A Spontaneous, Recurrent Mutation in Divalent Metal Transporter-1 Exposes a Calcium Entry Pathway

Divalent metal transporter-1 (DMT1/DCT1/Nramp2) is the major Fe2+ transporter mediating cellular iron uptake in mammals. Phenotypic analyses of animals with spontaneous mutations in DMT1 indicate that it functions at two distinct sites, transporting dietary iron across the apical membrane of intestinal absorptive cells, and transporting endosomal iron released from transferrin into the cytoplasm of erythroid precursors. DMT1 also acts as a proton-dependent transporter for other heavy metal ions including Mn2+, Co2+, and Cu2, but not for Mg2+ or Ca2+. A unique mutation in DMT1, G185R, has occurred spontaneously on two occasions in microcytic (mk) mice and once in Belgrade (b) rats. This mutation severely impairs the iron transport capability of DMT1, leading to systemic iron deficiency and anemia. The repeated occurrence of the G185R mutation cannot readily be explained by hypermutability of the gene. Here we show that G185R mutant DMT1 exhibits a new, constitutive Ca2+ permeability, suggesting a gain of function that contributes to remutation and the mk and b phenotypes.


Introduction
Spontaneous mutations in mice and rats have provided important information about mammalian iron homeostasis (reviewed in Andrews 2000). Interestingly, three independent, autosomal recessive mutants have been shown to have the same amino acid substitution in a key iron transport molecule. Two strains of mutant microcytic (mk) mice (MK/ ReJ-mk, SEC/1ReJ-mk) and Belgrade (b) rats have severe iron deficiency attributable to a G185R mutation in divalent metal transporter-1 (DMT1) (Fleming et al. 1997;Andrews 2000). Based on the phenotypes of these animals and the properties of DMT1 detailed below, we and others concluded that DMT1 is essential for intestinal absorption of Fe 2þ and for unloading of transferrin-derived iron from transferrin cycle endosomes (Fleming et al. 1997Gunshin et al. 1997;Picard et al. 2000). It is intriguing that no other DMT1 mutations have been described in mammals, and no features of the DNA sequence suggest that the G185 codon would be hypermutable in two species. We speculated that a novel characteristic of the G185R DMT1 protein might account for this remarkable pattern of remutation.
Trace metal ions including Fe 2þ , Mn 2þ , Cu 2þ , Zn 2þ , and Co 2þ are required cofactors for many essential cellular enzymes. They cannot cross the plasma membrane through simple diffusion, and active uptake requires specific transporters. DMT1 is the only molecule known to mediate cellular iron uptake in higher eukaryotes. It is structurally unrelated to known Zn 2þ and Cu 2þ transporters, but DMT1 can transport those and other divalent metal ions (Gunshin et al. 1997), and it appears to be the major mammalian Mn 2þ transporter (Chua and Morgan 1997). DMT1 is predicted to have 12 transmembrane (TM) segments ( Figure 1A). It is expressed on the apical brush border of the proximal duodenum ) and in transferrin cycle endosomes Gruenheid et al. 1999). It appears to function by coupling a metal entry pathway to a downhill proton gradient, taking advantage of the acidic pH in both of those sites. An earlier study proposed a 1:1 stoichiometry of metal ion and proton cotransport (Gunshin et al. 1997).
Ca 2þ is not a measurable substrate for wild-type DMT1 (Gunshin et al. 1997;Tandy et al. 2000), even though it is at least 1,000 times more abundant in plasma than trace metals. Surprisingly, we found that the G185R mutation ( Figure 1A) dramatically increases the Ca 2þ -permeability of DMT1, functionally converting DMT1 into a Ca 2þ channel. In light of the important and ubiquitous role of Ca 2þ in cell signaling (Berridge et al. 2003), this gain of function offers a likely explanation for the remutation.
Interpretations of recent structural data have already suggested that permeation pathways exist within some transporters (Hirai et al. 2002), blurring the distinction between transporters and ion channels (DeFelice and Blakely 1996). Our finding, that a single amino acid substitution in a presumed transporter can expose a channel pathway, strongly supports this notion and provides new insight into what must be viewed as a continuum between transporter and channel activities.

