LmrP is a major facilitator superfamily multidrug transporter from Lactococcus lactis that mediates the efflux of cationic amphiphilic substrates from the cell in a proton-motive force-dependent fashion. Interestingly, motif searches and docking studies suggested the presence of a putative Ca(2+)-binding site close to the interface between the two halves of inward facing LmrP. Binding experiments with radioactive (45)Ca(2+) demonstrated the presence of a high affinity Ca(2+)-binding site in purified LmrP, with an apparent K(d) of 7.2 μm, which is selective for Ca(2+) and Ba(2+) but not for Mn(2+), Mg(2+), or Co(2+). Consistent with our structure model and analogous to crystal structures of EF hand Ca(2+)-binding proteins, two carboxylates (Asp-235 and Glu-327) were found to be critical for (45)Ca(2+) binding. Using (45)Ca(2+) and a fluorescent Ca(2+)-selective probe, calcium transport measurements in intact cells, inside-out membrane vesicles, and proteoliposomes containing functionally reconstituted purified protein provided strong evidence for active efflux of Ca(2+) by LmrP with an apparent K(t) of 8.6 μm via electrogenic exchange with three or more protons. These observations demonstrate for the first time that LmrP mediates selective calcium/proton antiport and raise interesting questions about the functional and physiological links between this reaction and that of multidrug transport.
