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The CALHM proteins constitute a family of large pore channels that contains six closely related paralogs in humans. Two family members, CALHM1 and 3, have been associated with the release of ATP during taste sensation. Both proteins form heteromeric channels that activate at positive potential and decreased extracellular Ca 2+ concentration. Although the structures of several family members displayed large oligomeric organizations of different size, their function has in most cases remained elusive. Our previous study has identified the paralogs CALHM2, 4 and, 6 to be highly expressed in the placenta and defined their structural properties as membrane proteins exhibiting features of large pore channels with unknown activation properties (Drożdżyk et al., 2020). Here, we investigated whether these placental paralogs would form heteromers and characterized heteromeric complexes consisting of CALHM2 and CALHM4 subunits using specific binders as fiducial markers. Both proteins assemble with different stoichiometries with the largest population containing CALHM2 as the predominant component. In these oligomers, the subunits segregate and reside in their preferred conformation found in homomeric channels. Our study has thus revealed the properties that govern the formation of CALHM heteromers in a process of potential relevance in a cellular context., Competing Interests: KD, MP, RD No competing interests declared, (© 2024, Drożdżyk et al.)

Members of the SLC26 family constitute a conserved class of anion transport proteins, which encompasses uncoupled transporters with channel-like properties, coupled exchangers and motor proteins. Among the 10 functional paralogs in humans, several participate in the secretion of bicarbonate in exchange with chloride and thus play an important role in maintaining pH homeostasis. Previously, we have elucidated the structure of murine SLC26A9 and defined its function as an uncoupled chloride transporter (Walter et al., 2019). Here we have determined the structure of the closely related human transporter SLC26A6 and characterized it as a coupled exchanger of chloride with bicarbonate and presumably also oxalate. The structure defines an inward-facing conformation of the protein that generally resembles known structures of SLC26A9. The altered anion selectivity between both paralogs is a consequence of a remodeled ion binding site located in the center of a mobile unit of the membrane-inserted domain, which also accounts for differences in the coupling mechanism., Competing Interests: DT, CB, SB, MS, RD No competing interests declared, (© 2023, Tippett et al.)

The transport of transition metal ions by members of the SLC11/NRAMP family constitutes a ubiquitous mechanism for the uptake of Fe 2+ and Mn 2+ across all kingdoms of life. Despite the strong conservation of the family, two of its branches have evolved a distinct substrate preference with one mediating Mg 2+ uptake in prokaryotes and another the transport of Al 3+ into plant cells. Our previous work on the SLC11 transporter from Eggerthella lenta revealed the basis for its Mg 2+ selectivity (Ramanadane et al., 2022). Here, we have addressed the structural and functional properties of a putative Al 3+ transporter from Setaria italica . We show that the protein transports diverse divalent metal ions and binds the trivalent ions Al 3+ and Ga 3+ , which are both presumable substrates. Its cryo-electron microscopy (cryo-EM) structure displays an occluded conformation that is closer to an inward- than an outward-facing state, with a binding site that is remodeled to accommodate the increased charge density of its transported substrate., Competing Interests: KR, ML, DM, MS, GR, AU, RD, CM No competing interests declared, (© 2023, Ramanadane et al.)

A central regulatory mechanism of iron homeostasis in humans involves ferroportin (FPN), the sole cellular iron exporter, and the peptide hormone hepcidin, which inhibits Fe 2+ transport and induces internalization and degradation of FPN. Dysregulation of the FPN/hepcidin axis leads to diverse pathological conditions, and consequently, pharmacological compounds that inhibit FPN-mediated iron transport are of high clinical interest. Here, we describe the cryo-electron microscopy structures of human FPN in complex with synthetic nanobodies and vamifeport (VIT-2763), the first clinical-stage oral FPN inhibitor. Vamifeport competes with hepcidin for FPN binding and is currently in clinical development for β-thalassemia and sickle cell disease. The structures display two distinct conformations of FPN, representing outward-facing and occluded states of the transporter. The vamifeport site is located in the center of the protein, where the overlap with hepcidin interactions underlies the competitive relationship between the two molecules. The introduction of point mutations in the binding pocket of vamifeport reduces its affinity to FPN, emphasizing the relevance of the structural data. Together, our study reveals conformational rearrangements of FPN that are of potential relevance for transport, and it provides initial insight into the pharmacological targeting of this unique iron efflux transporter., Competing Interests: EL, ML, KD, CL, RD, CM No competing interests declared, PA P.A. is employee of CSL Vifor and is inventor in patents related to the publication (WO2021013771A1), VM V.M. is employee of CSL Vifor and is inventor in patents related to the publication (WO2017068089A9, WO2017068090, WO2021013771A1, WO2021013772A1, WO2021078889A1, WO2022157185A1), HS H.S. is employee of CSL Vifor, FD F.D. is employee of CSL Vifor and is inventor in patents related to the publication (WO2017068089A9, WO2017068090, WO2021013771A1, WO2021013772A1, WO2021078889A1, WO2022157185A1), (© 2023, Lehmann, Liziczai et al.)

