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J. Biol. Chem., Vol. 283, Issue 15, 9651-9658, April 11, 2008

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Solute Carrier 11 Cation Symport Requires Distinct Residues in Transmembrane Helices 1 and 6^*

Pascal Courville, Eva Urbankova, Christopher Rensing^¶, Roman Chaloupka, Matthias Quick^||, and Mathieu F. M. Cellier¹

From the INRS-Institut Armand-Frappier, 531 Bd des prairies, Laval, Québec H7V 1B7, Canada, the Faculty of Mathematics and Physics, Charles University, Institute of Physics, Ke Karlovu 5, 121 16 Prague 2, Czech Republic, the ^¶Department of Soil, Water, and Environmental Science, University of Arizona, Tucson, Arizona 85721, and the ^||Center for Molecular Recognition, Columbia University, College of Physicians and Surgeons 11-401, New York, New York 10032

Ubiquitous solute carriers 11 (SLC11) contribute to metal-ion homeostasis by importing Me²⁺ and H⁺ into the cytoplasm. To identify residues mediating cation symport, Escherichia coli proton-dependent manganese transporter (MntH) was mutated at five SLC11-specific transmembrane (TM) sites; each mutant activity was compared with wild-type MntH, and the biochemical results were tested by homology threading. Cd²⁺ and H⁺ uptake kinetics were analyzed in whole cells as a function of pH and temperature, and right-side out membrane vesicles were used to detail energy requirements and to probe site accessibility by Cys replacement and thiol modification. This approach revealed that TM segment 1 (TMS1) residue Asp³⁴ couples H⁺ and Me²⁺ symport and contributes to MntH forward transport electrogenicity, whereas the TMS6 His²¹¹ residue mediates pH-dependent Me²⁺ uptake; MntH Asn³⁷, Asn²⁵⁰, and Asn⁴⁰¹ in TMS1, TMS7, and TMS11 participate in transporter cycling and/or helix packing interactions. These biochemical results fit the LeuT/SLC6 structural fold, which suggests that conserved peptide motifs Asp³⁴-Pro-Gly (TMS1) and Met-Pro-His²¹¹ (TMS6) form antiparallel "TM helix/extended peptide" boundaries, lining a "pore" cavity and enabling H⁺-dependent Me²⁺ import.

Received for publication, December 4, 2007 , and in revised form, January 18, 2008.

^* This work was supported in part by Canadian Institutes of Health Research Grant MOP-78014-MI (to M. C.), Czech Science Foundation Grant 204/07/0558 (to R. C.), and the Fondation Armand-Frappier (to P. C.). 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 supplemental Figs. S1–S5, Table S1, and supplemental Methods 1 and 2.

¹ To whom correspondence should be addressed: INRS-Institut Armand-Frappier, 531, Bd des prairies, Laval, QC H7V 1B7, Canada. Fax: 450-686-5301; E-mail: mathieu.cellier{at}iaf.inrs.ca.