Results
We studied wild-type DMT1 and the G185R mutant proteins by whole-cell patch-clamp in transiently expressing CHO-K1 and HEK-293T cells and in doxycycline-inducible DMT1-HEK-On and G185R-HEK-On cells. Consistent with previous studies, DMT1 expression significantly increased cellular 55 Fe 2þ uptake at low pH ( Figure 1B). As reported in Xenopus oocytes (Gunshin et al. 1997), reduction of extracellular pH in the absence of metal (nominal free [Fe 2þ ] o of approximately 0.05 lM) induced large inward currents in DMT1-expressing cells ( Figure 1C and 1D). This current is referred to as a substrate-free ''leak'' pathway and is representative of ''drive-slip'' phenomena seen in DMT1 and a related yeast metal transporter, SMF1p (Sacher et al. 2001), as well as many other transporters (Nelson et al. 2002). Because we found that protons also activated an endogenous diisothiocyanostilbene 2,2-disolphonic acid (DIDS)-sensitive anion conductance (unpublished data) that was strongly outwardly rectifying ( Figure S1), we used SO 4 2to replace most of the Cl -([Cl -] o = 5 mM) in low-pH bath solutions. With elimination of the background Clcurrent, the proton-evoked current was inwardly rectifying (hyperbolic) ( Figure  1E). The large proton-induced current caused significant DMT1-specific intracellular acidification (Gunshin et al. 1997). In whole-cell recordings of DMT1 currents, we routinely observed slow inactivation (or decay) after a proton-induced current reached its peak (see Figure 1C). While the extent of the slow inactivation varied from cell to cell, it usually reached a relative steady state within 100 s. Addition of 100 lM Fe 2þ (data not shown) or Mn 2þ induced an additional current with less pronounced slow inactivation ( Figure 1C). Because Fe 2þ is readily oxidized to Fe 3þ in the absence of substantial concentrations of reducing agents (e.g., ascorbate), and Fe 3þ is not transported by DMT1 (Gunshin et al. 1997;Picard et al. 2000), we have used Mn 2þ as an Fe 2þ surrogate since both metals induced similar currents (Gunshin et al. 1997;unpublished data). The observed Mn 2þ deficiency of b rats in vivo (Chua and Morgan 1997) also supports its use in this role.
H þ alone or H þ /Mn 2þ induce distinct currents in DMT1. No significant voltage-or time-dependent fast inactivation was seen when the DMT1-mediated H þ /Mn 2þ current (I DMT1 ) was recorded ( Figure S2). The amplitude of additional Mn 2þinduced current was dependent on [Mn 2þ ] o , with a measurable response at [Mn 2þ ] o , 1 lM (pH 4.2). In the presence Figure 1. Wild-Type DMT1-Expressing Cells Exhibit a Proton Current and a Proton-Dependent Mn 2þ -Induced Current (A) The G185R mutation is in the fourth of 12 putative TM domains in both mouse (shown) and rat DMT1 proteins. (B) 55 Fe 2þ uptake was greatly reduced for G185R in comparison to wild-type DMT1, although the protein expression levels were comparable (inset). (C-E) Representative currents induced by protons (pH 4.2) and Mn 2þ (100 lM) at þ50 mV (open triangles; some of the datapoints have been removed for clarity) and À130 mV (open circles) in a wild-type DMT1-transfected CHO-K1 cell. Whole-cell currents were elicited by repeated voltage ramps (À140 to þ60 mV, 1,000 ms), shown in (E), with a 4 s interval between ramps. Holding potential (HP) was þ20 mV. of 100 lM Mn 2þ (pH 4.2), the additional Mn 2þ -induced current was typically half the amplitude of the proton-induced current. Addition of Mn 2þ alone (100 lM) at pH 7.4 did not induce any additional current. Since H þ or H þ /Mn 2þ induced two currents with distinct kinetics in DMT1-expressing cells, the underlying charge-carrying ion species and their relative contributions to the macroscopic currents were investigated. We monitored the reversal potential (E rev ) and the current amplitude in ion-substitution experiments. Replacement of Na þ with N-methyl-D-glucamine (NMDG þ ) did not significantly change the E rev of H þ or H þ /Mn 2þ -induced currents, although the net current amplitude was slightly increased ( Figure S3). On the other hand, the current amplitude (data not shown) and E rev of the proton current were strongly affected by [H þ ] o (see Figure 1F). The slope of E rev versus pH was 58 mV/decade, is consistent with an H þ -permeable pore. The large positive displacement in E rev from E H (see Figure 1F) may result in part from leak and capacitance-charging, but the carrier mechanism is not well understood.
