DMT1 (divalent metal transporter; also known as SLC11A2, DCT1 or Nramp2) is responsible for ferrous iron uptake in the duodenum, iron exit from endosomes during the transferrin cycle and some transferrin-independent iron uptake in many cells. Four protein isoforms differ by starting in exon 1A or 2 and ending with alternative peptides encoded by mRNA that contains or lacks an IRE (iron responsive element; +/-IRE). We have compared 1A/+IRE and 2/-IRE DMT1 during regulated ectopic expression. HEK-293-F (human embryonic kidney-293-fast growing variant) cells were stably transfected with each construct expressed from a tetracycline-regulated CMV promoter. Reverse transcriptase-PCR analysis showed that construct expression responded to doxycycline. Immunofluorescence staining of cells, using antibodies specific for DMT1 isoforms, confirmed an increase in expression in the plasma membrane and cytosolic vesicles after doxycycline treatment, but with isoform specific distributions. Immunoblotting also revealed stimulation of expression. Nevertheless, both DMT1 isoforms performed similarly in assays for functional properties based on 54Mn2+ and 59Fe2+ uptake. Mn incorporation after doxycycline treatment was approximately 10-fold greater than that of untreated cells, while expression in the untreated cells was approximately 5-fold greater than in the untransfected cells. Uptake of Mn depended on addition of doxycycline, with half maximal response at approximately 1 nM doxycycline. Doxycycline-stimulated Mn and Fe uptake was linear with time for 10 min but not over longer periods. Transport exhibited a pH optimum at approximately 5.5 and dependence on incubation temperature and Mn or Fe concentration. The new cell lines should prove useful for research on metal homoeostasis, toxicological studies and efforts to identify distinctive properties of the isoforms.

It has been shown recently that polypeptide toxins that modulate the gating properties of voltage-sensitive cation channels are able to bind to phospholipid membranes, leading to the suggestion that these toxins are able to access a channel-binding site that remains membrane-restricted (Lee, S.-Y., and MacKinnon, R. (2004) Nature 430, 232-235). We therefore examined the ability of anthopleurin B (ApB), a sea anemone toxin that selectively modifies inactivation kinetics of Na V 1.x channels, and ProTx-II, a spider toxin that modifies activation kinetics of the same channels, to bind to liposomes. Whereas ProTx-II can be quantitatively depleted from solution upon incubation with phosphatidylcholine/ phosphatidylserine liposomes, ApB displays no discernible phospholipid binding activity. We therefore examined the activities of structurally unrelated site 3 and site 4 toxins derived from Leiurus and Centruroides venoms, respectively, in the same assay. Like ApB, the site 3 toxin LqqV shows no lipid binding activity, whereas the site 4 toxin Centruroides toxin II, like ProTx-II, is completely bound. We conclude that toxins that modify inactivation kinetics via binding to Na V 1.x site 3 lack the ability to bind phospholipids, whereas site 4 toxins, which modify activation, have this activity. This inherent difference suggests that the conformation of domain II more closely resembles that of the K V AP channel than does the conformation of domain IV.Chemically diverse neurotoxins have historically been of great value in defining the overall architecture of voltage-dependent Na ϩ (Na V ) 1 and K ϩ (K V ) channels. The pores of such channels have been mapped by analysis of their interactions with conotoxins (Na V ) and a variety of polypeptides from scorpion venoms, such as charybdotoxin and agitoxins (1-4). More recently, regions of these channels involved in gating have been identified by using gating modifier toxins derived from scorpion (5), sea anemone (6), and spider (7-9) venoms. Most interestingly, gating modifier toxins appear to interact with the same channel region, designated the S3-S4 linker, irrespective of the type of channel being studied (10).Gating modifier toxins can also be important probes for the accessibility of defined regions of a given channel. Very recently, the MacKinnon laboratory has employed a novel spider toxin, VSTX, to probe the accessibility of defined regions of the voltage sensor of the archaebacterial K V AP channel (9). The resulting data were interpreted in the context of a channel three-dimensional structure (11) in which the K V AP S3-S4 linker was located either near the cytoplasmic surface or buried within the bilayer, depending on whether the channel was in the resting or activated state (9). The observation that VSTX possessed phospholipid binding activity (12) provided a potential explanation for the ability of this toxin to modify channels via interaction with sequences that were never exposed at the extracellular surface.To understand the extent to which this model could ...

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Voltage-gated Na؉ channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Na v and Ca v , but not K v , channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Na v 1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly K v channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Na v 1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Na v channels with potential to shed new light on this process.

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