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Mg2+-dependent gating of bacterial MgtE channel underlies Mg2+ homeostasis

2009; Springer Nature; Volume: 28; Issue: 22 Linguagem: Inglês

10.1038/emboj.2009.288

1460-2075

Motoyuki Hattori, N. Iwase, Noritaka Furuya, Yoshiki Tanaka, Tomoya Tsukazaki, Ryuichiro Ishitani, Michael E. Maguire, Koichi Ito, Andrés D. Maturana, Osamu Nureki,

Ion Transport and Channel Regulation

Article1 October 2009free access Mg2+-dependent gating of bacterial MgtE channel underlies Mg2+ homeostasis Motoyuki Hattori Motoyuki Hattori Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Norihiko Iwase Norihiko Iwase Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama-shi, Kanagawa, Japan Search for more papers by this author Noritaka Furuya Noritaka Furuya Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama-shi, Kanagawa, Japan Search for more papers by this author Yoshiki Tanaka Yoshiki Tanaka Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Tomoya Tsukazaki Tomoya Tsukazaki Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Ryuichiro Ishitani Ryuichiro Ishitani Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Michael E Maguire Michael E Maguire Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Koichi Ito Corresponding Author Koichi Ito Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Minato-ku, Tokyo, Japan Search for more papers by this author Andres Maturana Corresponding Author Andres Maturana Global Edge Institute, Tokyo Institute of Technology, E31, Tokyo, Japan Search for more papers by this author Osamu Nureki Corresponding Author Osamu Nureki Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama-shi, Kanagawa, Japan Search for more papers by this author Motoyuki Hattori Motoyuki Hattori Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Norihiko Iwase Norihiko Iwase Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama-shi, Kanagawa, Japan Search for more papers by this author Noritaka Furuya Noritaka Furuya Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama-shi, Kanagawa, Japan Search for more papers by this author Yoshiki Tanaka Yoshiki Tanaka Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Tomoya Tsukazaki Tomoya Tsukazaki Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Ryuichiro Ishitani Ryuichiro Ishitani Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Michael E Maguire Michael E Maguire Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Koichi Ito Corresponding Author Koichi Ito Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Minato-ku, Tokyo, Japan Search for more papers by this author Andres Maturana Corresponding Author Andres Maturana Global Edge Institute, Tokyo Institute of Technology, E31, Tokyo, Japan Search for more papers by this author Osamu Nureki Corresponding Author Osamu Nureki Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama-shi, Kanagawa, Japan Search for more papers by this author Author Information Motoyuki Hattori1,‡, Norihiko Iwase2,‡, Noritaka Furuya2,‡, Yoshiki Tanaka1, Tomoya Tsukazaki1, Ryuichiro Ishitani1, Michael E Maguire3, Koichi Ito 1,4, Andres Maturana 5 and Osamu Nureki 1,2 1Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan 2Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama-shi, Kanagawa, Japan 3Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA 4Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Minato-ku, Tokyo, Japan 5Global Edge Institute, Tokyo Institute of Technology, E31, Tokyo, Japan ‡These authors contributed equally to this work *Corresponding authors. Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: +81 3 6409 2125; Fax: +81 3 6409 2127; E-mail: [email protected] or Tel.: +81 258 47 9445; Fax: +81 47 258 9400; E-mail: [email protected] or Tel.: +81 3 5449 5309; Fax: +81 3 5449 5415; E-mail: [email protected] The EMBO Journal (2009)28:3602-3612https://doi.org/10.1038/emboj.2009.288 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The MgtE family of Mg2+ transporters is ubiquitously distributed in all phylogenetic domains. Recent crystal structures of the full-length MgtE and of its cytosolic domain in the presence and absence of Mg2+ suggested a Mg2+-homeostasis mechanism, in which the MgtE cytosolic domain acts as a ‘Mg2+ sensor’ to regulate the gating of the ion-conducting pore in response to the intracellular Mg2+ concentration. However, complementary functional analyses to confirm the proposed model have been lacking. Moreover, the limited resolution of the full-length structure precluded an unambiguous characterization of these regulatory divalent-cation-binding