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. 2011 Jun;1808(6):1587-91.
doi: 10.1016/j.bbamem.2010.10.017. Epub 2010 Nov 11.

Functional analysis of the conserved hydrophobic gate region of the magnesium transporter CorA

Soňa Svidová  1 , Gerhard Sponder, Rudolf J Schweyen, Kristina Djinović-Carugo
Affiliations

Functional analysis of the conserved hydrophobic gate region of the magnesium transporter CorA

Soňa Svidová et al. Biochim Biophys Acta. 2011 Jun.

Abstract

The Leu294 residue in the cytoplasmic neck of Thermotoga maritima CorA is considered to be the main gate for Mg2+ transport. We created three site-directed mutants at this position: in the Leu294Asp and Leu294Gly mutants we observed a defect in closing of the pore, while in the Leu294Arg mutant not only gating, but also the regulation of Mg2+ uptake was affected. Our results confirmed the importance of the Leu294 for gating of Mg2+ transport and in addition revealed the influence of the charge and structural features of the amino acid residues on the gating mechanism.

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Figures

Fig. 1
Fig. 1
Structure of the TmCorA Mg2+ transporter. (a) Single monomer: green — transmembrane domains TM1 and TM2, blue — α7 helix, red — N-terminal domain. (b) Side view of the homopentamer: cyan — DCS sites 1 and 2. (c) View from the top: blue — basic sphincter, yellow — aromatic ring, green — the gate forming residues Leu294 and Met291.
Fig. 2
Fig. 2
Growth complementation assay of the gating mutants. S. typhimurium strain MM281 which lacks all three known magnesium transport systems was transformed with plasmids indicated. The pQE TmCorA and pQE80L empty constructs served as positive and negative control.
Fig. 3
Fig. 3
Representative recordings of Mg2+ uptake. S. typhimurium strain MM281 which lacks all three known magnesium transport systems was transformed with plasmids indicated. The representative recordings show changes in fluorescence intensity of Mag-Fura-2 monitored over 10 min time after adding 10 mM MgCl2. The pQE TmCorA and pQE80L empty constructs served as positive and negative control.
Fig. 4
Fig. 4
Effect of gating mutations in different liquid medium. Growth curves of the S. typhimurium strain MM281 which lacks all three known magnesium transport systems, transformed with plasmids indicated. Cells were grown over night in LB amp medium containing 10 mM MgCl2. Cultures were diluted to an OD600 of 0.1 and grown over 5 h. The data were averaged from three independent experiments.
Fig. 5
Fig. 5
Effect of Co(III)hexamine, inhibitor of the CorA-mediated magnesium transport. Growth curves of the S. typhimurium strains LB5010 (wild-type strain) transformed with pQE80L parental vector and MM281 (corAΔ, mgtAΔ, mgtBΔ) transformed with pQE80 TmCorA and pQE80 TmCorA L294G in the presence or absence of cobalt hexamine. The data were averaged from three independent experiments.
Fig. 6
Fig. 6
Western blot analysis of whole cell samples. S. typhimurium strain MM281 which lacks all three known magnesium transport systems was transformed with plasmids indicated. The synthesis of CorA protein and variants thereof was induced by addition 0.05 mM IPTG for 3 h.
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References

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