In contrast, when Mn 2þ was introduced, the slope of the curve fitted to E rev versus pH deviated considerably from the theoretical slope for a H þ -permeable electrode (see Figure  1F). Replacement of Na þ by NMDG þ did not significantly affect the Mn 2þ -induced response (see Figure S3). Our interpretation of this deviation is that DMT1 transport stoichiometry is variable (Chen et al. 1999;Sacher et al. 2001;Adams and DeFelice 2002) or has a fixed but very low permeation ratio (P Mn /P H ) (Hodgkin and Horowicz 1959). P Mn /P H can be estimated from the slope of E rev versus pH based on an extended Goldman-Hodgkin-Katz equation (Lewis 1979) with two permeable ions (H þ and Mn 2þ ). At pH 4.2, the slope of E rev versus pH did not differ significantly with or without Mn 2þ (see Figure 1F). Therefore, we estimate that at pH 4.2 the contribution of H þ to I DMT1 is much larger than that of Fe 2þ /Mn 2þ (P Mn /P H , 0.01), in contrast to the 1:1 stoichiometry proposed previously (Gunshin et al. 1997). Importantly, no Ca 2þ permeability was observed, even in isotonic (105 mM) Ca 2þ solution (see Figure 1C).
In G185R-expressing cells, we observed a large inward current in control bath solution (10 mM Ca 2þ and 140 mM Na þ ) at pH 7.4 (Figure 2A), though no significant current was detected with wild-type DMT1 under similar conditions (see Figure 1E). This inward current mediated by G185R mutant DMT1 (I G185R ) was stable over minutes with no slow inactivation (see Figure 2A), in contrast to the DMT1mediated proton current (see Figure 1C). We observed I G185R in more than 85% of enhanced green fluorescent protein (EGFP)-positive cells transfected with the pTracer-G185R construct and in stable, doxycycline-induced G185R-HEK-On cells, but never in cells transfected with wild-type DMT1 ( Figure 1C) or with 30 DMT1 mutations at other positions (n . 300 cells; unpublished data). The inwardly rectifying current was cationic, since Ca 2þ and Na þ substitution by NMDG þ completely abrogated the current (see Figure 2A and 2B). The current and rectification profiles were not significantly changed when ATP and Mg 2þ were omitted from the intracellular solution, or when Na þ or K þ replaced Cs þ as the primary intracellular cation.
We found that low pH strongly inhibited I G185R (by approximately 90% at pH 5.8; Figure 2A), in contrast to both wild-type DMT1 currents, which were activated at low pH. However, further reduction to pH 4.2 revealed a current (Figure 2A and 2B) that was similar to the proton current of wild-type DMT1. Addition of Mn 2þ at pH 4.2 enhanced the inward current, as with wild-type DMT1 (Figure 2A and 2B). The proton current and Mn 2þ -induced response displayed similar patterns of inactivation and further activation as in wild-type DMT1-transfected cells, but both currents were much smaller than their wild-type counterparts. Consistent with this result and our previous uptake studies ), we found that G185R cells had much lower Fe 2þ uptake (approximately 10% measured at 16 min) compared to wildtype DMT1 at similar protein expression levels (see Figure  1B). I G185R rectified more steeply with voltage than I DMT1 , probably due to pronounced time-and voltage-dependent fast inactivation ( Figure 2C; see Figure S2 for comparison).
