Publications by authors named "Thomas J Jentsch"

111 Publications

Renal Deletion of LRRC8/VRAC Channels Induces Proximal Tubulopathy.

J Am Soc Nephrol 2022 Jul 1. Epub 2022 Jul 1.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany

Background: Volume-regulated anion channels (VRACs) are heterohexamers of LRRC8A with LRRC8B, -C, -D, or -E in various combinations. Depending on the subunit composition, these swelling-activated channels conduct chloride, amino acids, organic osmolytes, and drugs. Despite VRACs' role in cell volume regulation, and large osmolarity changes in the kidney, neither the localization nor the function of VRACs in the kidney is known.

Methods: Mice expressing epitope-tagged LRRC8 subunits were used to determine the renal localization of all VRAC subunits. Mice carrying constitutive deletions of -, or with inducible or cell-specific ablation of , were analyzed to assess renal functions of VRACs. Analysis included histology, urine and serum parameters in different diuresis states, and metabolomics.

Results: The kidney expresses all five VRAC subunits with strikingly distinct localization. Whereas LRRC8C is exclusively found in vascular endothelium, all other subunits are found in the nephron. LRRC8E is specific for intercalated cells, whereas LRRC8A, LRRC8B, and LRRC8D are prominent in basolateral membranes of proximal tubules. Conditional deletion of LRRC8A in proximal but not distal tubules and constitutive deletion of LRRC8D cause proximal tubular injury, increased diuresis, and mild Fanconi-like symptoms.

Conclusions: VRAC/LRRC8 channels are crucial for the function and integrity of proximal tubules, but not for more distal nephron segments in spite of their larger need for volume regulation. LRRC8A/D channels may be required for the basolateral exit of many organic compounds, including cellular metabolites, in proximal tubules. Proximal tubular injury likely results from combined accumulation of several transported molecules in the absence of VRAC channels.
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http://dx.doi.org/10.1681/ASN.2021111458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9342636PMC
July 2022

Proton-gated anion transport governs macropinosome shrinkage.

Nat Cell Biol 2022 06 19;24(6):885-895. Epub 2022 May 19.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.

Intracellular organelles change their size during trafficking and maturation. This requires the transport of ions and water across their membranes. Macropinocytosis, a ubiquitous form of endocytosis of particular importance for immune and cancer cells, generates large vacuoles that can be followed optically. Shrinkage of macrophage macropinosomes depends on TPC-mediated Na efflux and Cl exit through unknown channels. Relieving osmotic pressure facilitates vesicle budding, positioning osmotic shrinkage upstream of vesicular sorting and trafficking. Here we identify the missing macrophage Cl channel as the proton-activated Cl channel ASOR/TMEM206. ASOR activation requires Na-mediated depolarization and luminal acidification by redundant transporters including H-ATPases and CLC 2Cl/H exchangers. As corroborated by mathematical modelling, feedback loops requiring the steep voltage and pH dependencies of ASOR and CLCs render vacuole resolution resilient towards transporter copy numbers. TMEM206 disruption increased albumin-dependent survival of cancer cells. Our work suggests a function for the voltage and pH dependence of ASOR and CLCs, provides a comprehensive model for ion-transport-dependent vacuole maturation and reveals biological roles of ASOR.
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http://dx.doi.org/10.1038/s41556-022-00912-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9203271PMC
June 2022

Gating choreography and mechanism of the human proton-activated chloride channel ASOR.

Sci Adv 2022 Feb 2;8(5):eabm3942. Epub 2022 Feb 2.

Structural Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.

The proton-activated chloride channel ASOR (TMEM206/PAC) permeates anions across cellular membranes in response to acidification, thereby enhancing acid-induced cell death and regulating endocytosis. The molecular mechanisms of pH-dependent control are not understood, in part because structural information for an activated conformation of ASOR is lacking. Here, we reconstitute function from purified protein and present a 3.1-Å-resolution cryo-electron microscopy structure of human ASOR at acidic pH in an activated conformation. The work contextualizes a previous acidic pH structure as a desensitized conformation. Combined with electrophysiological studies and high-resolution structures of resting and desensitized states, the work reveals mechanisms of proton sensing and ion pore gating. Clusters of extracellular acidic residues function as pH sensors and coalesce when protonated. Ensuing conformational changes induce metamorphosis of transmembrane helices to fashion an ion conduction pathway unique to the activated conformation. The studies identify a new paradigm of channel gating in this ubiquitous ion channel.
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http://dx.doi.org/10.1126/sciadv.abm3942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8809534PMC
February 2022

Place fields of single spikes in hippocampus involve Kcnq3 channel-dependent entrainment of complex spike bursts.

Nat Commun 2021 08 10;12(1):4801. Epub 2021 Aug 10.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.

Hippocampal pyramidal cells encode an animal's location by single action potentials and complex spike bursts. These elementary signals are believed to play distinct roles in memory consolidation. The timing of single spikes and bursts is determined by intrinsic excitability and theta oscillations (5-10 Hz). Yet contributions of these dynamics to place fields remain elusive due to the lack of methods for specific modification of burst discharge. In mice lacking Kcnq3-containing M-type K channels, we find that pyramidal cell bursts are less coordinated by the theta rhythm than in controls during spatial navigation, but not alert immobility. Less modulated bursts are followed by an intact post-burst pause of single spike firing, resulting in a temporal discoordination of network oscillatory and intrinsic excitability. Place fields of single spikes in one- and two-dimensional environments are smaller in the mutant. Optogenetic manipulations of upstream signals reveal that neither medial septal GABA-ergic nor cholinergic inputs alone, but rather their joint activity, is required for entrainment of bursts. Our results suggest that altered representations by bursts and single spikes may contribute to deficits underlying cognitive disabilities associated with KCNQ3-mutations in humans.
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http://dx.doi.org/10.1038/s41467-021-24805-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8355348PMC
August 2021

Unique variants in CLCN3, encoding an endosomal anion/proton exchanger, underlie a spectrum of neurodevelopmental disorders.

