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Sulfate plays a pivotal role in numerous physiological processes in the human body, including bone and cartilage health. A role of the anion transporter SLC26A1 (Sat1) for sulfate reabsorption in the kidney is supported by the observation of hyposulfatemia and hypersulfaturia in Slc26a1-knockout mice. The impact of SLC26A1 on sulfate homeostasis in humans remains to be defined. By combining clinical genetics, functional expression assays, and population exome analysis, we identify SLC26A1 as a sulfate transporter in humans and experimentally validate several loss-of-function alleles. Whole-exome sequencing from a patient presenting with painful perichondritis, hyposulfatemia, and renal sulfate wasting revealed a homozygous mutation in SLC26A1, which has not been previously described to the best of our knowledge. Whole-exome data analysis of more than 5,000 individuals confirmed that rare, putatively damaging SCL26A1 variants were significantly associated with lower plasma sulfate at the population level. Functional expression assays confirmed a substantial reduction in sulfate transport for the SLC26A1 mutation of our patient, which we consider to be novel, as well as for the additional variants detected in the population study. In conclusion, combined evidence from 3 complementary approaches supports SLC26A1 activity as a major determinant of sulfate homeostasis in humans. In view of recent evidence linking sulfate homeostasis with back pain and intervertebral disc disorder, our study identifies SLC26A1 as a potential target for modulation of musculoskeletal health.

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.

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 which 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(op/op) 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.

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(op) allele strongly increases the chloride conductance of zona glomerulosa cells, provoking a strong depolarization and increasing cytoplasmic Ca(2+) concentration. Clcn2(op) 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(op/op) than in heterozygous Clcn2+/op mice. This difference is attributed to the unexpected finding that only ~50 % of Clcn2(+/op) zona glomerulosa cells are depolarized. By reproducing essential features of human PA, Clcn2(op) mice are a valuable model to study the pathological mechanisms underlying this disease.

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 LRRC8 (leucine-rich repeat-containing 8) 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 ICl,vol 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. Cd2+-mediated blocking of ICl,vol 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.

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 Lrrc8a leads to abnormal sperm and male infertility in mice. LRRC8A (leucine-rich repeat containing 8 A) is the only obligatory subunit of heteromeric volume-regulated VRAC anion channels. Its ablation severely compromises cell volume regulation by completely abolishing the transport of anions and osmolytes through VRAC. 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 Lrrc8a(-/-) mice and the spontaneous mouse mutant ébouriffé.

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.

In response to swelling, mammalian cells release chloride and organic osmolytes through VRAC volume-regulated anion channels. VRACs are heteromers of LRRC8A and other LRRC8 isoforms (B-E) 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 myo-inositol, LRRC8 channels transport the neurotransmitters glutamate, aspartate and 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 myo-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 transport rates of all investigated substrates, but their inclusion into LRRC8 heteromers influenced VRAC's substrate preference. 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.

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 (Lys98 and Asp100 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 carboxy-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.

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.

After a personal description of the convoluted path leading 25 years ago to the molecular identification of the Torpedo Cl(-) channel ClC-0 and the discovery of the CLC gene family, I succinctly describe general structural and functional features of these ion transporters before giving a short overview of mammalian CLCs. These can be categorized into plasma membrane Cl(-) channels and vesicular Cl(-) /H(+) -exchangers. They are involved in the regulation of membrane excitability, transepithelial transport, extracellular ion homeostasis, endocytosis, and lysosomal function. Diseases caused by CLC dysfunction include myotonia, neurodegeneration, deafness, blindness, leukodystrophy, male infertility, renal salt loss, kidney stones and osteopetrosis, revealing a surprisingly broad spectrum of biological roles for chloride transport that was unsuspected when I set out to clone the first voltage-gated chloride channel.