Fast inactivation was enhanced when [Ca 2þ ] o was increased to 105 mM ( Figure 2E), strengthening the notion that I G185R was fundamentally distinct from the currents mediated by wildtype DMT1. In control bath solution (10 mM Ca 2þ , 140 mM Na þ [pH 7.4]), I G185R was 64 6 7 pA/pF at À140 mV (mean 6 SEM, n = 15; Figure 2D) compared to less than 2 pA/pF in mock and DMT1-transfected cells. I G185R reversed at approximately þ20 mV with very little current above 0 mV ( Figure  2D), whereas the E rev of I DMT1 was approximately þ50 mV at pH 4.2. The dependence of I G185R on holding potential was also distinct from I DMT1 (see below).
We next investigated the cation selectivity of I G185R . The amplitude of I G185R was strongly dependent on [Ca 2þ ] o ( Figure  2F). With 10 mM Ca 2þ in the bath, replacement of 140 mM NMDG þ by 140 mM Na þ only slightly (by approximately 15%) increased the current, indicating that Ca 2þ permeated the plasma membrane of G185R-transfected cells much more readily than Na þ . As shown in Figure 3A and 3B, increasing [Ca 2þ ] o not only augmented the current amplitude but also shifted E rev toward depolarized potentials. The slope of this shift (25 mV per decade) was close to the slope of 29 mV per decade predicted by the Nernst equation for a Ca 2þ -selective electrode ( Figure 3C). The relative permeability of various divalent cations was studied under bi-ionic conditions (pipette solution containing Na þ and Glutamate; see Materi- als and Methods). After adding 10 mM test divalent cations to the NMDG þ solution, we recorded currents using step voltages from two holding potentials (-60 mV and þ40 mV). We determined G185R-specific currents by measuring the reversal potentials of the currents subtracted from two holding potentials (see Figure 4A and 4B) and corrected for the junction potential. The permeability sequence was Ca 2þ . Sr 2þ . Ba 2þ as calculated (Equation 2; see Materials and Methods) and illustrated in Figure 3E. For divalent cations, we found that the highest conductance was to Ca 2þ , followed by Sr 2þ and Ba 2þ ( Figure 3D). While Ca 2þ , Sr 2þ , and Ba 2þ currents were relatively stable over time, currents mediated by Mn 2þ and Mg 2þ were transient ( Figure 3D), the simplest explanation for this behavior being a block by these two weakly permeant ions. The monovalent permeability was calculated using Equation 1 (see Materials and Methods), yielding a selectivity sequence Li þ . Na þ . K þ . Cs þ ( Figure 3E). Under these conditions, P Mn was insignificant. The cationic permeability sequence ( Figure 3E) of I G185R was similar to L-type voltagegated Ca 2þ channels (VGCCs) (Sather and McCleskey 2003), but I G185R was less Ca 2þ -selective (P Ca /P Na of approximately 10) than VGCCs (P Ca /P Na of approximately 1,000). Single i G185R channels were not observed in cell-attached patches. Analysis of membrane current noise at À100 mV predicted a single-channel chord conductance of 0.4 6 0.1 pS (n = 5; unpublished data), too small to be observed under most patch-clamp conditions.
Using the Ca 2þ indicator dye Fura-2, we demonstrated G185R-mediated Ca 2þ influx by monitoring intracellular Ca 2þ levels in response to an elevation of [Ca 2þ ] o ( Figure 3F) (Bakowski and Parekh 2000). The halfmaximal activation voltages (V 1/2 ) were À33 mV and À10 mV for control and isotonic Ca 2þ solutions, respectively ( Figure  4B). The voltage-dependence of I G185R was Ca 2þ -independent, since the Na þ and Li þ currents in nominal [Ca 2þ ] o also exhibited a similar voltage dependence. By contrast, I DMT1 lacked this voltage dependence ( Figure 4B). I G185R was not enhanced under low-divalent conditions (less than 10 nM), nor was it blocked by antagonists of known Ca 2þ -permeant channels. In particular, the current was not blocked by ruthenium red (RR), Ca 2þ -release activated Ca 2þ channel (CRAC) blockers SKF96365 and 2-APB (Kozak et al. 2002;Prakriya and Lewis 2002) (Figure 4C), or the L-type VGCC blocker nifedepine (10 lM). Divalent cations, including DMT1 substrates (Cd 2þ , Ni 2þ , Co 2þ ), inhibited I G185R . La 3þ (1 mM; Figure 4C) and Cd 2þ (1 mM) blocked I G185R in a similar voltage-dependent manner ( Figure 4D). Thus, I G185R is distinct from known Ca 2þ -permeant channels such as VGCCs, transient receptor potentials (TRPs), and CRAC currents, based on its current-voltage (I-V) relation, kinetics, permeation properties, and pharmacological sensitivity.