Am J Hum Genet 2021 08 28;108(8):1450-1465. Epub 2021 Jun 28.

Erasmus University Medical Center, Department of Clinical Genetics, 3000 CA Rotterdam, the Netherlands.

The genetic causes of global developmental delay (GDD) and intellectual disability (ID) are diverse and include variants in numerous ion channels and transporters. Loss-of-function variants in all five endosomal/lysosomal members of the CLC family of Cl channels and Cl/H exchangers lead to pathology in mice, humans, or both. We have identified nine variants in CLCN3, the gene encoding CIC-3, in 11 individuals with GDD/ID and neurodevelopmental disorders of varying severity. In addition to a homozygous frameshift variant in two siblings, we identified eight different heterozygous de novo missense variants. All have GDD/ID, mood or behavioral disorders, and dysmorphic features; 9/11 have structural brain abnormalities; and 6/11 have seizures. The homozygous variants are predicted to cause loss of ClC-3 function, resulting in severe neurological disease similar to the phenotype observed in Clcn3 mice. Their MRIs show possible neurodegeneration with thin corpora callosa and decreased white matter volumes. Individuals with heterozygous variants had a range of neurodevelopmental anomalies including agenesis of the corpus callosum, pons hypoplasia, and increased gyral folding. To characterize the altered function of the exchanger, electrophysiological analyses were performed in Xenopus oocytes and mammalian cells. Two variants, p.Ile607Thr and p.Thr570Ile, had increased currents at negative cytoplasmic voltages and loss of inhibition by luminal acidic pH. In contrast, two other variants showed no significant difference in the current properties. Overall, our work establishes a role for CLCN3 in human neurodevelopment and shows that both homozygous loss of ClC-3 and heterozygous variants can lead to GDD/ID and neuroanatomical abnormalities.
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http://dx.doi.org/10.1016/j.ajhg.2021.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387284PMC
August 2021

A Recurrent Gain-of-Function Mutation in CLCN6, Encoding the ClC-6 Cl/H-Exchanger, Causes Early-Onset Neurodegeneration.

Am J Hum Genet 2020 12 19;107(6):1062-1077. Epub 2020 Nov 19.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), 13125 Berlin, Germany; Max-Delbrück-Centrum für Molekulare Medizin (MDC), 13125 Berlin, Germany; NeuroCure Cluster of Excellence, Charité Universitätsmedizin, 10117 Berlin, Germany. Electronic address:

Dysfunction of the endolysosomal system is often associated with neurodegenerative disease because postmitotic neurons are particularly reliant on the elimination of intracellular aggregates. Adequate function of endosomes and lysosomes requires finely tuned luminal ion homeostasis and transmembrane ion fluxes. Endolysosomal CLC Cl/H exchangers function as electric shunts for proton pumping and in luminal Cl accumulation. We now report three unrelated children with severe neurodegenerative disease, who carry the same de novo c.1658A>G (p.Tyr553Cys) mutation in CLCN6, encoding the late endosomal Cl/H-exchanger ClC-6. Whereas Clcn6 mice have only mild neuronal lysosomal storage abnormalities, the affected individuals displayed severe developmental delay with pronounced generalized hypotonia, respiratory insufficiency, and variable neurodegeneration and diffusion restriction in cerebral peduncles, midbrain, and/or brainstem in MRI scans. The p.Tyr553Cys amino acid substitution strongly slowed ClC-6 gating and increased current amplitudes, particularly at the acidic pH of late endosomes. Transfection of ClC-6, but not ClC-6, generated giant LAMP1-positive vacuoles that were poorly acidified. Their generation strictly required ClC-6 ion transport, as shown by transport-deficient double mutants, and depended on Cl/H exchange, as revealed by combination with the uncoupling p.Glu200Ala substitution. Transfection of either ClC-6 or ClC-6 generated slightly enlarged vesicles, suggesting that p.Glu200Ala, previously associated with infantile spasms and microcephaly, is also pathogenic. Bafilomycin treatment abrogated vacuole generation, indicating that H-driven Cl accumulation osmotically drives vesicle enlargement. Our work establishes mutations in CLCN6 associated with neurological diseases, whose spectrum of clinical features depends on the differential impact of the allele on ClC-6 function.
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http://dx.doi.org/10.1016/j.ajhg.2020.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820737PMC
December 2020

Cellular basis of ClC-2 Cl channel-related brain and testis pathologies.

J Biol Chem 2021 Jan-Jun;296:100074. Epub 2020 Nov 23.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany; Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany; NeuroCure Cluster of Excellence, Charité Universitätsmedizin, Berlin, Germany. Electronic address:

The ClC-2 chloride channel is expressed in the plasma membrane of almost all mammalian cells. Mutations that cause the loss of ClC-2 function lead to retinal and testicular degeneration and leukodystrophy, whereas gain-of-function mutations cause hyperaldosteronism. Leukodystrophy is also observed with a loss of GlialCAM, a cell adhesion molecule that binds to ClC-2 in glia. GlialCAM changes the localization of ClC-2 and opens the channel by altering its gating. We now used cell type-specific deletion of ClC-2 in mice to show that retinal and testicular degeneration depend on a loss of ClC-2 in retinal pigment epithelial cells and Sertoli cells, respectively, whereas leukodystrophy was fully developed only when ClC-2 was disrupted in both astrocytes and oligodendrocytes. The leukodystrophy of Glialcam mice could not be rescued by crosses with Clcn2 mice in which a mutation mimics the "opening" of ClC-2 by GlialCAM. These data indicate that GlialCAM-induced changes in biophysical properties of ClC-2 are irrelevant for GLIALCAM-related leukodystrophy. Taken together, our findings suggest that the pathology caused by Clcn2 disruption results from disturbed extracellular ion homeostasis and identifies the cells involved in this process.
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http://dx.doi.org/10.1074/jbc.RA120.016031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949093PMC
September 2021

Cryo-EM structure of the volume-regulated anion channel LRRC8D isoform identifies features important for substrate permeation.

Commun Biol 2020 05 15;3(1):240. Epub 2020 May 15.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.

Members of the leucine-rich repeat-containing 8 (LRRC8) protein family, composed of the five LRRC8A-E isoforms, are pore-forming components of the volume-regulated anion channel (VRAC). LRRC8A and at least one of the other LRRC8 isoforms assemble into heteromers to generate VRAC transport activities. Despite the availability of the LRRC8A structures, the structural basis of how LRRC8 isoforms other than LRRC8A contribute to the functional diversity of VRAC has remained elusive. Here, we present the structure of the human LRRC8D isoform, which enables the permeation of organic substrates through VRAC. The LRRC8D homo-hexamer structure displays a two-fold symmetric arrangement, and together with a structure-based electrophysiological analysis, revealed two key features. The pore constriction on the extracellular side is wider than that in the LRRC8A structures, which may explain the increased permeability of organic substrates. Furthermore, an N-terminal helix protrudes into the pore from the intracellular side and may be critical for gating.
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http://dx.doi.org/10.1038/s42003-020-0951-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229184PMC
May 2020

Transfer of cGAMP into Bystander Cells via LRRC8 Volume-Regulated Anion Channels Augments STING-Mediated Interferon Responses and Anti-viral Immunity.

Immunity 2020 05 10;52(5):767-781.e6. Epub 2020 Apr 10.

The Center for Microbes, Development and Health, CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, CAS Center for Excellence in Molecular Cell Science, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China. Electronic address:

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA in infected and malignant cells and catalyzes the formation of 2'3'cGMP-AMP (cGAMP), which in turn triggers interferon (IFN) production via the STING pathway. Here, we examined the contribution of anion channels to cGAMP transfer and anti-viral defense. A candidate screen revealed that inhibition of volume-regulated anion channels (VRACs) increased propagation of the DNA virus HSV-1 but not the RNA virus VSV. Chemical blockade or genetic ablation of LRRC8A/SWELL1, a VRAC subunit, resulted in defective IFN responses to HSV-1. Biochemical and electrophysiological analyses revealed that LRRC8A/LRRC8E-containing VRACs transport cGAMP and cyclic dinucleotides across the plasma membrane. Enhancing VRAC activity by hypotonic cell swelling, cisplatin, GTPγS, or the cytokines TNF or interleukin-1 increased STING-dependent IFN response to extracellular but not intracellular cGAMP. Lrrc8e mice exhibited impaired IFN responses and compromised immunity to HSV-1. Our findings suggest that cell-to-cell transmission of cGAMP via LRRC8/VRAC channels is central to effective anti-viral immunity.
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http://dx.doi.org/10.1016/j.immuni.2020.03.016DOI Listing
May 2020

Uncoupling endosomal CLC chloride/proton exchange causes severe neurodegeneration.

EMBO J 2020 05 2;39(9):e103358. Epub 2020 Mar 2.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.

CLC chloride/proton exchangers may support acidification of endolysosomes and raise their luminal Cl concentration. Disruption of endosomal ClC-3 causes severe neurodegeneration. To assess the importance of ClC-3 Cl /H exchange, we now generate Clcn3 mice in which ClC-3 is converted into a Cl channel. Unlike Clcn3 mice, Clcn3 mice appear normal owing to compensation by ClC-4 with which ClC-3 forms heteromers. ClC-4 protein levels are strongly reduced in Clcn3 , but not in Clcn3 mice because ClC-3 binds and stabilizes ClC-4 like wild-type ClC-3. Although mice lacking ClC-4 appear healthy, its absence in Clcn3 /Clcn4 mice entails even stronger neurodegeneration than observed in Clcn3 mice. A fraction of ClC-3 is found on synaptic vesicles, but miniature postsynaptic currents and synaptic vesicle acidification are not affected in Clcn3 or Clcn3 mice before neurodegeneration sets in. Both, Cl /H -exchange activity and the stabilizing effect on ClC-4, are central to the biological function of ClC-3.
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http://dx.doi.org/10.15252/embj.2019103358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196918PMC
May 2020

Pathogenesis of hypertension in a mouse model for human CLCN2 related hyperaldosteronism.

Nat Commun 2019 10 15;10(1):4678. Epub 2019 Oct 15.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.

Human primary aldosteronism (PA) can be caused by mutations in several ion channel genes but mouse models replicating this condition are lacking. We now show that almost all known PA-associated CLCN2 mutations markedly increase ClC-2 chloride currents and generate knock-in mice expressing a constitutively open ClC-2 Cl channel as mouse model for PA. The Clcn2 allele strongly increases the chloride conductance of zona glomerulosa cells, provoking a strong depolarization and increasing cytoplasmic Ca concentration. Clcn2 mice display typical features of human PA, including high serum aldosterone in the presence of low renin activity, marked hypertension and hypokalemia. These symptoms are more pronounced in homozygous Clcn2 than in heterozygous Clcn2 mice. This difference is attributed to the unexpected finding that only ~50 % of Clcn2 zona glomerulosa cells are depolarized. By reproducing essential features of human PA, Clcn2 mice are a valuable model to study the pathological mechanisms underlying this disease.
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http://dx.doi.org/10.1038/s41467-019-12113-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794291PMC
October 2019

Identification of TMEM206 proteins as pore of PAORAC/ASOR acid-sensitive chloride channels.