Defects in the astrocytic membrane protein MLC1, the adhesion molecule GlialCAM or the chloride channel ClC-2 underlie human leukoencephalopathies. Whereas GlialCAM binds ClC-2 and MLC1, and modifies ClC-2 currents in vitro, no functional connections between MLC1 and ClC-2 are known. Here we investigate this by generating loss-of-function Glialcam and Mlc1 mouse models manifesting myelin vacuolization. We find that ClC-2 is unnecessary for MLC1 and GlialCAM localization in brain, whereas GlialCAM is important for targeting MLC1 and ClC-2 to specialized glial domains in vivo and for modifying ClC-2's biophysical properties specifically in oligodendrocytes (OLs), the cells chiefly affected by vacuolization. Unexpectedly, MLC1 is crucial for proper localization of GlialCAM and ClC-2, and for changing ClC-2 currents. Our data unmask an unforeseen functional relationship between MLC1 and ClC-2 in vivo, which is probably mediated by GlialCAM, and suggest that ClC-2 participates in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts.

CLC anion transporters form dimers that function either as Cl(-) channels or as electrogenic Cl(-)/H(+)-exchangers. CLC channels display two different types of 'gates', 'protopore' gates that open and close the two pores of a CLC dimer independently of each other, and common gates that act on both pores simultaneously. ClC-7/Ostm1 is a lysosomal 2Cl(-)/1H(+)-exchanger that is slowly activated by depolarization. This gating process is drastically accelerated by many CLCN7 mutations underlying human osteopetrosis. Making use of some of these mutants, we now investigate whether slow voltage-activation of plasma membrane-targeted ClC-7/Ostm1 involves protopore or common gates. Voltage-activation of wild-type ClC-7 subunits was accelerated by co-expressing an excess of ClC-7 subunits carrying an accelerating mutation together with a point mutation rendering these subunits transport-deficient. Conversely, voltage-activation of a fast ClC-7 mutant could be slowed by co-expressing an excess of a transport-deficient mutant. These effects did not depend on whether the accelerating mutation localized to the transmembrane part or to cytoplasmic CBS domains of ClC-7. Combining accelerating mutations in the same subunit did not speed up gating further. No currents were observed when ClC-7 was truncated after the last intramembrane helix. Currents and slow gating were restored when the carboxy-terminus was co-expressed by itself or fused to the C-terminus of the {beta}-subunit Ostm1. We conclude that common gating underlies the slow voltage-activation of ClC-7. It depends on the CBS domain-containing carboxy-terminus that does not require covalent binding to the membrane domain of ClC-7.

Mutations in the ClC-7/Ostm1 ion transporter lead to osteopetrosis and lysosomal storage disease. Its lysosomal localization hitherto precluded detailed functional characterization. Using a mutated ClC-7 that reaches the plasma membrane, we now show that both the aminoterminus and transmembrane span of the Ostm1 β-subunit are required for ClC-7 Cl(-)/H(+)-exchange, whereas the Ostm1 transmembrane domain suffices for its ClC-7-dependent trafficking to lysosomes. ClC-7/Ostm1 currents were strongly outwardly rectifying owing to slow gating of ion exchange, which itself displays an intrinsically almost linear voltage dependence. Reversal potentials of tail currents revealed a 2Cl(-)/1H(+)-exchange stoichiometry. Several disease-causing CLCN7 mutations accelerated gating. Such mutations cluster to the second cytosolic cystathionine-β-synthase domain and potential contact sites at the transmembrane segment. Our work suggests that gating underlies the rectification of all endosomal/lysosomal CLCs and extends the concept of voltage gating beyond channels to ion exchangers.