To investigate whether G185R-induced Ca 2þ permeability might play a physiological role in the mutant animals, we recorded from intestinal enterocytes isolated from both wildtype and homozygous mk mice. We studied cells from the proximal 1 cm of the mouse duodenum, where DMT1 expression is highest and iron absorption is maximal (Gunshin et al. 1997;Canonne-Hergaux et al. 1999). Because DMT1 expression is very low in iron-replete, wild-type mice, but induced in iron-deficient mice (Canonne-Hergaux et al. 1999), we isolated enterocytes from mice that had been made iron-deficient by prolonged feeding of an iron-deficient diet, and confirmed DMT1 induction by Western blotting using a  [Ca 2þ ] o were dependent on holding potential before the voltage ramps (À140 to À120 mV shown). For clarity, only the first 20 ms of the 4 s-long holding potential is shown. (B) Voltage dependence of I G185R in control solution and 105 mM [Ca 2þ ] o . I DMT1 (dotted line) exhibited no dependence on the holding potential. Abbreviations: V 1/2 , half activation voltage. j, slope factor. (C and D) Sensitivity of I G185R to various pharmacological agents and cation channel blockers. I G185R was relatively insensitive to RR, 2-APB, or SKF96365, but was blocked by 1mM La 3þ or Cd 2þ (D). DOI: 10.1371/journal.pbio.0020050.g004 DMT1-specific antibody (unpublished data). We were able to record I DMT1 -like currents in mature enterocytes that stained positive for alkaline phosphatase (I . 80 pA at À130mV, n = 7 out of 20 cells; representative data shown in Figure 5A and 5B).
Mice homozygous for the mk mutation express large amounts of G185R DMT1 protein in the duodenum. Although much of it is mislocalized to the cytoplasm (Canonne-Hergaux et al. 2000), we expected that some would be present in the plasma membrane. Accordingly, and in contrast with wild-type enterocytes, we recorded a large, constitutive inward current in most mature mk enterocytes (n= 6 out of 8 cells; Figure 5C and 5D), which displayed the same conductance as seen in G185R-transfected cells. The I-V relationship, step current response, dependence on holding potential, ion selectivity and insensitivity to RR, and SKF96365 or 2-APB were indistinguishable from those of transfected I G185R . Furthermore, H þ inhibited the I G185R -like current in mk enterocytes, and the H þ /Mn 2þ -induced DMT1like current at pH 4.2 ( Figure 5C and 5D) was insensitive to holding potential, as observed in transfected cells. Based on these observations, we conclude that the major current observed in mk enterocytes was I G185R . Although our preparation did not allow us to distinguish apical versus basolateral localization, the large size of the current in mk cells was consistent with plasma membrane localization of G185R protein.

Discussion
We conclude that expression of G185R in transfected cells and in vivo in mk mice is associated with the appearance of a novel Ca 2þ permeation pathway that has the properties of a Ca 2þ channel. One interpretation is that a Ca 2þ channel pathway through the DMT1 protein is exposed or augmented by the G185R mutation. Another possibility is that Ca 2þ conduction occurs through an associated Ca 2þ -permeable protein. We favor the first possibility because the Ca 2þ conductance has been observed in diverse cell lines expressing G185R DMT1 (CHO-K1, HEK293T, and HEK-On cell lines) and in mk enterocytes. A putative associated protein, if present in these different cell types, would have to be activated in a G185R-dependent manner. We did not find evidence of an associated protein when we immunoprecipitated wild-type or G185R DMT1 from transfected CHO-K1 cells (unpublished data). Furthermore, a distinct DMT1 mutant, G185K, also displayed Ca 2þ permeability, but this mutant was less selective for Ca 2þ over Na þ (unpublished data).