sites. Here, we showed that MgtE is a highly Mg2+-selective channel gated by Mg2+ and elucidated the Mg2+-dependent gating mechanism of MgtE, using X-ray crystallographic, genetic, biochemical, and electrophysiological analyses. These structural and functional results have clarified the control of Mg2+ homeostasis through cooperative Mg2+ binding to the MgtE cytosolic domain. Introduction Magnesium ion is an essential metal element for life and has a crucial function in many biological process, such as ATP utilization, activation of and catalysis by hundreds of enzymes, and maintenance of genomic stability (Hartwig, 2001; Cowan, 2002). In human beings, abnormal Mg2+ homeostasis is reportedly associated with several diseases, including cardiovascular disease, diabetes, and high blood pressure (Alexander et al, 2008). However, among the major biological cations, the mechanisms of Mg2+ transport and homeostasis are only slowly being elucidated. Recently, three groups reported the crystal structures of the CorA Mg2+ transporter, which provided the first structural framework of a Mg2+ transporter (Nelson and Kennedy, 1971; Hmiel et al, 1986; Eshaghi et al, 2006; Lunin et al, 2006; Payandeh and Pai, 2006). The MgtE family of Mg2+ transporters is ubiquitously distributed in all kingdoms of life (Moomaw and Maguire, 2008). In Bacillus subtilis, the gene expression of MgtE is primarily regulated by a Mg2+-sensing riboswitch (M-box riboswitch) in response to intracellular Mg2+ levels, and probably also in other Gram-positive bacteria, which would contribute to relatively long-term Mg2+ homeostasis (Dann et al, 2007). In mammals, SLC41 transporters, which are distant MgtE homologues, were found to function in Mg2+ homeostasis (Goytain and Quamme, 2005a, 2005b; Sahni et al, 2007; Kolisek et al, 2008). In bacteria, MgtE consists of the N-terminal cytosolic domain and the C-terminal transmembrane domain. On the other hand, the SLC41 transporters lack the cytosolic domain. Despite its apparently significant function in Mg2+ homeostasis, the Mg2+-transport mechanism of MgtE is poorly understood, and it is not even clear whether MgtE functions as either a channel or an active transporter. Recently, we reported the crystal structures of the full-length MgtE of Thermus thermophilus at 3.5 Å resolution and of its cytosolic domain in the presence and absence of Mg2+ at 2.3 and 3.9 Å resolutions, respectively (Hattori et al, 2007a). The full-length structure of MgtE in the presence of Mg2+ revealed a homodimer. The putative ion-conducting pore in the TM domain is closed in this crystal form through interactions between the TM domains and the ‘plug helices’ connecting the cytosolic and TM domains. Several putative Mg2+ ions are bound to the interface between the dimeric cytosolic domains or between the cytosolic and TM domains. A structural comparison of the cytosolic domains in the presence and absence of Mg2+ revealed a drastic Mg2+-dependent structural transition involving the rotation of the plug helices. On the basis of these structures, we proposed a Mg2+-homeostasis mechanism in which the cytosolic domain of MgtE acts as a ‘Mg2+ sensor’ to regulate the gating of the ion-conducting pore through plug helix movement by sensing the intracellular Mg2+ concentration. Molecular dynamics simulations of the MgtE cytosolic domain also suggested that its multiple Mg2+-binding sites induce the conformational transition in a cooperative manner (Ishitani et al, 2008). The model comprehensively provided a short-period intracellular homeostasis mechanism for acute environmental changes. However, there has been no complementary functional analysis to substantiate the gating mechanism or even to determine whether MgtE is gated by the intracellular Mg2+ concentration. Moreover, the limited resolution of the full-length structure prevented the unambiguous identification of these divalent-cation-binding sites. Here, we established the channel properties and the Mg2+-dependent gating of MgtE by a combination of X-ray crystallographic, genetic, biochemical, and electrophysiological analyses to address the functional mechanism of the Mg2+ regulation of MgtE. Our data indicated that MgtE functions as a Mg2+-dependent gating channel and that the cooperative Mg2+ binding to the cytosolic domain stabilizes the closed conformation of the channel. On the basis of these results, we have proposed a novel structural transition mechanism of MgtE. Results MgtE structure with divalent-cation-binding sites In the earlier full-length MgtE structure, several putative Mg2+ ions apparently