RESUMO
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 Ca2+ 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 (Drozdzyk 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.
Assuntos
Multimerização Proteica , Feminino , Humanos , Canais de Cálcio/metabolismo , Canais de Cálcio/química , Canais de Cálcio/genética , Células HEK293 , Placenta/metabolismo , Conformação ProteicaRESUMO
Agonist binding in ligand-gated ion channels is coupled to structural rearrangements around the binding site, followed by the opening of the channel pore. In this process, agonist efficacy describes the equilibrium between open and closed conformations in a fully ligand-bound state. Calcium-activated chloride channels in the TMEM16 family are important sensors of intracellular calcium signals and are targets for pharmacological modulators, yet a mechanistic understanding of agonist efficacy has remained elusive. Using a combination of cryo-electron microscopy, electrophysiology, and autocorrelation analysis, we now show that agonist efficacy in the ligand-gated channel TMEM16A is dictated by the conformation of the pore-lining helix α6 around the Ca2+ -binding site. The closure of the binding site, which involves the formation of a π-helix below a hinge region in α6, appears to be coupled to the opening of the inner pore gate, thereby governing the channel's open probability and conductance. Our results provide a mechanism for agonist binding and efficacy and a structural basis for the design of potentiators and partial agonists in the TMEM16 family.
Assuntos
Canais de Cloreto , Ativação do Canal Iônico , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Anoctamina-1/genética , Anoctamina-1/química , Anoctamina-1/metabolismo , Ligantes , Microscopia Crioeletrônica , Sítios de Ligação , Cálcio/metabolismoRESUMO
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.
Assuntos
Antiporters , Bicarbonatos , Humanos , Animais , Camundongos , Antiporters/metabolismo , Bicarbonatos/metabolismo , Cloretos/metabolismo , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Transportadores de Sulfato/genéticaRESUMO
The transport of transition metal ions by members of the SLC11/NRAMP family constitutes a ubiquitous mechanism for the uptake of Fe2+ and Mn2+ 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 Mg2+ uptake in prokaryotes and another the transport of Al3+ into plant cells. Our previous work on the SLC11 transporter from Eggerthella lenta revealed the basis for its Mg2+ selectivity (Ramanadane et al., 2022). Here, we have addressed the structural and functional properties of a putative Al3+ transporter from Setaria italica. We show that the protein transports diverse divalent metal ions and binds the trivalent ions Al3+ and Ga3+, 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.
Assuntos
Alumínio , Proteínas de Membrana Transportadoras , Alumínio/metabolismo , Microscopia Crioeletrônica , Transporte Biológico , Proteínas de Membrana Transportadoras/metabolismo , Sítios de LigaçãoRESUMO
A central regulatory mechanism of iron homeostasis in humans involves ferroportin (FPN), the sole cellular iron exporter, and the peptide hormone hepcidin, which inhibits Fe2+ 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 ß-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.
Assuntos
Proteínas de Transporte de Cátions , Hepcidinas , Humanos , Hepcidinas/metabolismo , Microscopia Crioeletrônica , Ferro/metabolismo , Proteínas de Transporte de Cátions/metabolismoRESUMO
Volume-regulated anion channels (VRACs) participate in the cellular response to osmotic swelling. These membrane proteins consist of heteromeric assemblies of LRRC8 subunits, whose compositions determine permeation properties. Although structures of the obligatory LRRC8A, also referred to as SWELL1, have previously defined the architecture of VRACs, the organization of heteromeric channels has remained elusive. Here we have addressed this question by the structural characterization of murine LRRC8A/C channels. Like LRRC8A, these proteins assemble as hexamers. Despite 12 possible arrangements, we find a predominant organization with an A:C ratio of two. In this assembly, four LRRC8A subunits cluster in their preferred conformation observed in homomers, as pairs of closely interacting proteins that stabilize a closed state of the channel. In contrast, the two interacting LRRC8C subunits show a larger flexibility, underlining their role in the destabilization of the tightly packed A subunits, thereby enhancing the activation properties of the protein.
Assuntos
Proteínas de Membrana , Camundongos , Animais , Proteínas de Membrana/metabolismo , Ânions/metabolismoRESUMO
TMEM16F, a member of the conserved TMEM16 family, plays a central role in the initiation of blood coagulation and the fusion of trophoblasts. The protein mediates passive ion and lipid transport in response to an increase in intracellular Ca2+. However, the mechanism of how the protein facilitates both processes has remained elusive. Here we investigate the basis for TMEM16F activation. In a screen of residues lining the proposed site of conduction, we identify mutants with strongly activating phenotype. Structures of these mutants determined herein by cryo-electron microscopy show major rearrangements leading to the exposure of hydrophilic patches to the membrane, whose distortion facilitates lipid diffusion. The concomitant opening of a pore promotes ion conduction in the same protein conformation. Our work has revealed a mechanism that is distinct for this branch of the family and that will aid the development of a specific pharmacology for a promising drug target.
Assuntos
Anoctaminas , Proteínas de Transferência de Fosfolipídeos , Anoctaminas/genética , Anoctaminas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Microscopia Crioeletrônica , Conformação Proteica , Lipídeos , Cálcio/metabolismoRESUMO
TMEM16A, a calcium-activated chloride channel involved in multiple cellular processes, is a proposed target for diseases such as hypertension, asthma, and cystic fibrosis. Despite these therapeutic promises, its pharmacology remains poorly understood. Here, we present a cryo-EM structure of TMEM16A in complex with the channel blocker 1PBC and a detailed functional analysis of its inhibition mechanism. A pocket located external to the neck region of the hourglass-shaped pore is responsible for open-channel block by 1PBC and presumably also by its structural analogs. The binding of the blocker stabilizes an open-like conformation of the channel that involves a rearrangement of several pore helices. The expansion of the outer pore enhances blocker sensitivity and enables 1PBC to bind at a site within the transmembrane electric field. Our results define the mechanism of inhibition and gating and will facilitate the design of new, potent TMEM16A modulators.
Assuntos
Cálcio , Canais de Cloreto , Anoctamina-1/genética , Anoctamina-1/metabolismo , Cálcio/metabolismo , Canais de Cloreto/metabolismoRESUMO
Members of the LRRC8 family participate in the response of vertebrate cells to osmotic changes in their environment. These proteins form heteromeric assemblies composed of the obligatory subunit LRRC8A and at least one of the other four homologs, which together function as anion-selective channels with distinct properties that are activated upon cell-swelling. The hexameric complexes share a conserved architecture consisting of a membrane-inserted pore domain with an ion permeation path located at the axis of symmetry and cytoplasmic leucine-rich repeat domains that regulate the open probability of the channel. In this review, we summarize the current understanding of structure-function relationships of these unusual ion channels whose mechanisms are, despite their large physiological importance, still poorly understood.
Assuntos
Canais Iônicos , Proteínas de Membrana , Ânions/metabolismo , Tamanho Celular , Canais Iônicos/metabolismo , Proteínas de Membrana/química , Domínios ProteicosRESUMO
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 Ca2+ and Mg2+, 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 Mg2+. The protein transports Mg2+ and Mn2+ but not Ca2+ 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.
Assuntos
Bactérias/genética , Proteínas de Transporte de Cátions/genética , Magnésio/metabolismo , Bactérias/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismoRESUMO
The SLC11/NRAMP proteins constitute a conserved family of metal ion transporters that are expressed in all kingdoms of life. In humans, the two paralogs DMT1 and NRMP1 play an important role in iron homeostasis and the defense against pathogens. SLC11 transporters have evolved an exquisite selectivity for transition metal ions, which facilitates their efficient transport from a large background of Ca2+ and Mg2+. This is accomplished by the evolution of a conserved binding site, which contains besides promiscuous hard ligands, a methionine acting as soft ligand that exclusively coordinates transition metals and thus contributes to the exclusion of alkaline earth metal ions. This site is altered in a branch of prokaryotic family members, which are capable of transporting Mg2+, where the removal of the coordinating methionine and the accompanying expansion of the binding pocket captures this small ion in a hydrated state. The disposition of titratable residues in H+-coupled transition metal ion transporters, that are absent in uncoupled Mg2+ transporters, sheds light on potential coupling mechanisms. In combination, the discussed work has revealed detailed insight into transition metal ion transport and provides a basis for the development of inhibitors of DMT1 as strategy against iron overload disorders.
RESUMO
Members of the LRRC8 family form heteromeric assemblies, which function as volume-regulated anion channels. These modular proteins consist of a transmembrane pore and cytoplasmic leucine-rich repeat (LRR) domains. Despite their known molecular architecture, the mechanism of activation and the role of the LRR domains in this process has remained elusive. Here we address this question by generating synthetic nanobodies, termed sybodies, which target the LRR domain of the obligatory subunit LRRC8A. We use these binders to investigate their interaction with homomeric LRRC8A channels by cryo-electron microscopy and the consequent effect on channel activation by electrophysiology. The five identified sybodies either inhibit or enhance activity by binding to distinct epitopes of the LRR domain, thereby altering channel conformations. In combination, our work provides a set of specific modulators of LRRC8 proteins and reveals the role of their cytoplasmic domains as regulators of channel activity by allosteric mechanisms.
Assuntos
Epitopos/química , Canais Iônicos/química , Proteínas de Membrana/química , Anticorpos de Domínio Único/química , Regulação Alostérica , Sítio Alostérico , Sequência de Aminoácidos , Animais , Clonagem Molecular , Epitopos/genética , Epitopos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , Transporte de Íons , Cinética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo , Especificidade por SubstratoRESUMO
The Tweety homologs (TTYHs) are members of a conserved family of eukaryotic membrane proteins that are abundant in the brain. The three human paralogs were assigned to function as anion channels that are either activated by Ca2+ or cell swelling. To uncover their unknown architecture and its relationship to function, we have determined the structures of human TTYH1-3 by cryo-electron microscopy. All structures display equivalent features of a dimeric membrane protein that contains five transmembrane segments and an extended extracellular domain. As none of the proteins shows attributes reminiscent of an anion channel, we revisited functional experiments and did not find any indication of ion conduction. Instead, we find density in an extended hydrophobic pocket contained in the extracellular domain that emerges from the lipid bilayer, which suggests a role of TTYH proteins in the interaction with lipid-like compounds residing in the membrane.