Elife 2019 07 18;8. Epub 2019 Jul 18.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.

Acid-sensing ion channels have important functions in physiology and pathology, but the molecular composition of acid-activated chloride channels had remained unclear. We now used a genome-wide siRNA screen to molecularly identify the widely expressed acid-sensitive outwardly-rectifying anion channel PAORAC/ASOR. ASOR is formed by TMEM206 proteins which display two transmembrane domains (TMs) and are expressed at the plasma membrane. Ion permeation-changing mutations along the length of TM2 and at the end of TM1 suggest that these segments line ASOR's pore. While not belonging to a gene family, TMEM206 has orthologs in probably all vertebrates. Currents from evolutionarily distant orthologs share activation by protons, a feature essential for ASOR's role in acid-induced cell death. TMEM206 defines a novel class of ion channels. Its identification will help to understand its physiological roles and the diverse ways by which anion-selective pores can be formed.
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http://dx.doi.org/10.7554/eLife.49187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6663466PMC
July 2019

LRRC8 N termini influence pore properties and gating of volume-regulated anion channels (VRACs).

J Biol Chem 2018 08 20;293(35):13440-13451. Epub 2018 Jun 20.

From the Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), D-13125 Berlin, Germany,

Volume-regulated anion channels (VRACs) are crucial for cell volume regulation and have various roles in physiology and pathology. VRACs were recently discovered to be formed by heteromers of leucine-rich repeat-containing 8 (LRRC8) proteins. However, the structural determinants of VRAC permeation and gating remain largely unknown. We show here that the short stretch preceding the first LRRC8 transmembrane domain determines VRAC conductance, ion permeability, and inactivation gating. Substituted-cysteine accessibility studies revealed that several of the first 15 LRRC8 residues are functionally important and exposed to a hydrophilic environment. Substituting glutamate 6 with cysteine decreased the amplitudes of swelling-activated currents, strongly increased iodide-over-chloride permeability, and markedly shifted the voltage dependence of channel inactivation. Importantly, these effects were reversed by 2-sulfonatoethyl methanethiosulfonate, which restores the negative charge at this amino acid position. Cd-mediated blocking of in cysteine variants suggested that the LRRC8 N termini come close together in the multimeric channel complex and might form part of the pore. We propose a model in which the N termini of the LRRC8 subunits line the cytoplasmic portion of the VRAC pore, possibly by folding back into the ion permeation pathway.
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http://dx.doi.org/10.1074/jbc.RA118.002853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120214PMC
August 2018

LRRC8/VRAC anion channels are required for late stages of spermatid development in mice.

J Biol Chem 2018 07 7;293(30):11796-11808. Epub 2018 Jun 7.

From the Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), D-13125 Berlin, Germany,

Spermatogenesis is a highly complex developmental process that occurs primarily in seminiferous tubules of the testes and requires additional maturation steps in the epididymis and beyond. Mutations in many different genes can lead to defective spermatozoa and hence to male infertility. Some of these genes encode for ion channels and transporters that play roles in various processes such as cellular ion homeostasis, signal transduction, sperm motility, and the acrosome reaction. Here we show that germ cell-specific, but not Sertoli cell-specific, disruption of leads to abnormal sperm and male infertility in mice. LRRC8A (leucine-rich repeat containing 8A) is the only obligatory subunit of heteromeric volume-regulated anion channels (VRACs). Its ablation severely compromises cell volume regulation by completely abolishing the transport of anions and osmolytes through VRACs. Consistent with impaired volume regulation, the cytoplasm of late spermatids appeared swollen. These cells failed to properly reduce their cytoplasm during further development into spermatozoa and later displayed severely disorganized mitochondrial sheaths in the midpiece region, as well as angulated or coiled flagella. These changes, which progressed in severity on the way to the epididymis, resulted in dramatically reduced sperm motility. Our work shows that VRAC, probably through its role in cell volume regulation, is required in a cell-autonomous manner for proper sperm development and explains the male infertility of mice and the spontaneous mouse mutant .
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http://dx.doi.org/10.1074/jbc.RA118.003853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066314PMC
July 2018

CLC Chloride Channels and Transporters: Structure, Function, Physiology, and Disease.

Physiol Rev 2018 07;98(3):1493-1590

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin , Germany ; and Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Genova , Italy.

