The ClC chloride channels catalyse the selective flow of Cl- ions across cell membranes, thereby regulating electrical excitation in skeletal muscle and the flow of salt and water across epithelial barriers. Genetic defects in ClC Cl- channels underlie several familial muscle and kidney diseases. Here we present the X-ray structures of two prokaryotic ClC Cl- channels from Salmonella enterica serovar typhimurium and Escherichia coli at 3.0 and 3.5 A, respectively. Both structures reveal two identical pores, each pore being formed by a separate subunit contained within a homodimeric membrane protein. Individual subunits are composed of two roughly repeated halves that span the membrane with opposite orientations. This antiparallel architecture defines a selectivity filter in which a Cl- ion is stabilized by electrostatic interactions with alpha-helix dipoles and by chemical coordination with nitrogen atoms and hydroxyl groups. These findings provide a structural basis for further understanding the function of ClC Cl- channels, and establish the physical and chemical basis of their anion selectivity.

Cl- channels reside both in the plasma membrane and in intracellular organelles. Their functions range from ion homeostasis to cell volume regulation, transepithelial transport, and regulation of electrical excitability. Their physiological roles are impressively illustrated by various inherited diseases and knock-out mouse models. Thus the loss of distinct Cl- channels leads to an impairment of transepithelial transport in cystic fibrosis and Bartter's syndrome, to increased muscle excitability in myotonia congenita, to reduced endosomal acidification and impaired endocytosis in Dent's disease, and to impaired extracellular acidification by osteoclasts and osteopetrosis. The disruption of several Cl- channels in mice results in blindness. Several classes of Cl- channels have not yet been identified at the molecular level. Three molecularly distinct Cl- channel families (CLC, CFTR, and ligand-gated GABA and glycine receptors) are well established. Mutagenesis and functional studies have yielded considerable insights into their structure and function. Recently, the detailed structure of bacterial CLC proteins was determined by X-ray analysis of three-dimensional crystals. Nonetheless, they are less well understood than cation channels and show remarkably different biophysical and structural properties. Other gene families (CLIC or CLCA) were also reported to encode Cl- channels but are less well characterized. This review focuses on molecularly identified Cl- channels and their physiological roles.

Recent advances in single-cell genomics provide an alternative to largely gene-centric metagenomics studies, enabling whole-genome sequencing of uncultivated bacteria. However, single-cell assembly projects are challenging due to (i) the highly nonuniform read coverage and (ii) a greatly elevated number of chimeric reads and read pairs. While recently developed single-cell assemblers have addressed the former challenge, methods for assembling highly chimeric reads remain poorly explored. We present algorithms for identifying chimeric edges and resolving complex bulges in de Bruijn graphs, which significantly improve single-cell assemblies. We further describe applications of the single-cell assembler SPAdes to a new approach for capturing and sequencing "microbial dark matter" that forms small pools of randomly selected single cells (called a mini-metagenome) and further sequences all genomes from the mini-metagenome at once. On single-cell bacterial datasets, SPAdes improves on the recently developed E+V-SC and IDBA-UD assemblers specifically designed for single-cell sequencing. For standard (cultivated monostrain) datasets, SPAdes also improves on A5, ABySS, CLC, EULER-SR, Ray, SOAPdenovo, and Velvet. Thus, recently developed single-cell assemblers not only enable single-cell sequencing, but also improve on conventional assemblers on their own turf. SPAdes is available for free online download under a GPLv2 license.

ClC channels conduct chloride (Cl-) ions across cell membranes and thereby govern the electrical activity of muscle cells and certain neurons, the transport of fluid and electrolytes across epithelia, and the acidification of intracellular vesicles. The structural basis of ClC channel gating was studied. Crystal structures of wild-type and mutant Escherichia coli ClC channels bound to a monoclonal Fab fragment reveal three Cl- binding sites within the 15-angstrom neck of an hourglass-shaped pore. The Cl- binding site nearest the extracellular solution can be occupied either by a Cl- ion or by a glutamate carboxyl group. Mutations of this glutamate residue in Torpedo ray ClC channels alter gating in electrophysiological assays. These findings reveal a form of gating in which the glutamate carboxyl group closes the pore by mimicking a Cl- ion.

Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein-tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ∼ 2 s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappear following scission. We measured the recruitment dynamics of 34 types of endocytic protein to scission events: Abp1, ACK1, amphiphysin1, APPL1, Arp3, BIN1, CALM, CIP4, clathrin light chain (Clc), cofilin, coronin1B, cortactin, dynamin1/2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2β1, and syndapin2. For each protein we aligned ∼ 1,000 recruitment profiles to their respective scission events and constructed characteristic "recruitment signatures" that were grouped, as for yeast, to reveal the modular organization of mammalian CME. A detailed analysis revealed the unanticipated recruitment dynamics of SNX9, FBP17, and CIP4 and showed that the same set of proteins was recruited, in the same order, to scission events at CCSs of different sizes and lifetimes. Collectively these data reveal the fine-grained temporal structure of CME and suggest a simplified canonical model of mammalian CME in which the same core mechanism of CME, involving actin, operates at CCSs of diverse sizes and lifetimes.

CBS domains are defined as sequence motifs that occur in several different proteins in all kingdoms of life. Although thought to be regulatory, their exact functions have been unknown. However, their importance was underlined by findings that mutations in conserved residues within them cause a variety of human hereditary diseases, including (with the gene mutated in parentheses): Wolff-Parkinson-White syndrome (gamma 2 subunit of AMP-activated protein kinase); retinitis pigmentosa (IMP dehydrogenase-1); congenital myotonia, idiopathic generalized epilepsy, hypercalciuric nephrolithiasis, and classic Bartter syndrome (<em>CLC</em> chloride channel family members); and homocystinuria (cystathionine beta-synthase). AMP-activated protein kinase is a sensor of cellular energy status that is activated by AMP and inhibited by ATP, but the location of the regulatory nucleotide-binding sites (which are prime targets for drugs to treat obesity and diabetes) was not characterized. We now show that tandem pairs of CBS domains from AMP-activated protein kinase, IMP dehydrogenase-2, the chloride channel <em>CLC</em>2, and cystathionine beta-synthase bind AMP, ATP, or S-adenosyl methionine,while mutations that cause hereditary diseases impair this binding. This shows that tandem pairs of CBS domains act, in most cases, as sensors of cellular energy status and, as such, represent a newly identified class of binding domain for adenosine derivatives.

In myotonic dystrophy (dystrophia myotonica, DM), expression of RNAs that contain expanded CUG or CCUG repeats is associated with degeneration and repetitive action potentials (myotonia) in skeletal muscle. Using skeletal muscle from a transgenic mouse model of DM, we show that expression of expanded CUG repeats reduces the transmembrane chloride conductance to levels well below those expected to cause myotonia. The expanded CUG repeats trigger aberrant splicing of pre-mRNA for ClC-1, the main chloride channel in muscle, resulting in loss of ClC-1 protein from the surface membrane. We also have identified a similar defect in ClC-1 splicing and expression in two types of human DM. We propose that a transdominant effect of mutant RNA on RNA processing leads to chloride channelopathy and membrane hyperexcitability in DM.

Chloride channels play important roles in the plasma membrane and in intracellular organelles. Mice deficient for the ubiquitously expressed ClC-7 Cl(-) channel show severe osteopetrosis and retinal degeneration. Although osteoclasts are present in normal numbers, they fail to resorb bone because they cannot acidify the extracellular resorption lacuna. ClC-7 resides in late endosomal and lysosomal compartments. In osteoclasts, it is highly expressed in the ruffled membrane, formed by the fusion of H(+)-ATPase-containing vesicles, that secretes protons into the lacuna. We also identified CLCN7 mutations in a patient with human infantile malignant osteopetrosis. We conclude that ClC-7 provides the chloride conductance required for an efficient proton pumping by the H(+)-ATPase of the osteoclast ruffled membrane.

