Submitted on February 20, 2008
Revised on April 23, 2008
Accepted on May 7, 2008
Mouse Ae1 E699Q mediates SO42-i/aniono exchange with [SO42-]i-dependent reversal of wildtype pHo sensitivity
Marina N. Chernova1, Andrew K. Stewart1, Parul N. Barry2, Michael L. Jennings Ph.D.3, and Seth L. Alper1*
1 Beth Israel Deaconess Med Ctr
2 Wellesley College
3 University of Arkansas for Medical Science
* To whom correspondence should be addressed. E-mail: salper{at}caregroup.harvard.edu.
The SLC4A1/AE1 gene encodes the electroneutral Cl-/HCO3- exchanger of erythrocytes and renal Type A intercalated cells. AE1 mutations cause familial spherocytic and stomatocytic anemias, ovalocytosis, and distal renal tubular acidosis. The mutant mouse Ae1 polypeptide E699Q expressed in Xenopus oocytes cannot mediate Cl-/HCO3- exchange or 36Cl- efflux, but exhibits enhanced dual sulfate efflux mechanisms: electroneutral exchange of intracellular sulfate for extracellular sulfate (SO42-(i)/ SO42-(o) exchange), and electrogenic exchange of intracellular sulfate for extracellular chloride (SO42-(i)/Cl-(o) exchange). Whereas wildtype AE1 mediates 1:1 H+/SO42- cotransport in exchange for either Cl- or for the H+/SO42- pair, mutant Ae1 E699Q transports sulfate without cotransport of protons, similar to human erythrocyte AE1 in which the corresponding E681 carboxylate has been chemically converted to the alcohol (hAE1 E681OH). We now show that in contrast to the normal cis-stimulation by protons of wildtype AE1-mediated SO42- transport, both SO42-(i)/Cl-(o) exchange and SO42-(i)/ SO42-(o) exchange mediated by mutant Ae1 E699Q are inhibited by acidic pHo and activated by alkaline pHo. hAE1 E681OH displays a similarly altered pHo-dependence of SO42-(i)/Cl-(o) exchange. Elevated [SO42-](i) increases the K1/2 of Ae1 E699Q for both extracellular Cl- and SO42-, while reducing inhibition of both exchange mechanisms by acid pHo. The E699Q mutation also leads to increased potency of self-inhibition by extracellular SO42-. Study of the Ae1 E699Q mutation has revealed the existence of a novel pH-regulatory site of the Ae1 polypeptide, and should continue to provide valuable paths towards understanding substrate selectivity and self-inhibition in SLC4 anion transporters.
