Structure-function relationships of AE2 regulation by Ca(i)(2+)-sensitive stimulators NH(4+) and hypertonicity.
We showed previously that the nonerythroid anion exchanger AE2 and the erythroid anion exchanger AE1 differ greatly in their regulation by acute changes in intracellular pH (pH(i)) and extracellular pH (pH(o)). We have now examined how AE2, but not AE1, is activated by two stimuli with opposing effects on oocyte pH(i): an alkalinizing stimulus, hypertonicity, and an acidifying stimulus, NH(4)(+). We find that both NH(2)-terminal cytoplasmic and COOH-terminal transmembrane domains of AE2 are required for activation by either stimulus. Directed by initial deletion mutagenesis studies of the NH(2)-terminal cytoplasmic domain, an alanine scan of AE2 amino acids 336-347 identified residues whose individual mutation abolished or severely attenuated sensitivity to both or only one activating stimulus. Chelation of cytoplasmic Ca(2+) (Ca(i)(2+)) diminished or abolished AE2 stimulation by NH(4)(+) and by hypertonicity. Calmidazolium inhibited AE2 activity, but not that of AE1. AE2 was insensitive to many other modifiers of Ca(2+) signaling. Unlike AE2 stimulation by NH(4)(+) and by hypertonicity, AE2 inhibition by calmidazolium required only AE2's COOH-terminal transmembrane domain.
Chernova
Marina N
MN
Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA.
Stewart
Andrew K
AK
Jiang
Lianwei
L
Friedman
David J
DJ
Kunes
Yune Z
YZ
Alper
Seth L
SL
D000818:Animals; D027321:Anion Transport Proteins; D017920:Antiporters; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D006863:Hydrogen-Ion Concentration; D006982:Hypertonic Solutions / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000502:physiology; D009865:Oocytes; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000502:physiology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D064606:SLC4A Proteins; D013329:Structure-Activity Relationship; D014981:Xenopus
D027321:Anion Transport Proteins; D017920:Antiporters; D002614:Chelating Agents; D006982:Hypertonic Solutions; D007093:Imidazoles; D008565:Membrane Proteins; D000644:Quaternary Ammonium Compounds; D064606:SLC4A Proteins; C031938:calmidazolium; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium