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Chloride is one of the most abundant anions in the human body; it is implicated in several physiological processes such as the transmission of action potentials, transepithelial salt transport, maintenance of cellular homeostasis, regulation of osmotic pressure and intracellular pH, and synaptic transmission. The balance between the extracellular and intracellular chloride concentrations is controlled by the interplay of ion channels and transporters embedded in the cellular membranes. Vesicular members of the CLC chloride protein family (vCLCs) are chloride/proton exchangers expressed in the membrane of the intracellular organelles, where they control vesicular acidification and luminal chloride concentration. It is well known that mutations in CLCs cause bone, kidney, and lysosomal genetic diseases. However, the role of CLC exchangers in neurological disorders is only now emerging with the identification of pathogenic CLCN gene variants in patients with severe neuronal and intellectual dysfunctions. This review will provide an overview of the recent advances in understanding the role of the vesicular CLC chloride/proton exchangers in human pathophysiology.

Ca ²⁺-activated Cl ⁻ channels (CaCCs) are key regulators of numerous physiological functions, ranging from electrolyte secretion in airway epithelia to cellular excitability in sensory neurons and muscle fibers. Recently, TMEM16A (ANO1) and -B were shown to be critical components of CaCCs. It is still unknown whether they are also sufficient to form functional CaCCs, or whether association with other subunits is required. Recent reports suggest that the Ca ²⁺ sensitivity of TMEM16A is mediated by its association with calmodulin, suggesting that functional CaCCs are heteromultimers. To test whether TMEM16A is necessary and sufficient to form functional CaCCs, we expressed, purified, and reconstituted human TMEM16A. The purified protein mediates Ca ²⁺-dependent Cl ⁻ transport with submicromolar sensitivity to Ca ²⁺, consistent with what is seen in patch–clamp experiments. The channel is synergistically gated by Ca ²⁺ and voltage, so that opening is promoted by depolarizing potentials. Mutating two conserved glutamates in the TM6-7 intracellular loop selectively abolishes the Ca ²⁺ dependence of reconstituted TMEM16A, in a manner similar to what was reported for the heterologously expressed channel. Well-characterized CaCC blockers inhibit Cl ⁻ transport with K ᵢs comparable to those measured for native and heterologously expressed CaCCs. Finally, direct physical interactions between calmodulin and TMEM16A could not be detected in copurification experiments or in functional assays. Our results demonstrate that purified TMEM16A is necessary and sufficient to recapitulate the biophysical and pharmacological properties of native and heterologously expressed CaCCs. Our results also show that association of TMEM16A with other proteins, such as calmodulin, is not required for function.

ClC-4 and ClC-5 are members of the CLC gene family 1 , with ClC-5 mutated in Dent's disease 2 , a nephropathy associated with low-molecular-mass proteinuria and eventual renal failure. ClC-5 has been proposed to be an electrically shunting Cl - channel in early endosomes, facilitating intraluminal acidification 3 , 4 . Motivated by the discovery that certain bacterial CLC proteins are secondary active Cl - /H + antiporters 5 , we hypothesized that mammalian CLC proteins might not be classical Cl - ion channels but might exhibit Cl - -coupled proton transport activity. Here we report that ClC-4 and ClC-5 carry a substantial amount of protons across the plasma membrane when activated by positive voltages, as revealed by measurements of pH close to the cell surface. Both proteins are able to extrude protons against their electrochemical gradient, demonstrating secondary active transport. H + , but not Cl - , transport was abolished when a pore glutamate was mutated to alanine (E211A). ClC-0, ClC-2 and ClC-Ka proteins showed no significant proton transport. The muscle channel ClC-1 exhibited a small H + transport that might be physiologically relevant. For ClC-5, we estimated that Cl - and H + transport contribute about equally to the total charge movement, raising the possibility that the coupled Cl - /H + transport of ClC-4 and ClC-5 is of significant magnitude in vivo .

Phospholipid (PL) scramblases disrupt the lipid asymmetry of the plasma membrane, externalizing phosphatidylserine to trigger blood coagulation and mark apoptotic cells. Recently, members of the TMEM16 family of Ca 2+ -gated channels have been shown to be involved in Ca 2+ -dependent scrambling. It is however controversial whether they are scramblases or channels regulating scrambling. Here we show that purified afTMEM16, from Aspergillus fumigatus , is a dual-function protein: it is a Ca 2+ -gated channel, with characteristics of other TMEM16 homologues, and a Ca 2+ -dependent scramblase, with the expected properties of mammalian PL scramblases. Remarkably, we find that a single Ca 2+ site regulates separate transmembrane pathways for ions and lipids. Two other purified TMEM16-channel homologues do not mediate scrambling, suggesting that the family diverged into channels and channel/scramblases. We propose that the spatial separation of the ion and lipid pathways underlies the evolutionary divergence of the TMEM16 family, and that other homologues, such as TMEM16F, might also be dual-function channel/scramblases. TMEM16-channel family members have been shown to be involved in Ca2 + -dependent lipid scrambling, but whether they have intrinsic scramblase activity remains controversial. Malvezzi et al . identify a TMEM16 family member in Aspergillus in which a single Ca2 + -binding site regulates intrinsic channel and scramblase activities.