bind to the cytosolic domains, which would induce the stabilization of the closed conformation of MgtE. However, the limited resolution (3.5 Å) of the full-length structure did not allow an unambiguous identification of these divalent-cation-binding sites. To test the Mg2+-dependent gating model, the precise divalent-cation-binding sites of MgtE should be clarified. Therefore, we determined a higher resolution structure of the full-length MgtE. By extensive screening of detergents for MgtE crystallization, we obtained a new crystal form of MgtE in the presence of Mg2+, which diffracts to a higher resolution (2.94 Å) than the earlier crystal form (3.5 Å). The updated structure of the full-length MgtE is shown in Figure 1. The overall structure of the new crystal form is quite similar to that of the earlier-reported MgtE, with an RMSD value of 0.9 Å for all of the Cα atoms, and represents the closed conformation. On the other hand, some parts of the electron density of the updated structure have been improved. As a result, we found significant differences between the current and earlier structures. Figure 1.The 2.9-Å resolution MgtE structure. (A) The MgtE dimer is viewed in the membrane plane, with the N domain (blue), the CBS domain (green), the plug helix (yellow), and the transmembrane (TM) domain (red) highlighted in one subunit. The other subunit is coloured grey. The TM helices of one subunit are numbered. The membrane surface is indicated. (B) Solvent-accessible surface of the ion-conducting pore. Residues lining the pore are indicated. Download figure Download PowerPoint First, in the earlier structure, the electron density around Pro321, lining the putative ion-conducting pore, was ambiguous, and the preceding portion of the TM2 helix apparently adopted a non-helical, flexible structure (Hattori et al, 2007a). On the other hand, in the current structure, this region is restructured to extend the TM2 helix, which is kinked at Pro321 (Figure 1B). It is noteworthy that the region around Pro321 is the narrowest part of the pore, which would prevent ion conduction in the closed state. In addition, the hydrophobic residues near the periplasmic side would also prevent the ion conduction (Figure 1B). These regions at the periplasmic side of MgtE may create a strong barrier to ion permeation. Therefore, in addition to the interaction between the plug helices and the TM domains on the cytosolic side of MgtE (Hattori et al, 2007a), the MgtE pore would be closed on both the periplasimic and cytosolic sides. Moreover, for MgtE to adopt an open conformation, the TM2 helices of both subunits should move apart at the position of Pro321, in addition to the movement of the plug helices. Such a putative ‘hydrophobic gate’ has also been observed in the closed-state structure of the CorA Mg2+ channel (Lunin et al, 2006), the inwards rectifying potassium channel (Kuo et al, 2003) and the nicotinic acetylcholine receptor (Miyazawa et al, 2003). Interestingly, although these other channels use bulky hydrophobic residues to form the gates, MgtE seems to use small hydrophobic residues with a kinked main chain structure. Second, in the new crystal form, an additional strong electron density peak was observed between the conserved acidic residues Glu59 in the N domain of one monomer and Asp226 in the CBS domain from the other subunit (Figure 2D; Supplementary Figure S1). We interpreted the electron density as an additional Mg2+ ion (Mg7), based on the same criteria as earlier used to assign the other putative-bound Mg2+ ions (Figure 2A–D) (Hattori et al, 2007a). Figure 2.Divalent-cation-binding sites. (A) Side view of the overall structure with the bound Mg2+. (B–D) Close-up view of the respective Mg2+ (Mg1–7)-binding sites, with an Fo−Fc omit map contoured at 4.0 σ, calculated with the full-length structure excluding Mg2+. (E–G) Anomalous difference Fourier map derived from Co2+-soaked crystals, contoured at 5.0 σ, at the Mg1–7-binding sites. Download figure Download PowerPoint Certain divalent cations, such as Co2+ or Ni2+, bind to Mg2+-binding sites by mimicking the octahedral coordination geometry of Mg2+ (Eshaghi et al, 2006). To confirm that the earlier-assigned bound Mg2+ were indeed Mg2+, this new crystal form was used for soaking experiments with Co2+ and Ni2+, to distinguish the divalent-cation-binding sites by the anomalous signals derived from these heavier cations. The anomalous difference Fourier maps of the Co2+-soaked crystals clearly supported our assignment of all of the cytosolic Mg2+-binding sites (for Mg2–7) (Figure 2F and G). The data