Solute carrier 11 cation symport requires distinct residues in transmembrane helices 1 and 6

Abstract

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 Asp 34 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. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.