G185R mutations have occurred at least three times in rodents, which suggests that G185R not only inactivates DMT1, but may confer an unknown selective advantage. Because it has arisen in inbred colonies, the postulated selective advantage must either make the animals more viable than other DMT1 mutants with impaired iron transport or more likely to be noticed by those managing the animal colonies. In parallel with these studies, we have generated knockout mice homozygous for a null DMT1 allele (Dmt1 -/-; H. Gunshin and N. C. Andrews, personal communication). Although detailed phenotypic characterization has not yet been completed, we have noted that Dmt1 -/mice invariably die by the end of the first week of life, in contrast to mk/mk mice, which are poorly viable but can survive for more than a year (H. Gunshin and N. C. Andrews, personal communication). This suggests that the small amount of residual function of G185R DMT1, perhaps in combination with its gain-offunction Ca 2þ conductance, contributes to viability.
Two previous studies support the notion that the gain-offunction reported here is an advantage. Elevated intracellular [Ca 2þ ] has been reported to increase nontransferrin-bound iron uptake through an undefined transport system that has characteristics distinct from DMT1 (Kaplan et al. 1991). This Figure 5. DMT1-Like and G185R-Like Currents in Enterocytes Isolated from Wild-Type and mk/mk Mice, Respectively (A) Enterocyte currents isolated from an iron-deficient wild-type mouse (ÀFe). Reducing bath pH (140 mM NaCl) induced a slowly desensitizing inward current that was further enhanced by addition of Mn 2þ . (B) Both proton and H þ /Mn 2þ currents were inwardly rectifying. (C and D) An mk enterocyte expressed a large constitutive inward current in control bath solution. Reducing the bath pH (140 mM NaCl) first inhibited and then activated another inward current insensitive to the holding potential. This slowly-desensitizing current displayed a less steeply rectifying I-V as shown in (D). DOI: 10.1371/journal.pbio.0020050.g005 might ameliorate the iron-transport defect caused by inactivation of DMT1, either in the intestine or in erythroid precursors. The transferrin cycle is essential for iron uptake by erythroid precursor cells (Levy et al. 1999), and DMT1 mediates at least some of the transfer of iron from transferrin cycle endosomes to the cytoplasm Gruenheid et al. 1999;Touret et al. 2003). Elevated [Ca 2þ ] i has been reported to accelerate iron uptake through the transferrin cycle, apparently through activation of protein kinase C (Ci et al. 2003). Thus, the influx of Ca 2þ might potentiate the residual DMT1 iron-transport activity. Accordingly, 55 Fe uptake by mk/mk reticulocytes has been reported to be approximately 45% of the level observed in wild-type reticulocytes (Canonne-Hergaux et al. 2001), higher than expected for a severe loss-of-function mutation.
In summary, we have found that a single point mutation (G185R) in a 12-TM transporter protein conferred new Ca 2þselective permeability. Previous studies have suggested that channels, pumps, and transporters may share some common mechanisms for ion translocation (Gadsby et al. 1993;Fairman et al. 1995;Cammack and Schwartz 1996; for review see references in Lester and Dougherty 1998;Nelson et al. 2002). The ''channel mode'' has been proposed to explain the ''drive-slip'' mechanism as part of the transport cycle. In this sense, wild-type DMT1 may simply be a proton channel with limited permeability for certain divalent metal ions. By mutating a single residue, G185R, it becomes an unambiguously Ca 2þ -permeant ion channel. Our findings may add new insight into mechanisms of Ca 2þ entry and transporter function. The notion that the 12-TM proteins can be ion channels may inform the search for candidate Ca 2þ and/or cationic channels and facilitate the molecular characterization of many unidentified native conductances.