from the Ni2+-soaked crystal provided results exactly consistent with those from the Co2+-soaked crystal (Supplementary Figure S2). In contrast to the Mg2–7-binding sites in the cytosolic domain, the Mg1-binding site in the TM domain could not be confirmed with either the Co2+ or Ni2+ anomalous signal (Figure 2E; Supplementary Figure S2A). This result indicates that Co2+ and Ni2+ ions might not be able to access the Mg1-binding site at the centre of the pore in the crystalline state, because of the current closed structure. This is consistent with the idea that the ion-conducting pore is closed on both the periplasmic and cytosolic sides in the closed state of MgtE. Altogether, we could confirm 12 Mg2+ (Mg2–7 for two monomers)-binding sites in the cytosolic domain of MgtE. MgtE acts as a highly selective Mg2+ channel To functionally verify our structure-based mechanism of ion conduction and Mg2+-dependent gating by MgtE, we have developed two types of analyses to evaluate its Mg2+-transport activity: in vivo complementation and in vitro patch-clamp analyses. Although a Salmonella strain lacking two major Mg2+-transporter genes, mgtA and corA, reportedly exhibits Mg2+ auxotrophy and has been used for in vivo functional analyses of Mg2+ transporters (Kehres and Maguire, 2002), Escherichia coli K12 strains with the same gene knockouts unexpectedly did not show Mg2+ auxotrophy (data not shown). This fact, unfortunately, prevented researchers from adopting the many useful analytical tools established in E. coli genetics. By a systematic search for additional component(s) involved in major Mg2+ transport in the mgtA and corA knocked-out E. coli background, the novel gene yhiD, encoding an integral membrane protein related to MgtC, was successfully assigned as the candidate of the third component. We generated a Mg2+-auxotrophic E. coli K12 strain, devoid of the major set of genes for Mg2+ transport, corA, mgtA, and yhiD, which can survive only when supplemented with a sufficiently high concentration (100 mM) of Mg2+. The result newly indicated that the yhiD gene would be an essential component for the Mg2+ uptake system in E. coli. When we transformed the strain with the full-length wild type (WT) MgtE gene, the cells could survive in Mg2+-free LB medium, whereas the control empty vector could not complement the Mg2+ auxotroph (Figure 3A), suggesting that the exogenously expressed WT MgtE is clearly capable of reconstituting Mg2+-transport activity in vivo. The expression of the MgtE proteins in the membrane fraction was confirmed by anti-His tag western blotting (Supplementary Figure S3). Figure 3.Mg2+ channel activity of MgtE. (A) Growth complementation of the Mg2+-auxotrophic strains harbouring either WT or empty vector in the presence (+Mg2+, 100 mM MgSO4) or absence (−Mg2+, 0 mM MgSO4) of supplementary Mg2+. Growth of each transformant is indicated as ‘+++’ (similar to that of empty vector in the presence of 100 mM MgSO4), ‘++’ (less), ‘+’ (much less), ‘+/−’ (severe growth retardation), and ‘−’ (no growth at all). (B) Single channel currents were measured in the inside–out configuration from E. coli spheroplasts. An example of current traces recorded from control (upper) and MgtE-overexpressing triple knock-out E. coli spheroplasts (bottom) at −70 mV. (C) The current–voltage relationship between −70 and +40 mV was determined from the single current amplitude at the indicated potentials. Values are mean and standard error of the mean (s.e.m.) of 10 experiments. (D) The Co2+ permeability of the MgtE channel was determined by measuring the current in the presence of 90 mM CoCl2. The current–voltage relationship was determined by the single current amplitude. (E) The permeabilities of nickel, manganese, and calcium were also tested. Traces are examples of currents recorded at −40 mV. Each experiment was performed four times. Download figure Download PowerPoint Then, we generated giant spheroplasts (Martinac et al, 1987; Kuo et al, 2007) of the same transformants of the Mg2+-auxotrophic E. coli strain for the patch-clamp experiments (Supplementary Figure S4). Our newly generated Mg2+-auxotrophic E. coli strain has the advantage of expressing the genes encoded in the pET vector under the control of the strong T7 promotor, which enables us to overexpress MgtE in the generated giant spheroplasts. Thus, the giant spheroplasts transformed with the mgtE-pET plasmid could be efficiently used for a patch-clamp analysis, because of the high expression of the MgtE channel. Using a Mg2+-sensitive dye, mag-fluo4, we confirmed that MgtE is functionally