The multidrug transporter LmrP protein mediates selective calcium efflux.
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.
Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.
Theresia A
TA
Schaedler
Zhen
Z
Tong
Hendrik W
HW
van Veen
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D024901:Drug Resistance, Multiple, Bacterial / Q000502:physiology*; D017136:Ion Transport / Q000502:physiology; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
D001426:Bacterial Proteins; C096807:LmrP protein, Lactococcus lactis; D026901:Membrane Transport Proteins; D002118:Calcium