Variants in and , encoding the neuronal endosomal Cl /H antiporters ClC-3 and ClC-4, are linked to neurodevelopmental disorders with broad phenotypic variability. Over sixty variants have been functionally characterized, showing gain- or loss-of-function (GoF or LoF) effects. While ClC-3 can function as a homodimer, ClC-4 depends on heterodimerization with ClC-3 for efficient endosomal trafficking. , located on the X chromosome, exhibits diverse pathogenic outcomes: complete LoF variants often cause non-syndromic presentations in hemizygous males and are asymptomatic in heterozygous females, whereas certain missense variants with partial or complete LoF produce severe syndromic phenotypes in both sexes. Here, we demonstrate dominant effects of three variants within ClC-3/ClC-4 heterodimers using two-electrode voltage-clamp recordings in Xenopus laevis oocytes and whole-cell patch-clamp recordings in mammalian cells co-expressing both proteins via a bicistronic IRES construct. Our findings provide the first evidence of dominant-negative effects within ClC-3/ClC-4 complexes and establish a platform for functional analysis of additional disease-associated variants.

Mechanistic studies of active exchangers suggest that substrate binding in active exchangers is antagonistic, and coupling is maintained by preventing shuttling of the empty transporter. However, isothermal titration calorimetry and free energy calculations now show that substrate binding of H + and Cl − to the prokaryotic CLC-ec1 exchanger is synergistic and occurs simultaneously. Active exchangers dissipate the gradient of one substrate to accumulate nutrients, export xenobiotics and maintain cellular homeostasis. Mechanistic studies have suggested that two fundamental properties are shared by all exchangers: substrate binding is antagonistic, and coupling is maintained by preventing shuttling of the empty transporter. The CLC H + /Cl − exchangers control the homeostasis of cellular compartments in most living organisms, but their transport mechanism remains unclear. We show that substrate binding to CLC-ec1 is synergistic rather than antagonistic: chloride binding induces protonation of a crucial glutamate. The simultaneous binding of H + and Cl − gives rise to a fully loaded state that is incompatible with conventional transport mechanisms. Mutations in the Cl − transport pathway identically alter the stoichiometries of H + /Cl − exchange and binding. We propose that the thermodynamics of synergistic substrate binding, rather than the kinetics of conformational changes and ion binding, determine the stoichiometry of transport.

The nine-member CLC gene family of Cl− chloride-transporting membrane proteins is divided into plasma membrane-localized Cl− channels and endo-/lysosomal Cl−/H+ antiporters. Accessory proteins have been identified for ClC-K and ClC-2 channels and for the lysosomal ClC-7, but not the other CLCs. Here, we identified TMEM9 Domain Family Member B (TMEM9B), a single-span type I transmembrane protein of unknown function, to strongly interact with the neuronal endosomal ClC-3 and ClC-4 transporters. Co-expression of TMEM9B with ClC-3 or ClC-4 dramatically reduced transporter activity in Xenopus oocytes and transfected HEK cells. For ClC-3, TMEM9B also induced a slow component in the kinetics of the activation time course, suggesting direct interaction. Currents mediated by ClC-7 were hardly affected by TMEM9B, and ClC-1 currents were only slightly reduced, demonstrating specific interaction with ClC-3 and ClC-4. We obtained strong evidence for direct interaction by detecting significant Förster Resonance Energy Transfer (FRET), exploiting fluorescence lifetime microscopy-based (FLIM-FRET) techniques between TMEM9B and ClC-3 and ClC-4, but hardly any FRET with ClC-1 or ClC-7. The discovery of TMEM9B as a novel interaction partner of ClC-3 and ClC-4 might have important implications for the physiological role of these transporters in neuronal endosomal homeostasis and for a better understanding of the pathological mechanisms in CLCN3- and CLCN4-related pathological conditions.