ClC Cl- channels make up a large molecular family, ubiquitous with respect to both organisms and cell types. In eukaryotes, these channels fulfill numerous biological roles requiring gated anion conductance, from regulating skeletal muscle excitability to facilitating endosomal acidification by (H+)ATPases. In prokaryotes, ClC functions are unknown except in Escherichia coli, where the ClC-ec1 protein promotes H+ extrusion activated in the extreme acid-resistance response common to enteric bacteria. Recently, the high-resolution structure of ClC-ec1 was solved by X-ray crystallography. This primal prokaryotic ClC structure has productively guided understanding of gating and anion permeation in the extensively studied eukaryotic ClC channels. We now show that this bacterial homologue is not an ion channel, but rather a H+-Cl- exchange transporter. As the same molecular architecture can support two fundamentally different transport mechanisms, it seems that the structural boundary separating channels and transporters is not as clear cut as generally thought.

Reactive oxygen species are ubiquitous signaling molecules in biological systems. Four members of the NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species in the vasculature: Nox1, Nox2, Nox4, and Nox5. Signaling cascades triggered by stresses, hormones, vasoactive agents, and cytokines control the expression and activity of these enzymes and of their regulatory subunits, among which p22phox, p47phox, Noxa1, and p67phox are present in blood vessels. Vascular Nox enzymes are also regulated by Rac, ClC-3, Poldip2, and protein disulfide isomerase. Multiple Nox subtypes, simultaneously present in different subcellular compartments, produce specific amounts of superoxide, some of which is rapidly converted to hydrogen peroxide. The identity and location of these reactive oxygen species, and of the enzymes that degrade them, determine their downstream signaling pathways. Nox enzymes participate in a broad array of cellular functions, including differentiation, fibrosis, growth, proliferation, apoptosis, cytoskeletal regulation, migration, and contraction. They are involved in vascular pathologies such as hypertension, restenosis, inflammation, atherosclerosis, and diabetes. As our understanding of the regulation of these oxidases progresses, so will our ability to alter their functions and associated pathologies.

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder caused by a CTG expansion in the 3' untranslated region of the DMPK gene. A predominant characteristic of DM1 is myotonia resulting from skeletal muscle membrane hyperexcitability. Here we demonstrate loss of the muscle-specific chloride channel (ClC-1) mRNA and protein in DM1 skeletal muscle tissue due to aberrant splicing of the ClC-1 pre-mRNA. The splicing regulator, CUG binding protein (CUG-BP), which is elevated in DM1 striated muscle, binds to the ClC-1 pre-mRNA, and overexpression of CUG-BP in normal cells reproduces the aberrant pattern of ClC-1 splicing observed in DM1 skeletal muscle. We propose that disruption of alternative splicing regulation causes a predominant pathological feature of DM1.

Eukaryotic members of the CLC gene family function as plasma membrane chloride channels, or may provide neutralizing anion currents for V-type H(+)-ATPases that acidify compartments of the endosomal/lysosomal pathway. Loss-of-function mutations in the endosomal protein ClC-5 impair renal endocytosis and lead to kidney stones, whereas loss of function of the endosomal/lysosomal protein ClC-7 entails osteopetrosis and lysosomal storage disease. Vesicular CLCs have been thought to be Cl- channels, in particular because ClC-4 and ClC-5 mediate plasma membrane Cl- currents upon heterologous expression. Here we show that these two mainly endosomal CLC proteins instead function as electrogenic Cl-/H+ exchangers (also called antiporters), resembling the transport activity of the bacterial protein ClC-e1, the crystal structure of which has already been determined. Neutralization of a critical glutamate residue not only abolished the steep voltage-dependence of transport, but also eliminated the coupling of anion flux to proton counter-transport. ClC-4 and ClC-5 may still compensate the charge accumulation by endosomal proton pumps, but are expected to couple directly vesicular pH gradients to Cl- gradients.

ClC-4 and ClC-5 are members of the CLC gene family, with ClC-5 mutated in Dent's disease, 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. Motivated by the discovery that certain bacterial CLC proteins are secondary active Cl-/H+ antiporters, 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.

CLC genes are expressed in species from bacteria to human and encode Cl(-)-channels or Cl(-)/H(+)-exchangers. CLC proteins assemble to dimers, with each monomer containing an ion translocation pathway. Some mammalian isoforms need essential beta -subunits (barttin and Ostm1). Crystal structures of bacterial CLC Cl(-)/H(+)-exchangers, combined with transport analysis of mammalian and bacterial CLCs, yielded surprising insights into their structure and function. The large cytosolic carboxy-termini of eukaryotic CLCs contain CBS domains, which may modulate transport activity. Some of these have been crystallized. Mammals express nine CLC isoforms that differ in tissue distribution and subcellular localization. Some of these are plasma membrane Cl(-) channels, which play important roles in transepithelial transport and in dampening muscle excitability. Other CLC proteins localize mainly to the endosomal-lysosomal system where they may facilitate luminal acidification or regulate luminal chloride concentration. All vesicular CLCs may be Cl(-)/H(+)-exchangers, as shown for the endosomal ClC-4 and -5 proteins. Human diseases and knockout mouse models have yielded important insights into their physiology and pathology. Phenotypes and diseases include myotonia, renal salt wasting, kidney stones, deafness, blindness, male infertility, leukodystrophy, osteopetrosis, lysosomal storage disease and defective endocytosis, demonstrating the broad physiological role of CLC-mediated anion transport.

Lysosomes, the terminal organelles on the endocytic pathway, digest macromolecules and make their components available to the cell as nutrients. Hydrolytic enzymes specific to a wide range of targets reside within the lysosome; these enzymes are activated by the highly acidic pH (between 4.5 and 5.0) in the organelles' interior. Lysosomes generate and maintain their pH gradients by using the activity of a proton-pumping V-type ATPase, which uses metabolic energy in the form of ATP to pump protons into the lysosome lumen. Because this activity separates electric charge and generates a transmembrane voltage, another ion must move to dissipate this voltage for net pumping to occur. This so-called counterion may be either a cation (moving out of the lysosome) or an anion (moving into the lysosome). Recent data support the involvement of ClC-7, a Cl(-)/H(+) antiporter, in this process, although many open questions remain as to this transporter's involvement. Although functional results also point to a cation transporter, its molecular identity remains uncertain. Both the V-ATPase and the counterion transporter are likely to be important players in the mechanisms determining the steady-state pH of the lysosome interior. Exciting new results suggest that lysosomal pH may be dynamically regulated in some cell types.

Renal salt loss in Bartter's syndrome is caused by impaired transepithelial transport in the loop of Henle. Sodium chloride is taken up apically by the combined activity of NKCC2 (Na+-K--2Cl- cotransporters) and ROMK potassium channels. Chloride ions exit from the cell through basolateral ClC-Kb chloride channels. Mutations in the three corresponding genes have been identified that correspond to Bartter's syndrome types 1-3. The gene encoding the integral membrane protein barttin is mutated in a form of Bartter's syndrome that is associated with congenital deafness and renal failure. Here we show that barttin acts as an essential beta-subunit for ClC-Ka and ClC-Kb chloride channels, with which it colocalizes in basolateral membranes of renal tubules and of potassium-secreting epithelia of the inner ear. Disease-causing mutations in either ClC-Kb or barttin compromise currents through heteromeric channels. Currents can be stimulated further by mutating a proline-tyrosine (PY) motif on barttin. This work describes the first known beta-subunit for CLC chloride channels and reveals that heteromers formed by ClC-K and barttin are crucial for renal salt reabsorption and potassium recycling in the inner ear.

Maintenance of a constant cell volume in the face of osmotic stress is an evolutionarily ancient homeostatic process. Over the last two decades physiologists have gained an impressive understanding of the "volume-sensitive" channels, cotransporters, exchangers, metabolic pathways, and genes that are responsible for modulating intracellular solute content and cell volume. This review focuses on one part of this story, the characteristics and osmoregulatory functions of volume-sensitive anion channels. Three distinct types of swelling-activated anion channels have been observed and studied extensively in animal cells. These channels include 1) ClC-2, which is a member of the ClC family of voltage-gated anion channels, 2) an outwardly rectifying intermediate conductance channel, and 3) a large-conductance or "maxi" channel. In addition to these three channels, several other less well-characterized anion channels have been observed. This review discusses the electrophysiological and molecular biological characteristics and regulation of these channels. The possible roles different types of anion channels might play in cell volume homeostasis are also discussed.

