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The Multidrug Transporter LmrP Protein Mediates Selective Calcium Efflux*

Theresia A. Schaedler,Zhen Tong,H. V. van Veen

Published 2012 in Journal of Biological Chemistry

ABSTRACT

Background: The multidrug transporter LmrP confers drug resistance on cells by mediating efflux of structurally dissimilar cytotoxic substrates. Results: Surprisingly, LmrP catalyzes the selective, high affinity binding and extrusion of Ca2+, which inhibits multidrug transport by LmrP. Conclusion: LmrP can act as a calcium/proton antiporter. Significance: Multidrug transporters might fulfill additional physiological roles, which could promote their persistence in the absence of antibiotics. 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 Ca2+-binding site close to the interface between the two halves of inward facing LmrP. Binding experiments with radioactive 45Ca2+ demonstrated the presence of a high affinity Ca2+-binding site in purified LmrP, with an apparent Kd of 7.2 μm, which is selective for Ca2+ and Ba2+ but not for Mn2+, Mg2+, or Co2+. Consistent with our structure model and analogous to crystal structures of EF hand Ca2+-binding proteins, two carboxylates (Asp-235 and Glu-327) were found to be critical for 45Ca2+ binding. Using 45Ca2+ and a fluorescent Ca2+-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 Ca2+ by LmrP with an apparent Kt 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.

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