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J. Biol. Chem., Vol. 283, Issue 26, 18402-18410, June 27, 2008

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Entry to " Tunnel" Revealed by SLC4A4 Human Mutation and Structural Model^*

Min-Hwang Chang¹, Jennifer DiPiero, Frank D. Sönnichsen², and Michael F. Romero³

From the Department Physiology & Biophysics and Biology, Case Western Reserve University, Cleveland, Ohio 44106 and the Department Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota 55905

Glaucoma, cataracts, and proximal renal tubular acidosis are diseases caused by point mutations in the human electrogenic Na⁺ bicarbonate cotransporter (NBCe1/SLC4A4) (1, 2). One such mutation, R298S, is located in the cytoplasmic N-terminal domain of NBCe1 and has only moderate (75%) function. As SLC transporters have high similarity in their membrane and N-terminal primary sequences, we homology-modeled NBCe1 onto the crystal structure coordinates of Band 3(AE1) (3). Arg-298 is predicted to be located in a solvent-inaccessible subsurface pocket and to associate with Glu-91 or Glu-295 via H-bonding and charge-charge interactions. We perturbed these putative interactions between Glu-91 and Arg-298 by site-directed mutagenesis and used expression in Xenopus oocyte to test our structural model. Mutagenesis of either residue resulted in reduced transport function. Function was "repaired" by charge reversal (E91R/R298E), implying that these two residues are interchangeable and interdependent. These results contrast the current understanding of the AE1 N terminus as protein-binding sites and propose that hkNBCe1 (and other SLC4) cytoplasmic N termini play roles in controlling permeation.

Received for publication, December 3, 2007 , and in revised form, April 23, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants DK056218 and EY017732 (to M. F. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.

¹ M-HC was supported by a postdoctoral fellowship from the American Heart Association Ohio Valley Affiliate.

² Present address: Otto Diels Institute for Organic Chemistry Christian Albrechts University, 24118 Kiel, Germany.

³ To whom correspondence should be addressed: Dept. Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, 200 First St. SW, Guggenheim 9-21D, Rochester, MN 55905. Tel.: 507-284-8127; E-mail: romero.michael{at}mayo.edu.

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