The maintenance of a constant volume in the face of extracellular and intracellular osmotic perturbation is essential for the normal function and survival of animal cells. Osmotically swollen cells restore their volume, exhibiting a regulatory volume decrease by releasing intracellular K+, Cl-, organic solutes, and obligated water. In many cell types, the volume regulatory effluxes of Cl- and some organic osmolytes are known to be induced by swelling-induced activation of anion channels that are characterized by their moderate outward rectification, cytosolic ATP dependency, and intermediate unitary conductance (10-100 pS). Recently, simultaneous measurements of cell size by light microscopy and whole cell Cl- current have shown that the Cl- current density is proportionally increased with an increase in the outer surface area, which is mainly achieved through unfolding of membrane invaginations by volume expansion. Thus this anion channel can somehow sense volume expansion and can be called the volume expansion-sensing outwardly rectifying (VSOR) anion channel. Its molecular identity and activation mechanism are yet to be elucidated. Three cloned proteins, ClC-2, P-glycoprotein, and pIcln, have been proposed as candidates for the VSOR anion channel. The unitary conductance, voltage dependency, anion selectivity, pH dependency, and pharmacology of the VSOR anion channel are distinct from the ClC-2 Cl- channel, which is also known to be sensitive to volume changes. Recent patch-clamp studies in combination with molecular biological techniques have shown that P-glycoprotein is not itself the channel protein but is a regulator of its volume sensitivity. Although there is still debate about another candidate protein, pIcln, the most recent study has suggested that this is likely to be a regulator of some other distinct Cl- channel. Identification of the VSOR anion channel protein per se, its volume-sensing mechanism, and its accessory/regulatory proteins at the molecular level is currently a subject of utmost physiological importance.

Autosomal recessive generalized myotonia (Becker's disease) (GM) and autosomal dominant myotonia congenita (Thomsen's disease) (MC) are characterized by skeletal muscle stiffness that is a result of muscle membrane hyperexcitability. For both diseases, alterations in muscle chloride or sodium currents or both have been observed. A complementary DNA for a human skeletal muscle chloride channel (CLC-1) was cloned, physically localized on chromosome 7, and linked to the T cell receptor beta (TCRB) locus. Tight linkage of these two loci to GM and MC was found in German families. An unusual restriction site in the CLC-1 locus in two GM families identified a mutation associated with that disease, a phenylalanine-to-cysteine substitution in putative transmembrane domain D8. This suggests that different mutations in CLC-1 may cause dominant or recessive myotonia.

Chloride channels have several functions, including the regulation of cell volume, stabilizing membrane potential, signal transduction and transepithelial transport. The plasma membrane Cl- channels already cloned belong to different structural classes: ligand-gated channels, voltage-gated channels, and possibly transporters of the ATP-binding-cassette type (if the cystic fibrosis transmembrane regulator is a Cl- channel). The importance of chloride channels is illustrated by the phenotypes that can result from their malfunction: cystic fibrosis, in which transepithelial transport is impaired, and myotonia, in which ClC-1, the principal skeletal muscle Cl- channel, is defective. Here we report the properties of ClC-2, a new member of the voltage-gated Cl- channel family. Its sequence is approximately 50% identical to either the Torpedo electroplax Cl- channel, ClC-0 (ref. 8), or the rat muscle Cl- channel, ClC-1 (ref. 9). Isolated initially from rat heart and brain, it is also expressed in pancreas, lung and liver, for example, and in pure cell lines of fibroblastic, neuronal, and epithelial origin, including tissues and cells affected by cystic fibrosis. Expression in Xenopus oocytes induces Cl- currents that activate slowly upon hyperpolarization and display a linear instantaneous current-voltage relationship. The conductivity sequence is Cl- greater than or equal to Br- greater than I-. The presence of ClC-2 in such different cell types contrasts with the highly specialized expression of ClC-1 (ref. 9) and also with the cloned cation channels, and suggests that its function is important for most cells.

