 as jointly responsible for the difference in pH(o)((50)) between AE2c1 and the longer AE2a, AE2b1, and AE2b2 polypeptides. Thus, AE2c1 exhibits a unique pH(o) sensitivity among the murine AE2 variant polypeptides, in addition to a unique tissue distribution. Physiological coexpression of AE2c1 with other AE2 variant polypeptides in the same cell should extend the range over which changing pH(o) can regulate AE2 transport activity.
Kurschat
Christine E
CE
Molecular and Vascular Medicine and Renal Units, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA.
Shmukler
Boris E
BE
Jiang
Lianwei
L
Wilhelm
Sabine
S
Kim
Edward H
EH
Chernova
Marina N
MN
Kinne
Rolf K H
RK
Stewart
Andrew K
AK
Alper
Seth L
SL
D000818:Animals; D027321:Anion Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D017920:Antiporters / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D002460:Cell Line; D027963:Chloride-Bicarbonate Antiporters; D002852:Chromatography, Ion Exchange / Q000379:methods; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D017353:Gene Deletion; D014644:Genetic Variation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D064606:SLC4A Proteins; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014981:Xenopus
D027321:Anion Transport Proteins; D017920:Antiporters; D027963:Chloride-Bicarbonate Antiporters; D018076:DNA, Complementary; D010455:Peptides; D012333:RNA, Messenger; D064606:SLC4A Proteins; C579496:SLC4A2 protein, human; C579495:Slc4a2 protein, mouse