We initiated these studies to investigate why a unique DMT1 mutation, G185R, has occurred independently at least twice in mice and once in rats (Fleming et al. 1997. The multiple occurrences of this spontaneous mutation suggested that it might confer some type of selective advantage. We speculate that the proposed Ca 2þ entry gain of function helps to account for this remarkable pattern of remutation. Further investigation of this hypothesis will require direct and detailed comparison of DMT1-null and mk mice.

Materials and Methods
Molecular biology. The DMT1 cDNA used in this study was derived from one of four alternatively-spliced DMT1 gene transcripts. The G185R mutation was generated by using M13 phage and the oligonucleotide GTCCCCCTGTGGGGCCGAGTCCTCATCACCA. Wild-type DMT1 and the G185R mutant were tagged with a Cterminal FLAG epitope and subcloned into pTracer-CMV2 (Invitrogen, Carlsbad, California, United States). CHO-K1 or HEK293T cells transiently transfected with DMT1 and G185R were used for the 55 Fe uptake assay and Western blot analysis. To obtain a stable G185Rexpressing cell line, the G185R-encoding DMT1 gene was subcloned into pRevTRE (Clontech, Palo Alto, California, United States), a retroviral vector that drives expression from a Tet-responsive element. All constructs were confirmed by sequencing. DMT1 Western blot analyses were performed with an anti-FLAG M2 monoclonal antibody (Sigma, St. Louis, Missouri, United States) and, in some cases, with a goat polyclonal antibody raised against human DMT1 (Santa Cruz Biotechnology, Santa Cruz, California, United States).
The permeability to monovalent cations (relative to P Na ) was estimated according to Equation 1 from the shift in E rev upon replacing [Na þ ] o in nominally Ca 2þ -free bath solution (150 mM XCl, 20 mM HEPES, 10 mM glucose [pH 7.4]]), where X þ was Na þ , K þ , Cs þ , or Li þ . For the permeability to divalent cations (relative to P Na ), biionic conditions were used; Y 2þ was Ca 2þ , Ba 2þ , or Sr 2þ (Equation 2). The internal pipette solution contained 100 mM Na-gluconate, 10 mM NaCl, 10 mM EGTA, 20 mM HEPES-Na (pH 7.4 adjusted with NaOH, [Na þ ] total = 140). The external solution was 140 mM NMDG-Cl, 10 mM Y 2þ Cl 2 , 20 mM HEPES (pH 7.4 adjusted with HCl). The permeability ratios of cations were estimated from the following equations (Lewis 1979): where R, T, F, V, and c are, respectively, the gas constant, absolute temperature, Faraday constant, E rev , and activity coefficient. The liquid junction potentials were measured and corrected as described by Neher (1992). Uptake assay. The assay buffer contained 25 mM Tris, 25 mM MES, 140 mM NaCl, 5.4 mM KCl, 5 mM glucose, 1.8 mM CaCl 2 , 0.8 mM MgCl 2 . Ascorbic acid was adjusted to 1 mM and the pH was adjusted to 5.8. Most assays were performed with 20 lM Fe 2þ at pH 5.8 unless otherwise indicated. A 50-fold 55 Fe stock was made immediately before the assay with 1 mM 55 Fe (with a 1:20 molar ratio for 55 FeCl 3 and FeSO 4 ) and 50 mM nitrilotriacetic acid. About 30 h after transient transfection, CHO-K1 or HEK293T cells were washed and harvested with PBS (for CHO-K1 cells, trypsin treatment was required). Cells were resuspended in glass test tubes at 0.5-1 million/ml in 490 ll assay buffer at 308C. The reaction was started by adding 10 ll of 55 Fe stock and stopped at 4, 8, and 16 min by quickly filtering the reaction mix on a nitrocellulose filter (HAWP02500; Millipore, Billerica, Massachusetts, United States). Filters were washed twice with 2 ml of assay buffer, dried, and radioactivity counted by liquid scintillation spectrometry.
Calcium imaging. Cells were loaded with 2 lM Fura-2 AM in culture medium at 378C for 30 min. Low levels of G185R protein were expressed in the absence of doxycycline in G185R HEK-On cells (Western blotting; unpublished data). Therefore, doxycycline-treated HEK-On cells not expressing DMT1 were used as controls in imaging experiments. We recorded Fura-2 ratios (F340/F380) on an Ultra-VIEW imaging system (Olympus, Tokyo, Japan). A standard curve for Fura-2 ratio versus [Ca 2þ ] was constructed according to Grynkiewicz et al. (1985).