Dent's disease is an X-linked disorder associated with the urinary loss of low-molecular-weight proteins, phosphate and calcium, which often leads to kidney stones. It is caused by mutations in ClC-5, a renal chloride channel that is expressed in endosomes of the proximal tubule. Here we show that disruption of the mouse clcn5 gene causes proteinuria by strongly reducing apical proximal tubular endocytosis. Both receptor-mediated and fluid-phase endocytosis are affected, and the internalization of the apical transporters NaPi-2 and NHE3 is slowed. At steady state, however, both proteins are redistributed from the plasma membrane to intracellular vesicles. This may be caused by an increased stimulation of luminal parathyroid hormone (PTH) receptors owing to the observed decreased tubular endocytosis of PTH. The rise in luminal PTH concentration should also stimulate the hydroxylation of 25(OH) vitamin D3 to the active hormone. However, this is counteracted by a urinary loss of the precursor 25(OH) vitamin D3. The balance between these opposing effects, both of which are secondary to the defect in proximal tubular endocytosis, probably determines whether there will be hypercalciuria and kidney stones.

BACKGROUND

Roche 454 pyrosequencing has become a method of choice for generating transcriptome data from non-model organisms. Once the tens to hundreds of thousands of short (250-450 base) reads have been produced, it is important to correctly assemble these to estimate the sequence of all the transcripts. Most transcriptome assembly projects use only one program for assembling 454 pyrosequencing reads, but there is no evidence that the programs used to date are optimal. We have carried out a systematic comparison of five assemblers (CAP3, MIRA, Newbler, SeqMan and CLC) to establish best practices for transcriptome assemblies, using a new dataset from the parasitic nematode Litomosoides sigmodontis.

RESULTS

Although no single assembler performed best on all our criteria, Newbler 2.5 gave longer contigs, better alignments to some reference sequences, and was fast and easy to use. SeqMan assemblies performed best on the criterion of recapitulating known transcripts, and had more novel sequence than the other assemblers, but generated an excess of small, redundant contigs. The remaining assemblers all performed almost as well, with the exception of Newbler 2.3 (the version currently used by most assembly projects), which generated assemblies that had significantly lower total length. As different assemblers use different underlying algorithms to generate contigs, we also explored merging of assemblies and found that the merged datasets not only aligned better to reference sequences than individual assemblies, but were also more consistent in the number and size of contigs.

CONCLUSIONS

Transcriptome assemblies are smaller than genome assemblies and thus should be more computationally tractable, but are often harder because individual contigs can have highly variable read coverage. Comparing single assemblers, Newbler 2.5 performed best on our trial data set, but other assemblers were closely comparable. Combining differently optimal assemblies from different programs however gave a more credible final product, and this strategy is recommended.

Chloride channels of the ClC family are important for the control of membrane excitability, cell volume regulation, and possibly transepithelial transport. Although lacking the typical voltage-sensor found in cation channels, gating of ClC channels is clearly voltage-dependent. For the prototype Torpedo channel ClC-0 (refs 11-15) we now show that channel opening is strongly facilitated by external chloride. Other less permeable anions can substitute for chloride with less efficiency. ClC-0 conductance shows an anomalous mole fraction behaviour with Cl-/NO3- mixtures, suggesting a multi-ion pore. Gating shows a similar anomalous behaviour, tightly linking permeation to gating. Eliminating a positive charge at the cytoplasmic end of domain D12 changes kinetics, concentration dependence and halide selectivity of gating, and alters pore properties such as ion selectivity, single-channel conductance and rectification. Taken together, our results strongly suggest that in these channels voltage-dependent gating is conferred by the permeating ion itself, acting as the gating charge.

Several plasma membrane chloride channels are well characterized, but much less is known about the molecular identity and function of intracellular Cl- channels. ClC-3 is thought to mediate swelling-activated plasma membrane currents, but we now show that this broadly expressed chloride channel is present in endosomal compartments and synaptic vesicles of neurons. While swelling-activated currents are unchanged in mice with disrupted ClC-3, acidification of synaptic vesicles is impaired and there is severe postnatal degeneration of the retina and the hippocampus. Electrophysiological analysis of juvenile hippocampal slices revealed no major functional abnormalities despite slightly increased amplitudes of miniature excitatory postsynaptic currents. Mice almost lacking the hippocampus survive and show several behavioral abnormalities but are still able to acquire motor skills.