Members of the ubiquitous SLC11/NRAMP family catalyze the uptake of divalent transition metal ions into cells. They have evolved to efficiently select these trace elements from a large pool of Ca 2+ and Mg 2+ , which are both orders of magnitude more abundant, and to concentrate them in the cytoplasm aided by the cotransport of H + serving as energy source. In the present study, we have characterized a member of a distant clade of the family found in prokaryotes, termed NRMTs, that were proposed to function as transporters of Mg 2+ . The protein transports Mg 2+ and Mn 2+ but not Ca 2+ by a mechanism that is not coupled to H + . Structures determined by cryo-EM and X-ray crystallography revealed a generally similar protein architecture compared to classical NRAMPs, with a restructured ion binding site whose increased volume provides suitable interactions with ions that likely have retained much of their hydration shell., Competing Interests: KR, MS, RD, CM No competing interests declared, (© 2022, Ramanadane et al.)

The exposure of the negatively charged lipid phosphatidylserine on the cell surface, catalyzed by lipid scramblases, is an important signal for the clearance of apoptotic cells by macrophages. The protein XKR9 is a member of a conserved family that has been associated with apoptotic lipid scrambling. Here, we describe structures of full-length and caspase-treated XKR9 from Rattus norvegicus in complex with a synthetic nanobody determined by cryo-electron microscopy. The 43 kDa monomeric membrane protein can be divided into two structurally related repeats, each containing four membrane-spanning segments and a helix that is partly inserted into the lipid bilayer. In the full-length protein, the C-terminus interacts with a hydrophobic pocket located at the intracellular side acting as an inhibitor of protein function. Cleavage by caspase-3 at a specific site releases 16 residues of the C-terminus, thus making the pocket accessible to the cytoplasm. Collectively, the work has revealed the unknown architecture of the XKR family and has provided initial insight into its activation by caspases., Competing Interests: MS, CA, MS, RD No competing interests declared, (© 2021, Straub et al.)

The transport of substances across the placenta is essential for the development of the fetus. Here, we were interested in the role of channels of the calcium homeostasis modulator (CALHM) family in the human placenta. By transcript analysis, we found the paralogs CALHM2, 4, and 6 to be highly expressed in this organ and upregulated during trophoblast differentiation. Based on electrophysiology, we observed that activation of these paralogs differs from the voltage- and calcium-gated channel CALHM1. Cryo-EM structures of CALHM4 display decameric and undecameric assemblies with large cylindrical pore, while in CALHM6 a conformational change has converted the pore shape into a conus that narrows at the intracellular side, thus describing distinct functional states of the channel. The pore geometry alters the distribution of lipids, which occupy the cylindrical pore of CALHM4 in a bilayer-like arrangement whereas they have redistributed in the conical pore of CALHM6 with potential functional consequences., Competing Interests: KD, MS, MB, ZJ, DD, CA, RD No competing interests declared, (© 2020, Drożdżyk et al.)

In humans, the divalent metal ion transporter-1 (DMT1) mediates the transport of ferrous iron across the apical membrane of enterocytes. Hence, its inhibition could be beneficial for the treatment of iron overload disorders. Here we characterize the interaction of aromatic bis-isothiourea-substituted compounds with human DMT1 and its prokaryotic homologue EcoDMT. Both transporters are inhibited by a common competitive mechanism with potencies in the low micromolar range. The crystal structure of EcoDMT in complex with a brominated derivative defines the binding of the inhibitor to an extracellular pocket of the transporter in direct contact with residues of the metal ion coordination site, thereby interfering with substrate loading and locking the transporter in its outward-facing state. Mutagenesis and structure-activity relationships further support the observed interaction mode and reveal species-dependent differences between pro- and eukaryotic transporters. Together, our data provide the first detailed mechanistic insight into the pharmacology of SLC11/NRAMP transporters., Competing Interests: CM, JP, MP, JR, MH, RD No competing interests declared, (© 2019, Manatschal et al.)