Canonical olfactory signal transduction involves the activation of cyclic AMP-activated cation channels that depolarize the cilia of receptor neurons and raise intracellular calcium. Calcium then activates Cl(-) currents that may be up to tenfold larger than cation currents and are believed to powerfully amplify the response. We identified Anoctamin2 (Ano2, also known as TMEM16B) as the ciliary Ca(2+)-activated Cl(-) channel of olfactory receptor neurons. Ano2 is expressed in the main olfactory epithelium (MOE) and in the vomeronasal organ (VNO), which also expresses the related Ano1 channel. Disruption of Ano2 in mice virtually abolished Ca(2+)-activated Cl(-) currents in the MOE and VNO. Ano2 disruption reduced fluid-phase electro-olfactogram responses by only approximately 40%, did not change air-phase electro-olfactograms and did not reduce performance in olfactory behavioral tasks. In contrast with the current view, cyclic nucleotide-gated cation channels do not need a boost by Cl(-) channels to achieve near-physiological levels of olfaction.

The profound luminal acidification essential for the degradative function of lysosomes requires a counter-ion flux to dissipate an opposing voltage that would prohibit proton accumulation. It has generally been assumed that a parallel anion influx is the main or only counter-ion transport that enables acidification. Indeed, defective anion conductance has been suggested as the mechanism underlying attenuated lysosome acidification in cells deficient in CFTR or ClC-7. To assess the individual contribution of counter-ions to acidification, we devised means of reversibly and separately permeabilizing the plasma and lysosomal membranes to dialyze the cytosol and lysosome lumen in intact cells, while ratiometrically monitoring lysosomal pH. Replacement of cytosolic Cl(-) with impermeant anions did not significantly alter proton pumping, while the presence of permeant cations in the lysosomal lumen supported acidification. Accordingly, the lysosomes were found to acidify to the same pH in both CFTR- and ClC-7-deficient cells. We conclude that cations, in addition to chloride, can support lysosomal acidification and defects in lysosomal anion conductance cannot explain the impaired microbicidal capacity of CF phagocytes.

A high intracellular chloride concentration in immature neurons leads to a depolarizing action of GABA that is thought to shape the developing neuronal network. We show that GABA-triggered depolarization and Ca2+ transients were attenuated in mice deficient for the Na-K-2Cl cotransporter NKCC1. Correlated Ca2+ transients and giant depolarizing potentials (GDPs) were drastically reduced and the maturation of the glutamatergic and GABAergic transmission in CA1 delayed. Brain morphology, synaptic density, and expression levels of certain developmental marker genes were unchanged. The expression of lynx1, a protein known to dampen network activity, was decreased. In mice deficient for the neuronal Cl(-)/HCO(3)(-) exchanger AE3, GDPs were also diminished. These data show that NKCC1-mediated Cl(-) accumulation contributes to GABAergic excitation and network activity during early postnatal development and thus facilitates the maturation of excitatory and inhibitory synapses.

ClC-2 is a broadly expressed plasma membrane chloride channel that is modulated by voltage, cell swelling, and pH. A human mutation leading to a heterozygous loss of ClC-2 has previously been reported to be associated with epilepsy, whereas the disruption of Clcn2 in mice led to testicular and retinal degeneration. We now show that the white matter of the brain and spinal cord of ClC-2 knock-out mice developed widespread vacuolation that progressed with age. Fluid-filled spaces appeared between myelin sheaths of the central but not the peripheral nervous system. Neuronal morphology, in contrast, seemed normal. Except for the previously reported blindness, neurological deficits were mild and included a decreased conduction velocity in neurons of the central auditory pathway. The heterozygous loss of ClC-2 had no detectable functional or morphological consequences. Neither heterozygous nor homozygous ClC-2 knock-out mice had lowered seizure thresholds. Sequencing of a large collection of human DNA and electrophysiological analysis showed that several ClC-2 sequence abnormalities previously found in patients with epilepsy most likely represent innocuous polymorphisms.