CLC anion transporters are found in all phyla and form a gene family of eight members in mammals. Two CLC proteins, each of which completely contains an ion translocation parthway, assemble to homo- or heteromeric dimers that sometimes require accessory β-subunits for function. CLC proteins come in two flavors: anion channels and anion/proton exchangers. Structures of these two CLC protein classes are surprisingly similar. Extensive structure-function analysis identified residues involved in ion permeation, anion-proton coupling and gating and led to attractive biophysical models. In mammals, ClC-1, -2, -Ka/-Kb are plasma membrane Cl channels, whereas ClC-3 through ClC-7 are 2Cl/H-exchangers in endolysosomal membranes. Biological roles of CLCs were mostly studied in mammals, but also in plants and model organisms like yeast and Caenorhabditis elegans. CLC Cl channels have roles in the control of electrical excitability, extra- and intracellular ion homeostasis, and transepithelial transport, whereas anion/proton exchangers influence vesicular ion composition and impinge on endocytosis and lysosomal function. The surprisingly diverse roles of CLCs are highlighted by human and mouse disorders elicited by mutations in their genes. These pathologies include neurodegeneration, leukodystrophy, mental retardation, deafness, blindness, myotonia, hyperaldosteronism, renal salt loss, proteinuria, kidney stones, male infertility, and osteopetrosis. In this review, emphasis is laid on biophysical structure-function analysis and on the cell biological and organismal roles of mammalian CLCs and their role in disease.
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http://dx.doi.org/10.1152/physrev.00047.2017DOI Listing
July 2018

LRRC8/VRAC anion channels enhance β-cell glucose sensing and insulin secretion.

Nat Commun 2018 05 17;9(1):1974. Epub 2018 May 17.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Robert-Rössle-Strasse 10, 13125, Berlin, Germany.

Glucose homeostasis depends critically on insulin that is secreted by pancreatic β-cells. Serum glucose, which is directly sensed by β-cells, stimulates depolarization- and Ca-dependent exocytosis of insulin granules. Here we show that pancreatic islets prominently express LRRC8A and LRRC8D, subunits of volume-regulated VRAC anion channels. Hypotonicity- or glucose-induced β-cell swelling elicits canonical LRRC8A-dependent VRAC currents that depolarize β-cells to an extent that causes electrical excitation. Glucose-induced excitation and Ca responses are delayed in onset, but not abolished, in β-cells lacking the essential VRAC subunit LRRC8A. Whereas Lrrc8a disruption does not affect tolbutamide- or high-K-induced insulin secretion from pancreatic islets, it reduces first-phase glucose-induced insulin secretion. Mice lacking VRAC in β-cells have normal resting serum glucose levels but impaired glucose tolerance. We propose that opening of LRRC8/VRAC channels increases glucose sensitivity and insulin secretion of β-cells synergistically with K closure. Neurotransmitter-permeable LRRC8D-containing VRACs might have additional roles in autocrine/paracrine signaling within islets.
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http://dx.doi.org/10.1038/s41467-018-04353-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5958052PMC
May 2018

Ca-activated Cl currents in the murine vomeronasal organ enhance neuronal spiking but are dispensable for male-male aggression.

J Biol Chem 2018 06 16;293(26):10392-10403. Epub 2018 May 16.

From the Leibniz-Forschungsinstitut für Molekulare Pharmakologie, D-13125 Berlin, Germany,

Ca-activated Cl currents have been observed in many physiological processes, including sensory transduction in mammalian olfaction. The olfactory vomeronasal (or Jacobson's) organ (VNO) detects molecular cues originating from animals of the same species or from predators. It then triggers innate behaviors such as aggression, mating, or flight. In the VNO, Ca-activated Cl channels (CaCCs) are thought to amplify the initial pheromone-evoked receptor potential by mediating a depolarizing Cl efflux. Here, we confirmed the co-localization of the Ca-activated Cl channels anoctamin 1 (Ano1, also called TMEM16A) and Ano2 (TMEM16B) in microvilli of apically and basally located vomeronasal sensory neurons (VSNs) and their absence in supporting cells of the VNO. Both channels were expressed as functional isoforms capable of giving rise to Ca-activated Cl currents. Although these currents persisted in the VNOs of mice lacking , they were undetectable in olfactory neuron-specific knockout mice irrespective of the presence of The loss of Ca-activated Cl currents resulted in diminished spontaneous and drastically reduced pheromone-evoked spiking of VSNs. Although this indicated an important role of anoctamin channels in VNO signal amplification, the lack of this amplification did not alter VNO-dependent male-male territorial aggression in olfactory / double knockout mice. We conclude that Ano1 mediates the bulk of Ca-activated Cl currents in the VNO and that Ano2 plays only a minor role. Furthermore, vomeronasal signal amplification by CaCCs appears to be dispensable for the detection of male-specific pheromones and for near-normal aggressive behavior in mice.
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http://dx.doi.org/10.1074/jbc.RA118.003153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028972PMC
June 2018

A gain-of-function mutation in the CLCN2 chloride channel gene causes primary aldosteronism.

Nat Genet 2018 03 5;50(3):355-361. Epub 2018 Feb 5.

INSERM, UMRS 970, Paris Cardiovascular Research Center, Paris, France.

Primary aldosteronism is the most common and curable form of secondary arterial hypertension. We performed whole-exome sequencing in patients with early-onset primary aldosteronism and identified a de novo heterozygous c.71G>A/p.Gly24Asp mutation in the CLCN2 gene, encoding the voltage-gated ClC-2 chloride channel , in a patient diagnosed at 9 years of age. Patch-clamp analysis of glomerulosa cells of mouse adrenal gland slices showed hyperpolarization-activated Cl currents that were abolished in Clcn2 mice. The p.Gly24Asp variant, located in a well-conserved 'inactivation domain', abolished the voltage- and time-dependent gating of ClC-2 and strongly increased Cl conductance at resting potentials. Expression of ClC-2 in adrenocortical cells increased expression of aldosterone synthase and aldosterone production. Our data indicate that CLCN2 mutations cause primary aldosteronism. They highlight the important role of chloride in aldosterone biosynthesis and identify ClC-2 as the foremost chloride conductor of resting glomerulosa cells.
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http://dx.doi.org/10.1038/s41588-018-0053-8DOI Listing
March 2018

K TASK-2 and KCNQ1-KCNE3 K channels are major players contributing to intestinal anion and fluid secretion.