expressed in the giant spheroplasts. Mg2+ influx is readily apparent in giant spheroplasts expressing MgtE under 10 mM sucrose condition as well as under 300 mM sucrose, but not in those carrying the control empty vector (Supplementary Figure S5). We measured the MgtE-associated currents in the patch-clamp analysis by excising membrane patches from the MgtE-expressing giant spheroplasts. Currents were recorded at test potentials ranging from −70 to +30 mV. The pipette solution (corresponding to the extracellular region, [Mg2+]out) included 90 mM MgCl2, whereas the bath solution (corresponding to the intracellular region, [Mg2+]in) contained 0.2 mM MgCl2. The results clearly showed representative currents reflecting the open and closed states of a single MgtE channel (Figure 3B). In contrast, cells carrying the control empty vector did not show this activity (Figure 3B). The current was strictly dependent on the presence of Mg2+ in the pipette, and its amplitude increased with increasingly negative membrane potentials (Figure 3C), which is consistent with Mg2+ permeation through the expressed MgtE channel. The current–voltage relationship determined at negative membrane potential yielded a slope conductance of 96±2.7 pS (Figure 3C) at −40 mV, corresponding to a transport efficiency of ∼3 × 107 ions/s at −60 mV, which is much higher that those of active transporters. The current did not depend on the bath pH (Supplementary Figure S6). These results strongly favour a channel-type mechanism for MgtE. To further characterize the single channel properties of MgtE, we examined the ion-conduction selectivity for other divalent cations (Co2+, Ni2+, Mn2+, and Ca2+) (Figure 3D and E). The pipette solution and the bath solution included 90 and 0.2 mM divalent cations, respectively. Currents were apparently observed for Co2+, but not for Ni2+, Mn2+, and Ca2+ (Figure 3D and E), whereas cells carrying the control empty vector did not show activity with any of these cations (data not shown). The current–voltage relationship determined at negative membrane potential yielded a conductance of 22±1.6 pS with 90 mM Co2+, which is much smaller than that with Mg2+. The low but considerable level of Co2+-transport activity, which coincides with the earlier studies on MgtE (Smith et al, 1995), suggests that MgtE might also be involved in the uptake of Co2+ as a micronutrient. On the other hand, Co(III) hexammine, an analogue of fully hydrated Mg2+ and a well-characterized inhibitor of the CorA Mg2+ channel (Kucharski et al, 2000; Kolisek et al, 2003; Schindl et al, 2007), completely blocked Mg2+ influx through MgtE (Supplementary Figure S7). This inhibition by Co(III) hexammine suggests that a fully hydrated Mg2+ ion initially binds to MgtE. Altogether, these results clearly show that MgtE functions as a highly selective Mg2+ channel. Ion-conducting pore in the MgtE structure To identify the ion-conducting pathway of the MgtE channel, we designed structure-based mutants of MgtE. To evaluate their ability to transport Mg2+, we used a growth-complementation system using the Mg2+-auxotrophic E. coli strain. The MgtE homodimer seems to be stabilized by electrostatic interactions between the monomers within the low-dielectric environment of the membrane. Mutations of the highly conserved polar residues involved in these dimer interactions (R285A, Q333A, and E359A) (Figure 4A; Supplementary Figure S1) abolished the Mg2+-transport activity, as shown by the complementation assay (Figure 4B). These results support our hypothesis that stable homodimer formation is requisite for ion conduction. Figure 4.Ion-conducting pore of MgtE. (A) TM domain residues involved in molecular dimerization. Hydrogen and ionic bonds are shown as dotted lines. The backbones of the two subunits are coloured green and blue, respectively. (B) Mg2+-auxotrophic growth complementation by mutated MgtE (Y273L, R285A, Q333A and E359A) genes on the −Mg2+ plates. (C) Mg2+-auxotrophic growth complementation by mutated MgtE (F318A, P321A, L324A, N329A, N332A, N424A, D432N, and D432A) genes on the −Mg2+ plates. (D) Mutations of the conserved hydrophilic residues in the pore inhibit the MgtE current. Examples of currents recorded at −40 mV from membrane patches of spheroplasts overexpressing the D432A and N329A mutants. The pipette solution and the bath solution included 90 and 0.2 mM MgCl2, respectively. Each experiment was repeated four times. Download figure Download PowerPoint We then mutated the conserved residues that line the putative ion-conducting pore. Mutations of the hydrophobic residues near the