K-Cl cotransport activity in rbc is a major determinant of rbc volume and density. Pathologic activation of erythroid K-Cl cotransport activity in sickle cell disease contributes to rbc dehydration and cell sickling. To address the roles of individual K-Cl cotransporter isoforms in rbc volume homeostasis, we disrupted the Kcc1 and Kcc3 genes in mice. As rbc K-Cl cotransport activity was undiminished in Kcc1(-/-) mice, decreased in Kcc3(-/-) mice, and almost completely abolished in mice lacking both isoforms, we conclude that K-Cl cotransport activity of mouse rbc is mediated largely by KCC3. Whereas rbc of either Kcc1(-/-) or Kcc3(-/-) mice were of normal density, rbc of Kcc1(-/-)Kcc3(-/-) mice exhibited defective volume regulation, including increased mean corpuscular volume, decreased density, and increased susceptibility to osmotic lysis. K-Cl cotransport activity was increased in rbc of SAD mice, which are transgenic for a hypersickling human hemoglobin S variant. Kcc1(-/-)Kcc3(-/-) SAD rbc lacked nearly all K-Cl cotransport activity and exhibited normalized values of mean corpuscular volume, corpuscular hemoglobin concentration mean, and K(+) content. Although disruption of K-Cl cotransport rescued the dehydration phenotype of most SAD rbc, the proportion of the densest red blood cell population remained unaffected.

CLC proteins are a large structurally defined family of Cl- ion channels and H+/Cl- antiporters with nine distinct genes in mammals. The membrane-embedded part of CLC proteins bears no obvious similarity to any other class of membrane proteins, while the cytoplasmic C-terminus of most eukaryotic and some prokaryotic CLCs contains two regions with homology to cystathionine beta synthase (CBS) domains that are found in other proteins as well. Different members serve a broad range of physiological roles, including stabilization of the membrane potential, transepithelial ion transport, and vesicular acidification. Their physiological importance is underscored by the causative involvement in at least four different human genetic diseases. From functional studies of the Torpedo homologue ClC-0, a homodimeric architecture with two physically separate ion conduction pathways was anticipated and fully confirmed by solving the crystal structure of prokaryotic CLC homologues. The structure revealed a complex fold of 18 alpha-helices per subunit with at least two Cl- ions bound in the center of each protopore. A critical glutamic acid residue was identified whose side-chain seems to occupy a third Cl- ion binding site in the closed state and that moves away to allow Cl- binding. While the overall architecture and pore structure is certainly conserved from bacteria to humans, the bacterial proteins that were crystallized are actually not Cl- ion channels, but coupled H+/Cl- antiporters. These recent breakthroughs will allow us to study in further detail the structure, function, and the physiological and pathophysiological role of CLC proteins.

All eukaryotic CLC Cl(-) channel subunits possess a long cytoplasmic carboxy-terminus that contains two so-called CBS (cystathionine beta-synthase) domains. These domains are found in various unrelated proteins from all phylae. The crystal structure of the CBS domains of inosine monophosphate dehydrogenase (IMPDH) is known, but it is not known whether this structure is conserved in CLC channels. Working primarily with ClC-1, we used deletion scanning mutagenesis, coimmunoprecipitation and electrophysiology to demonstrate that its CBS domains interact. The replacement of CBS domains of ClC-1 with the corresponding CBS domains from other CLC channels and even human IMPDH yielded functional channels, indicating a high degree of structural conservation. Based on a homology model of the pair of CBS domains of CLC channels, we identified some residues that, when mutated, affected the common gate which acts on both pores of the dimeric channel. Thus, we propose that the structure of CBS domains from CLC channels is highly conserved and that they play a functional role in the common gate.

Crystal structures of bacterial CLC proteins were solved recently, but it is unclear to which level of detail they can be extrapolated to mammalian chloride channels. Exploiting the difference in inhibition by 9-anthracene carboxylic acid (9-AC) between ClC-0, -1, and -2, we identified a serine between helices O and P as crucial for 9-AC binding. Mutagenesis based on the crystal structure identified further residues affecting inhibitor binding. They surround a partially hydrophobic pocket close to the chloride binding site that is accessible from the cytoplasm, consistent with the observed intracellular block by 9-AC. Mutations in presumably Cl--coordinating residues yield additional insights into the structure and function of ClC-1. Our work shows that the structure of bacterial CLCs can be extrapolated with fidelity to mammalian Cl- channels.