The epithelial anion transporter SLC26A9 contributes to airway surface hydration and gastric acid production. Colocalizing with CFTR, SLC26A9 has been proposed as a target for the treatment of cystic fibrosis. To provide molecular details of its transport mechanism, we present cryo-EM structures and a functional characterization of murine Slc26a9. These structures define the general architecture of eukaryotic SLC26 family members and reveal an unusual mode of oligomerization which relies predominantly on the cytosolic STAS domain. Our data illustrates conformational transitions of Slc26a9, supporting a rapid alternate-access mechanism which mediates uncoupled chloride transport with negligible bicarbonate or sulfate permeability. The characterization of structure-guided mutants illuminates the properties of the ion transport path, including a selective anion binding site located in the center of a mobile module within the transmembrane domain. This study thus provides a structural foundation for the understanding of the entire SLC26 family and potentially facilitates their therapeutic exploitation., Competing Interests: JW, MS, RD No competing interests declared, (© 2019, Walter et al.)

Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca 2+ -dependent lipid scramblase, its regulation mechanism has remained elusive. Here, we have used cryo-electron microscopy and functional assays to address this question. Ca 2+ -bound and Ca 2+ -free conformations of nhTMEM16 in detergent and lipid nanodiscs illustrate the interactions with its environment and they reveal the conformational changes underlying its activation. In this process, Ca 2+ binding induces a stepwise transition of the catalytic subunit cavity, converting a closed cavity that is shielded from the membrane in the absence of ligand, into a polar furrow that becomes accessible to lipid headgroups in the Ca 2+ -bound state. Additionally, our structures demonstrate how nhTMEM16 distorts the membrane at both entrances of the subunit cavity, thereby decreasing the energy barrier for lipid movement., Competing Interests: VK, VC, LB, GO, RD, CP No competing interests declared, (© 2019, Kalienkova et al.)

The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of murine TMEM16F (mTMEM16F) that underlie its function as a lipid scramblase and an ion channel. The cryo-EM data of mTMEM16F in absence and presence of Ca 2+ define the ligand-free closed conformation of the protein and the structure of a Ca 2+ -bound intermediate. Both conformations resemble their counterparts of the scrambling-incompetent anion channel mTMEM16A, yet with distinct differences in the region of ion and lipid permeation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, both functions appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state., Competing Interests: CA, NL, VC, GO, RD, CP No competing interests declared, (© 2019, Alvadia et al.)

TMEM16A is a ligand-gated anion channel that is activated by intracellular Ca 2+ . This channel comprises two independent pores and closely apposed Ca 2+ binding sites that are contained within each subunit of a homodimeric protein. Previously we characterized the influence of positively charged pore-lining residues on anion conduction (Paulino et al., 2017a). Here, we demonstrate the electrostatic control of permeation by the bound calcium ions in mouse TMEM16A using electrophysiology and Poisson-Boltzmann calculations. The currents of constitutively active mutants lose their outward rectification as a function of Ca 2+ concentration due to the alleviation of energy barriers for anion conduction. This phenomenon originates from Coulombic interactions between the bound Ca 2+ and permeating anions and thus demonstrates that an electrostatic gate imposed by the vacant binding site present in the sterically open pore, is released by Ca 2+ binding to enable an otherwise sub-conductive pore to conduct with full capacity., Competing Interests: AL, RD No competing interests declared, (© 2018, Lam et al.)

The calcium-activated chloride channel TMEM16A is a member of a conserved protein family that comprises ion channels and lipid scramblases. Although the structure of the scramblase nhTMEM16 has defined the architecture of the family, it was unknown how a channel has adapted to cope with its distinct functional properties. Here we have addressed this question by the structure determination of mouse TMEM16A by cryo-electron microscopy and a complementary functional characterization. The protein shows a similar organization to nhTMEM16, except for changes at the site of catalysis. There, the conformation of transmembrane helices constituting a membrane-spanning furrow that provides a path for lipids in scramblases has changed to form an enclosed aqueous pore that is largely shielded from the membrane. Our study thus reveals the structural basis of anion conduction in a TMEM16 channel and it defines the foundation for the diverse functional behavior in the TMEM16 family.