periplasmic side (F318A, P321A, L324A in Figure 1B) eliminated the growth-complementation activity (Figure 4C). Furthermore, mutations of the conserved hydrophilic residues at the centre of the pore (N329A, D432A, and D432N in Figure 1B; Supplementary Figure S1) also abolished the complementation activity (Figure 4C). Thus, Asp432, which binds Mg1, is proposed to be an ion-selective site of MgtE. The patch-clamp analyses of the D432A and N329A mutants revealed that they also lost transport activity in vitro (Figure 4D), supporting the proposal that this site is crucial for Mg2+ transport. The expression and integration of all of the MgtE mutants in the membrane fraction was confirmed by anti-His tag western blotting (Supplementary Figure S3). Furthermore, the gel filtration analysis of the purified D432A mutant showed a similar elution profile to that of the purified WT MgtE, regardless of the presence of Mg2+ (Supplementary Figure S8). The result implies that the D432A mutant, which lost the putative Mg2+-binding site in the TM domain and the transport activity, would retain the channel assembly ability. On the other hand, it is well established that the permeant ions of potassium channels have a very important function in the stabilization of the channel assembly (Splitt et al, 2000). Mutations of the polar residues at the cytosolic end (N332A and N424A in Figure 1B) had either a slight or no effect on the ability to rescue the Mg2+-auxotrophic growth (Figure 4C). These results are reasonable, as these residues are replaced with alanine in the MgtE channels from other organisms (Supplementary Figure S1). In the open-form structure, this part of the pore would be exposed to the cytosolic vestibule, suggesting that it might not be crucial for ion permeation. In addition, these in vivo complementation results were further supported by an in vivo transport assay of 57Co2+ (Supplementary Figure S9). In summary, these results support the proposal that the MgtE pore, formed by the dimerized TM domain, functions as the ‘ion-conducting pore’. Gating control of MgtE by the intracellular Mg2+ concentration On the basis of our earlier structures, we proposed that MgtE is involved in cytosolic Mg2+ homeostasis by an ability to sense the intracellular Mg2+ concentration through the cytosolic Mg2+-binding sites (Hattori et al, 2007a). To verify this hypothesis, we examined the effect of [Mg2+]in on the MgtE channel activity (Figure 5). As the [Mg2+]in in the bath solution was increased from 0.2 to 10 mM, the current progressively decreased (Figure 5A–D). The addition of Mg2+ to the bath will change the driving force for Mg2+. This means that the equilibrium potential will be shifted to a more positive voltage, thus reducing the current. It can be estimated that the reduction of the current because of the change in driving force should be about 17% for [Mg2+]in=1 mM, as compared with [Mg2+]in=0.2 mM, 25% for [Mg2+]in=5 mM, and 42% for [Mg2+]in=10 mM at −40 mV with a conductance of 96 pS. This means that currents of about 5.8 pA for [Mg2+]in=1 mM, about 5.3 pA for [Mg2+]in=5 mM, and about 4 pA for [Mg2+]in=10 mM should be measured at −40 mV, considering that a current of around 7 pA was measured at −40 mV in 0.2 mM MgCl2. For [Mg2+]in=1 and 5 mM, the measured amplitudes are consistent with those of the predicted amplitudes (Figure 5E). In contrast, for [Mg2+]in=10 mM, we could not measure any current (Figure 5D), which means that the channel is completely closed at least at 10 mM intracellular Mg2+ concentration. Quantitatively, an increase in [Mg2+]in drastically reduced the open probability (Figure 5F). Thus, the MgtE channel was completely inactivated between 5 and 10 mM [Mg2+]in (Figure 5C–F). The threshold for MgtE inactivation by [Mg2+]in is approximately consistent with the results from the earlier Mg2+-dependent protease protection analysis of MgtE (Ishitani et al, 2008). Altogether, these results indicate that the intracellular Mg2+ concentration is capable of regulating the MgtE channel activity. Figure 5.Mg2+ dependence of the MgtE channel activity. (A–D) The MgCl2 concentration in the bath was increased from 0.2 to 1 mM and 5 to 10 mM, and single currents were measured at each MgCl2 concentration with MgtE-overexpressing spheroplasts. Example of current traces recorded at −40 mV from a single membrane patch at different [Mg2+]in. (E) The current–voltage relationships of the MgtE current recorded at different [Mg2+]in were determined with a single current amplitude. (F) The open probability was determined at the different [Mg2+]in (n=5 for