KCNE-Hilfsuntereinheiten assoziieren mit Spannungs-abhängigen K+-Kanälen und verändern dadurch deren subzelluläre Lokalisation, Regulation sowie deren biophysikalische Eigenschaften. Bei heterologer Expression interagiert KCNE3 mit mehreren Poren-bildenden K+-Kanal-Hauptuntereinheiten, deren Ströme dadurch stark modifiziert werden. Aufgrund dieser in vitro-Experimente wurden verschiedenste Funktionen von KCNE3 in den verschiedenen Geweben, wie Gehirn, Herz, Muskel, Kolon und Niere, vermutet. Außerdem wurden Variationen im kcne3-Gen mit menschlichen Skelettmuskelpathologien in Verbindung gesetzt (Abbott et al. 2001). In der gegenwärtigen Literatur wird die physiologische Funktion von KCNE3 eher als komplex und heterogen dargestellt. Auch die direkte Beteiligung von KCNE3 an Krankheiten ist immer noch spekulativ. Zur Untersuchung der physiologischen Funktion von KCNE3 in vivo sowie der potentiellen Rolle bei Krankheiten generierten wir ein kcne3-/- Mausmodell. Die vorliegende Arbeit unterstützt die kritische Rolle der KCNQ1/KCNE3-Kanäle beim Salz- und Flüssigkeitstransport über intestinale und respiratorische Epithelien. Insbesondere fanden wir für die KCNQ1/KCNE3-Heteromere eine basolaterale Lokalisation in Darm- und Trachea-Epithelzellen, wo sie die transepitheliale Cl--Sekretion über basolaterales Recycling von K+-Ionen sowie über Erhöhung der elektrochemischen Triebkraft für apikalen Cl--Austritt fördern. Da weder Veränderungen in der KCNQ1-Expressionsmenge noch in dessen subzellulärer Lokalisation festgestellt wurden, ist die durch KCNE3 verursachte Modifikation der KCNQ1-Kanaleigenschaften essenziell für die hier beschriebene physiologische Rolle im Intestinal- und Trachealtransport. Ferner wird von unserer Arbeit die postulierte Funktion von KCNE3-Heteromeren im Skelettmuskel, Herz und zentralen Nervensystem nicht unterstützt und erweckt somit erhebliche Zweifel über den Beitrag von KCNE3 zu menschlichen Krankheiten, die mit diesen Organen in Verbindung stehen. When overexpressed in heterologous systems, KCNE3 is able to interact with several pore-forming K+ channel alpha subunits greatly modifying their currents. Based on these in vitro evidences, KCNE3 has been proposed to serve different roles in multiple tissues, including brain, heart, muscle, colon and kidney. Additional reports have also linked sequence variations in the KCNE3 gene to cardiac and skeletal muscle pathologies in human. Based on the literature, the overall picture of KCNE3 physiological function is rather complex and heterogeneous, and its direct involvement in pathologies is still speculative and far from being conclusively proven. In order to study the physiological role of KCNE3 in vivo and to address its potential pathological implications, we generated kcne3-/- mice. The present analysis of kcne3-/- mice strongly supports a crucial role of KCNQ1/KCNE3 channels in salt- and fluid secretion across intestinal and airway epithelia. In particular, we found that KCNQ1/KCNE3 heteromers are present in basolateral membranes of intestinal and tracheal epithelial cells where they facilitate transepithelial Cl- secretion through basolateral recycling of K+ ions and by increasing the electrochemical driving force for apical Cl- exit. Because the abundance and subcellular localization of KCNQ1 was unchanged in kcne3-/- mice, the modification of biophysical properties of KCNQ1 by KCNE3 is essential for its role in intestinal and tracheal transport. In addition, our work does not support the postulated role of KCNE3 heteromers in skeletal muscle, heart and CNS physiology, and raises considerable doubts concerning its implication in human pathologies which affect these tissues.