J Physiol 2018 02 18;596(3):393-407. Epub 2017 Dec 18.

Centro de Estudios Científicos (CECs), Avenida Arturo Prat 514, Valdivia, Chile.

Key Points: K channels are important in intestinal epithelium as they ensure the ionic homeostasis and electrical potential of epithelial cells during anion and fluid secretion. Intestinal epithelium cAMP-activated anion secretion depends on the activity of the (also cAMP dependent) KCNQ1-KCNE3 K channel, but the secretory process survives after genetic inactivation of the K channel in the mouse. Here we use double mutant mice to investigate which alternative K channels come into action to compensate for the absence of KCNQ1-KCNE3 K channels. Our data establish that whilst Ca -activated K 3.1 channels are not involved, K two-pore domain TASK-2 K channels are major players providing an alternative conductance to sustain the intestinal secretory process. Work with double mutant mice lacking both TASK-2 and KCNQ1-KCNE3 channels nevertheless points to yet-unidentified K channels that contribute to the robustness of the cAMP-activated anion secretion process.

Abstract: Anion and fluid secretion across the intestinal epithelium, a process altered in cystic fibrosis and secretory diarrhoea, is mediated by cAMP-activated CFTR Cl channels and requires the simultaneous activity of basolateral K channels to maintain cellular ionic homeostasis and membrane potential. This function is fulfilled by the cAMP-activated K channel formed by the association of pore-forming KCNQ1 with its obligatory KCNE3 β-subunit. Studies using mice show sizeable cAMP-activated intestinal anion secretion in the absence of either KCNQ1 or KCNE3 suggesting that an alternative K conductance must compensate for the loss of KCNQ1-KCNE3 activity. We used double mutant mouse and pharmacological approaches to identify such a conductance. Ca -dependent anion secretion can also be supported by Ca -dependent K 3.1 channels after independent CFTR activation, but cAMP-dependent anion secretion is not further decreased in the combined absence of K 3.1 and KCNQ1-KCNE3 K channel activity. We show that the K K channel TASK-2 is expressed in the epithelium of the small and large intestine. Tetrapentylammonium, a TASK-2 inhibitor, abolishes anion secretory current remaining in the absence of KCNQ1-KCNE3 activity. A double mutant mouse lacking both KCNQ1-KCNE3 and TASK-2 showed a much reduced cAMP-mediated anion secretion compared to that observed in the single KCNQ1-KCNE3 deficient mouse. We conclude that KCNQ1-KCNE3 and TASK-2 play major roles in the intestinal anion and fluid secretory phenotype. The persistence of an, admittedly reduced, secretory activity in the absence of these two conductances suggests that further additional K channel(s) as yet unidentified contribute to the robustness of the intestinal anion secretory process.
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http://dx.doi.org/10.1113/JP275178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792569PMC
February 2018

Loss of the Na/H exchanger NHE8 causes male infertility in mice by disrupting acrosome formation.

J Biol Chem 2017 06 5;292(26):10845-10854. Epub 2017 May 5.

From the Leibniz-Forschungsinstitut für Molekulare Pharmakologie, D-13125 Berlin and

Mammalian sperm feature a specialized secretory organelle on the anterior part of the sperm nucleus, the acrosome, which is essential for male fertility. It is formed by a fusion of Golgi-derived vesicles. We show here that the predominantly Golgi-resident Na/H exchanger NHE8 localizes to the developing acrosome of spermatids. Similar to wild-type mice, mice generated Golgi-derived vesicles positive for acrosomal markers and attached to nuclei, but these vesicles failed to form large acrosomal granules and the acrosomal cap. Spermatozoa from mice completely lacked acrosomes, were round-headed, exhibited abnormal mitochondrial distribution, and displayed decreased motility, resulting in selective male infertility. Of note, similar features are also found in globozoospermia, one of the causes of male infertility in humans. Germ cell-specific, but not Sertoli cell-specific disruption recapitulated the globozoospermia phenotype, demonstrating that NHE8's role in spermiogenesis is germ cell-intrinsic. Our work has uncovered a crucial role of NHE8 in acrosome biogenesis and suggests that some forms of human globozoospermia might be caused by a loss of function of this Na/H exchanger. It points to as a candidate gene for human globozoospermia and a possible drug target for male contraception.
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http://dx.doi.org/10.1074/jbc.M117.784108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491771PMC
June 2017

Selective transport of neurotransmitters and modulators by distinct volume-regulated LRRC8 anion channels.

J Cell Sci 2017 03 13;130(6):1122-1133. Epub 2017 Feb 13.

Leibniz-Institut für Molekulare Pharmakologie (FMP), D-13125 Berlin, Germany

In response to swelling, mammalian cells release chloride and organic osmolytes through volume-regulated anion channels (VRACs). VRACs are heteromers of LRRC8A and other LRRC8 isoforms (LRRC8B to LRRC8E), which are co-expressed in HEK293 and most other cells. The spectrum of VRAC substrates and its dependence on particular LRRC8 isoforms remains largely unknown. We show that, besides the osmolytes taurine and -inositol, LRRC8 channels transport the neurotransmitters glutamate, aspartate and γ-aminobutyric acid (GABA) and the co-activator D-serine. HEK293 cells engineered to express defined subsets of LRRC8 isoforms were used to elucidate the subunit-dependence of transport. Whereas LRRC8D was crucial for the translocation of overall neutral compounds like -inositol, taurine and GABA, and sustained the transport of positively charged lysine, flux of negatively charged aspartate was equally well supported by LRRC8E. Disruption of LRRC8B or LRRC8C failed to decrease the transport rates of all investigated substrates, but their inclusion into LRRC8 heteromers influenced the substrate preference of VRAC. This suggested that individual VRACs can contain three or more different LRRC8 subunits, a conclusion confirmed by sequential co-immunoprecipitations. Our work suggests a composition-dependent role of VRACs in extracellular signal transduction.
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http://dx.doi.org/10.1242/jcs.196253DOI Listing
March 2017

Disruption of Kcc2-dependent inhibition of olfactory bulb output neurons suggests its importance in odour discrimination.