ClC-Ka and ClC-Kb Cl⁻ channels are pivotal for renal salt reabsorption and water balance. There is growing interest in identifying ligands that allow pharmacological interventions aimed to modulate their activity. Starting from available ligands, we followed a rational chemical strategy, accompanied by computational modeling and electrophysiological techniques, to identify the molecular requisites for binding to a blocking or to an activating binding site on ClC-Ka. The major molecular determinant that distinguishes activators from blockers is the level of planarity of the aromatic portions of the molecules: only molecules with perfectly coplanar aromatic groups display potentiating activity. Combining several molecular features of various CLC-K ligands, we discovered that phenyl-benzofuran carboxylic acid derivatives yield the most potent ClC-Ka inhibitors so far described (affinity <10 μM). The increase in affinity compared with 3-phenyl-2-p-chlorophenoxy-propionic acid (3-phenyl-CPP) stems primarily from the conformational constraint provided by the phenyl-benzofuran ring. Several other key structural elements for high blocking potency were identified through a detailed structure-activity relationship study. Surprisingly, some benzofuran-based drugs inhibit ClC-Kb with a similar affinity of <10 μM, thus representing the first inhibitors for this CLC-K isoform identified so far. Based on our data, we established a pharmacophore model that will be useful for the development of drugs targeting CLC-K channels.

CLC-type H + /Cl − exchangers are known to be regulated by voltage and H + and Cl − concentrations, but their gating mechanism remains poorly understood. New data now suggest that transport by the CLCs is regulated by two gates that are functionally linked through structural rearrangements outside of the ion-transport pathway. CLC-type exchangers mediate transmembrane Cl − transport. Mutations altering their gating properties cause numerous genetic disorders. However, their transport mechanism remains poorly understood. In conventional models, two gates alternatively expose substrates to the intra- or extracellular solutions. A glutamate was identified as the only gate in the CLCs, suggesting that CLCs function by a nonconventional mechanism. Here we show that transport in CLC-ec1, a prokaryotic homolog, is inhibited by cross-links constraining movement of helix O far from the transport pathway. Cross-linked CLC-ec1 adopts a wild-type–like structure, indicating stabilization of a native conformation. Movements of helix O are transduced to the ion pathway via a direct contact between its C terminus and a tyrosine that is a constitutive element of the second gate of CLC transporters. Therefore, the CLC exchangers have two gates that are coupled through conformational rearrangements outside the ion pathway.

Calcium-activated chloride channels play key roles in physiology, from mediating sensory transduction and nociception to regulating mucine secretion in airway epithelia and controlling excitability of smooth muscle fibers. Recently, TMEM16A was identified as the pore-forming subunit of these channels. It remains unclear, however, whether this protein is sufficient to form calcium-activated chloride channels, or whether association with other subunits is required for function. Recently, association with calmodulin has been proposed to be required for the calcium-dependent activation and ion selectivity of these channels. Here we show that purified and reconstituted TMEM16A is necessary and sufficient to recapitulate the properties of native and heterologously expressed calcium-activated chloride currents. Thus, association of TMEM16A with other proteins is not required for function. Ca 2+ -activated Cl − channels (CaCCs) are key regulators of numerous physiological functions, ranging from electrolyte secretion in airway epithelia to cellular excitability in sensory neurons and muscle fibers. Recently, TMEM16A (ANO1) and -B were shown to be critical components of CaCCs. It is still unknown whether they are also sufficient to form functional CaCCs, or whether association with other subunits is required. Recent reports suggest that the Ca 2+ sensitivity of TMEM16A is mediated by its association with calmodulin, suggesting that functional CaCCs are heteromultimers. To test whether TMEM16A is necessary and sufficient to form functional CaCCs, we expressed, purified, and reconstituted human TMEM16A. The purified protein mediates Ca 2+ -dependent Cl − transport with submicromolar sensitivity to Ca 2+ , consistent with what is seen in patch–clamp experiments. The channel is synergistically gated by Ca 2+ and voltage, so that opening is promoted by depolarizing potentials. Mutating two conserved glutamates in the TM6-7 intracellular loop selectively abolishes the Ca 2+ dependence of reconstituted TMEM16A, in a manner similar to what was reported for the heterologously expressed channel. Well-characterized CaCC blockers inhibit Cl − transport with K i s comparable to those measured for native and heterologously expressed CaCCs. Finally, direct physical interactions between calmodulin and TMEM16A could not be detected in copurification experiments or in functional assays. Our results demonstrate that purified TMEM16A is necessary and sufficient to recapitulate the biophysical and pharmacological properties of native and heterologously expressed CaCCs. Our results also show that association of TMEM16A with other proteins, such as calmodulin, is not required for function.