Assuntos
Canais de Cloreto/ultraestrutura , Microscopia Crioeletrônica/métodos , Proteínas de Membrana/ultraestrutura , Proteínas de Neoplasias/ultraestrutura , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Humanos , Canais Iônicos/química , Canais Iônicos/metabolismo , Canais Iônicos/ultraestrutura , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
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.
Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Caspases/metabolismo , Microscopia Crioeletrônica/métodos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Caspase 3 , Membrana Celular/metabolismo , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/genética , Membranas/metabolismo , Modelos Moleculares , Fosfatidilserinas/metabolismo , RatosRESUMO
The binding of cytoplasmic Ca2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore. By combining mutagenesis, electrophysiology, and cryo-electron microscopy, we identified three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate. Mutation of each of these residues increases the potency of Ca2+ and results in pronounced basal activity. The structure of an activating mutant shows a conformational change of an α-helix that contributes to Ca2+ binding as a likely cause for the basal activity. Although not in physical contact, the three residues are functionally coupled to collectively contribute to the stabilization of the gate in the closed conformation of the pore, thus explaining the low open probability of the channel in the absence of Ca2+.
Assuntos
Anoctamina-1/metabolismo , Cálcio/metabolismo , Ativação do Canal Iônico , Proteínas de Neoplasias/metabolismo , Anoctamina-1/genética , Anoctamina-1/ultraestrutura , Sítios de Ligação/genética , Cátions Bivalentes/metabolismo , Cloretos/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutagênese , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/ultraestrutura , Ligação Proteica , Conformação Proteica em alfa-HéliceRESUMO
The anion channel TMEM16A is activated by intracellular Ca2+ in a highly cooperative process. By combining electrophysiology and autocorrelation analysis, we investigated the mechanism of channel activation and the concurrent rearrangement of the gate in the narrow part of the pore. Features in the fluctuation characteristics of steady-state current indicate the sampling of intermediate conformations that are successively occupied during gating. The initial step is related to conformational changes induced by Ca2+ binding, which is ensued by rearrangements that open the pore. Mutations in the gate shift the equilibrium of transitions in a manner consistent with a progressive destabilization of this region during pore opening. We come up with a mechanism of channel activation where the binding of Ca2+ induces conformational changes in the protein that, in a sequential manner, propagate from the binding site and couple to the gate in the narrow pore to allow ion permeation.
Assuntos
Anoctamina-1/metabolismo , Cálcio/metabolismo , Ativação do Canal Iônico , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Regulação Alostérica , Anoctamina-1/genética , Anoctamina-1/ultraestrutura , Sítios de Ligação/genética , Cátions Bivalentes/metabolismo , Cloretos/metabolismo , Células HEK293 , Humanos , Cinética , Método de Monte Carlo , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/ultraestrutura , Técnicas de Patch-Clamp , Distribuição de Poisson , Ligação Proteica/genética , Conformação Proteica em alfa-HéliceRESUMO
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.
Assuntos
Canais de Cálcio/metabolismo , Microscopia Crioeletrônica , Glicoproteínas de Membrana/metabolismo , Placenta/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/ultraestrutura , Feminino , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestrutura , Lipídeos de Membrana/metabolismo , Potenciais da Membrana , Modelos Moleculares , Placenta/ultraestrutura , Gravidez , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Cystic fibrosis is a hereditary disease that originates from mutations in the epithelial chloride channel CFTR. Whereas established therapies for the treatment of cystic fibrosis target CFTR to repair its function, alternative therapeutic strategies aim for the restoration of chloride transport by the activation of other chloride transport proteins such as TMEM16A or SLC26A9 or by the application of synthetic anionophores. TMEM16A is an anion-selective channel that is activated by the binding of Ca2+ from the cytoplasm. Pharmacological efforts aim for the increase of its open probability at resting Ca2+ concentrations. SLC26 is an uncoupled chloride transporter, which shuttles chloride across the membrane by an alternate-access mechanism. Its activation requires its mobilization from intracellular stores. Finally, anionophores are small synthetic molecules that bind chloride to form lipid-soluble complexes, which shuttle the anion across the membrane. All three approaches are currently pursued and have provided promising initial results.
Assuntos
Antiporters/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Moduladores de Transporte de Membrana/farmacologia , Anoctamina-1/genética , Anoctamina-1/metabolismo , Antiporters/genética , Antiporters/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Medicina Molecular/métodos , Medicina Molecular/tendências , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismoRESUMO
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.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Metais/metabolismo , Tioureia/farmacologia , Sítios de Ligação , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Transporte de Íons/efeitos dos fármacos , Cinética , Modelos Moleculares , Mutação/genética , Tioureia/químicaRESUMO
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.