Isolation of enterocytes. Homozygous mk mice (Fleming et al. 1997) were housed in the barrier facility at Children's Hospital (Boston, Massachusetts, United States). Husbandry and use were according to protocols approved by the Animal Care and Use Committee. Wildtype iron-deficient mice were provided by J.-J. Chen (Massachusetts Institute of Technology, Cambridge, Massachusetts, United States). Mouse enterocytes were isolated using a modified protocol provided by Dr. F. Sepulveda (Monaghan et al. 1997). In brief, 1 cm of the proximal duodenum was excised, rinsed with cold PBS, and soaked for 5 min at 378C in a solution containing 7 mM K 2 SO 4 , 44 mM K 2 HPO 4 , 9 mM NaHCO 3 , 15 mM Na 3 Citrate, 10 mM HEPES, and 180 mM glucose (pH 7.4). The tissue was then incubated with gentle shaking for 3 min in a similar solution containing 7 mM K 2 SO 4 , 44 mM K 2 HPO 4 , 9 mM NaHCO 3 , 10 mM HEPES, 180 mM glucose, 1 mM DTT, and 0.2 mM EDTA (pH 7.4). The mucosal cells were gently squeezed from the duodenum with forceps into 5 ml of ice-cold DMEM/F12 medium, pelleted at 800 3 g for 4 min, resuspended in 5 ml of prewarmed DMEM/F12 with 0.5 mg/ml collagenase type 1A, and incubated at 378C for 10 min. Cells isolated by this procedure have been shown previously to be primarily of villus origin and hence are mature enterocytes. We confirmed this by alkaline phosphatase staining. Diluted cells were filtered through a 40-lm nylon cell mesh (BD Biosciences, Palo Alto, California, United States). The cells were then washed with DMEM/F12, resuspended in 20 ml of ice-cold DMEM and kept at 48C. They were plated on coverslips coated with Cell-Tak TM (BD Biosciences) and maintained on ice before patchclamp recording at room temperature.
Data analysis. Group data are presented as mean 6 SEM. Statistical comparisons were made using analysis of variance and the t-test with Bonferroni correction. A two-tailed value of p , 0.05 was taken to be statistically significant. Figure S1. CHO-K1 Cells Express an Endogenous Proton-Activated Chloride Channel (A) Anion dependence of pH-induced response in a DMT1-expressing cell. Outward current usually appears later than the inward current. (B) Currents generated in response to a voltage ramp. (C) pH-induced outwardly rectifying current in a nontransfected CHO-K1 cell. A similar current was seen also in HEK293T and HEK-On cells, with properties similar to the cloned ClC-7 channel (Diewald et al. 2002). This current exhibits the same anion dependence as in (A) (data not shown). We attributed the outward currents shown in (A) and (B) to this endogenous Clcurrent. Therefore, for our recordings on DMT1, SO 4 2was usually used to replace most of the Cl -([Cl -] o = 5 mM) for all low-pH bath solutions. Found at DOI: 10.1371/journal.pbio.0020050.sg001 (718 KB PDF). Figure S2. Time-and Voltage-Dependent Kinetics of H þ /Mn 2þ Current of DMT1 Whole-cell currents were generated by voltage steps from À140 to þ80 mV in 20 mV steps, 400 ms. The interval between steps was 1,000 ms. Found at DOI: 10.1371/journal.pbio.0020050.sg002 (1 MB PDF). Figure S3. Na þ -Dependence of DMT1 H þ and H þ /Mn 2þ Currents Replacement of extracellular Na þ by NMDG þ slightly increased the proton current (approximately 20%) and this was further augmented by adding 300 lM Mn 2þ . The concentrations used were Na þ and NMDG þ , 140 mM, (pH 4.2); Mn 2þ , 300 lM. Found at DOI: 10.1371/ journal.pbio.0020050.sg003 (141 KB PDF).