Nat Commun 2016 07 8;7:12043. Epub 2016 Jul 8.

Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Roessle Str. 10, 13125 Berlin, Germany.

Synaptic inhibition in the olfactory bulb (OB), the first relay station of olfactory information, is believed to be important for odour discrimination. We interfered with GABAergic inhibition of mitral and tufted cells (M/T cells), the principal neurons of the OB, by disrupting their potassium-chloride cotransporter 2 (Kcc2). Roughly, 70% of mice died around 3 weeks, but surviving mice appeared normal. In these mice, the resulting increase in the intracellular Cl(-) concentration nearly abolished GABA-induced hyperpolarization of mitral cells (MCs) and unexpectedly increased the number of perisomatic synapses on MCs. In vivo analysis of odorant-induced OB electrical activity revealed increased M/T cell firing rate, altered phasing of action potentials in the breath cycle and disrupted separation of odour-induced M/T cell activity patterns. Mice also demonstrated a severely impaired ability to discriminate chemically similar odorants or odorant mixtures. Our work suggests that precisely tuned GABAergic inhibition onto M/T cells is crucial for M/T cell spike pattern separation needed to distinguish closely similar odours.
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http://dx.doi.org/10.1038/ncomms12043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941119PMC
July 2016

The ClC-K2 Chloride Channel Is Critical for Salt Handling in the Distal Nephron.

J Am Soc Nephrol 2017 Jan 22;28(1):209-217. Epub 2016 Jun 22.

Institut National de la Santé et de la Recherche Médicale U970, Paris Cardiovascular Research Center, Paris, France;

Chloride transport by the renal tubule is critical for blood pressure (BP), acid-base, and potassium homeostasis. Chloride uptake from the urinary fluid is mediated by various apical transporters, whereas basolateral chloride exit is thought to be mediated by ClC-Ka/K1 and ClC-Kb/K2, two chloride channels from the ClC family, or by KCl cotransporters from the SLC12 gene family. Nevertheless, the localization and role of ClC-K channels is not fully resolved. Because inactivating mutations in ClC-Kb/K2 cause Bartter syndrome, a disease that mimics the effects of the loop diuretic furosemide, ClC-Kb/K2 is assumed to have a critical role in salt handling by the thick ascending limb. To dissect the role of this channel in detail, we generated a mouse model with a targeted disruption of the murine ortholog ClC-K2. Mutant mice developed a Bartter syndrome phenotype, characterized by renal salt loss, marked hypokalemia, and metabolic alkalosis. Patch-clamp analysis of tubules isolated from knockout (KO) mice suggested that ClC-K2 is the main basolateral chloride channel in the thick ascending limb and in the aldosterone-sensitive distal nephron. Accordingly, ClC-K2 KO mice did not exhibit the natriuretic response to furosemide and exhibited a severely blunted response to thiazide. We conclude that ClC-Kb/K2 is critical for salt absorption not only by the thick ascending limb, but also by the distal convoluted tubule.
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http://dx.doi.org/10.1681/ASN.2016010085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198284PMC
January 2017

Inactivation and Anion Selectivity of Volume-regulated Anion Channels (VRACs) Depend on C-terminal Residues of the First Extracellular Loop.

J Biol Chem 2016 08 20;291(33):17040-8. Epub 2016 Jun 20.

From the Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), D-13125 Berlin, Neurocure, Charité Universitätsmedizin, D-10117 Berlin, Germany

Canonical volume-regulated anion channels (VRACs) are crucial for cell volume regulation and have many other important roles, including tumor drug resistance and release of neurotransmitters. Although VRAC-mediated swelling-activated chloride currents (ICl,vol) have been studied for decades, exploration of the structure-function relationship of VRAC has become possible only after the recent discovery that VRACs are formed by differently composed heteromers of LRRC8 proteins. Inactivation of ICl,vol at positive potentials, a typical hallmark of VRACs, strongly varies between native cell types. Exploiting the large differences in inactivation between different LRRC8 heteromers, we now used chimeras assembled from isoforms LRRC8C and LRRC8E to uncover a highly conserved extracellular region preceding the second LRRC8 transmembrane domain as a major determinant of ICl,vol inactivation. Point mutations identified two amino acids (Lys-98 and Asp-100 in LRRC8A and equivalent residues in LRRC8C and -E), which upon charge reversal strongly altered the kinetics and voltage dependence of inactivation. Importantly, charge reversal at the first position also reduced the iodide > chloride permeability of ICl,vol This change in selectivity was stronger when both the obligatory LRRC8A subunit and the other co-expressed isoform (LRR8C or -E) carried such mutations. Hence, the C-terminal part of the first extracellular loop not only determines VRAC inactivation but might also participate in forming its outer pore. Inactivation of VRACs may involve a closure of the extracellular mouth of the permeation pathway.
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http://dx.doi.org/10.1074/jbc.M116.739342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016109PMC
August 2016

VRACs and other ion channels and transporters in the regulation of cell volume and beyond.

Authors:
Thomas J Jentsch

Nat Rev Mol Cell Biol 2016 05 1;17(5):293-307. Epub 2016 Apr 1.

Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Robert-Rössle-Strasse 10, 13125 Berlin, Germany.

Cells need to regulate their volume to counteract osmotic swelling or shrinkage, as well as during cell division, growth, migration and cell death. Mammalian cells adjust their volume by transporting potassium, sodium, chloride and small organic osmolytes using plasma membrane channels and transporters. This generates osmotic gradients, which drive water in and out of cells. Key players in this process are volume-regulated anion channels (VRACs), the composition of which has recently been identified and shown to encompass LRRC8 heteromers. VRACs also transport metabolites and drugs and function in extracellular signal transduction, apoptosis and anticancer drug resistance.
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http://dx.doi.org/10.1038/nrm.2016.29DOI Listing
May 2016

KCNQ Potassium Channels Modulate Sensitivity of Skin Down-hair (D-hair) Mechanoreceptors.

J Biol Chem 2016 Mar 5;291(11):5566-5575. Epub 2016 Jan 5.

From the Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Robert-Rössle-Strasse 10, 13125 Berlin and; Neurocure Cluster of Excellence, Charité Universitätsmedizin Berlin, 10117 Berlin, Germany. Electronic address:

M-current-mediating KCNQ (Kv7) channels play an important role in regulating the excitability of neuronal cells, as highlighted by mutations in Kcnq2 and Kcnq3 that underlie certain forms of epilepsy. In addition to their expression in brain, KCNQ2 and -3 are also found in the somatosensory system. We have now detected both KCNQ2 and KCNQ3 in a subset of dorsal root ganglia neurons that correspond to D-hair Aδ-fibers and demonstrate KCNQ3 expression in peripheral nerve endings of cutaneous D-hair follicles. Electrophysiological recordings from single D-hair afferents from Kcnq3(-/-) mice showed increased firing frequencies in response to mechanical ramp-and-hold stimuli. This effect was particularly pronounced at slow indentation velocities. Additional reduction of KCNQ2 expression further increased D-hair sensitivity. Together with previous work on the specific role of KCNQ4 in rapidly adapting skin mechanoreceptors, our results show that different KCNQ isoforms are specifically expressed in particular subsets of mechanosensory neurons and modulate their sensitivity directly in sensory nerve endings.
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http://dx.doi.org/10.1074/jbc.M115.681098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786699PMC
March 2016

VRAC: molecular identification as LRRC8 heteromers with differential functions.

Pflugers Arch 2016 Mar 3;468(3):385-93. Epub 2015 Dec 3.

Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Robert-Rössle-Strasse 10, 13125, Berlin, Germany.

A major player of vertebrate cell volume regulation is the volume-regulated anion channel (VRAC), which conducts halide ions and organic osmolytes to counteract osmotic imbalances. The molecular entity of this channel was unknown until very recently, although its biophysical characteristics and diverse physiological roles have been extensively studied over the last 30 years. On the road to the molecular identification of VRAC, experimental difficulties led to the proposal of a variety of false candidates. In 2014, in a final breakthrough, two groups independently identified LRRC8A as indispensable component of VRAC. LRRC8A is part of the leucine-rich repeat containing 8 family, which is comprised of five members (LRRC8A-E). Of those, LRRC8A is an obligatory subunit of VRAC but it needs at least one of the other family members to mediate the swelling-induced Cl(-) current ICl,vol. This review discusses the remarkable journey which led to the molecular identification of VRAC, evidence for LRRC8 proteins forming the VRAC pore and their heteromeric assembly. Furthermore, first major insights on the role of LRRC8 proteins in cancer drug resistance and apoptosis and the role of LRRC8D in cisplatin and taurine transport will be summarized.
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http://dx.doi.org/10.1007/s00424-015-1766-5DOI Listing
March 2016

Optogenetic acidification of synaptic vesicles and lysosomes.

Nat Neurosci 2015 Dec 9;18(12):1845-1852. Epub 2015 Nov 9.

Neuroscience Research Center, Charité - Universitätsmedizin Berlin, Berlin, Germany.

Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes.
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http://dx.doi.org/10.1038/nn.4161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869830PMC
December 2015

Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs.

EMBO J 2015 Dec 3;34(24):2993-3008. Epub 2015 Nov 3.

Leibniz-Institut für Molekulare Pharmakologie (FMP) Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany NeuroCure Cluster of Excellence, Charité Universitätsmedizin, Berlin, Germany

Although platinum-based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume-regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E. Cell swelling strongly enhanced LRRC8-dependent cisplatin uptake, bolstering the notion that cisplatin enters cells through VRAC. LRRC8A disruption also suppressed drug-induced apoptosis independently from drug uptake, possibly by impairing VRAC-dependent apoptotic cell volume decrease. Hence, by mediating cisplatin uptake and facilitating apoptosis, VRAC plays a dual role in the cellular drug response. Incorporation of the LRRC8D subunit into VRAC substantially increased its permeability for cisplatin and the cellular osmolyte taurine, indicating that LRRC8 proteins form the channel pore. Our work suggests that LRRC8D-containing VRACs are crucial for cell volume regulation by an important organic osmolyte and may influence cisplatin/carboplatin responsiveness of tumors.
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http://dx.doi.org/10.15252/embj.201592409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687416PMC
December 2015

In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11.

PLoS Genet 2015 Aug 18;11(8):e1005454. Epub 2015 Aug 18.

Institute of Human Genetics, Jena University Hospital, Friedrich-Schiller-University Jena, Jena, Germany.

Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.
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http://dx.doi.org/10.1371/journal.pgen.1005454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4540459PMC
August 2015
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