Metabolic energy sensing by mammalian CLC anion/proton exchangers.

CLC anion/proton exchangers control the pH and [Cl- ] of the endolysosomal system that is essential for cellular nutrient uptake. Here, we use heterologous expression and whole-cell electrophysiology to investigate the regulation of the CLC isoforms ClC-3, ClC-4, and ClC-5 by the adenylic system components ATP, ADP, and AMP. Our results show that cytosolic ATP and ADP but not AMP and Mg2+ -free ADP enhance CLC ion transport. Biophysical analysis reveals that adenine nucleotides alter the ratio between CLC ion transport and CLC gating charge and shift the CLC voltage-dependent activation. The latter effect is suppressed by blocking the intracellular entrance of the proton transport pathway. We suggest, therefore, that adenine nucleotides regulate the internal proton delivery into the CLC transporter machinery and alter the probability of CLC transporters to undergo silent non-transporting cycles. Our findings suggest that the CBS domains in mammalian CLC transporters serve as energy sensors that regulate vesicular Cl- /H+ exchange by detecting changes in the cytosolic ATP/ADP/AMP equilibrium. Such sensing mechanism links the endolysosomal activity to the cellular metabolic state.

Barttin Regulates the Subcellular Localization and Posttranslational Modification of Human Cl-/H+ Antiporter ClC-5.

Dent disease 1 (DD1) is a renal salt-wasting tubulopathy associated with mutations in the Cl-/H+ antiporter ClC-5. The disease typically manifests with proteinuria, hypercalciuria, nephrocalcinosis, and nephrolithiasis but is characterized by large phenotypic variability of no clear origin. Several DD1 cases have been reported lately with additional atypical hypokalemic metabolic alkalosis and hyperaldosteronism, symptoms usually associated with another renal disease termed Bartter syndrome (BS). Expression of the Bartter-like DD1 mutant ClC-5 G261E in HEK293T cells showed that it is retained in the ER and lacks the complex glycosylation typical for ClC-5 WT. Accordingly, the mutant abolished CLC ionic transport. Such phenotype is not unusual and is often observed also in DD1 ClC-5 mutants not associated with Bartter like phenotype. We noticed, therefore, that one type of BS is associated with mutations in the protein barttin that serves as an accessory subunit regulating the function and subcellular localization of ClC-K channels. The overlapping symptomatology of DD1 and BS, together with the homology between the proteins of the CLC family, led us to investigate whether barttin might also regulate ClC-5 transport. In HEK293T cells, we found that barttin cotransfection impairs the complex glycosylation and arrests ClC-5 in the endoplasmic reticulum. As barttin and ClC-5 are both expressed in the thin and thick ascending limbs of the Henle's loop and the collecting duct, interactions between the two proteins could potentially contribute to the phenotypic variability of DD1. Pathologic barttin mutants differentially regulated trafficking and processing of ClC-5, suggesting that the interaction between the two proteins might be relevant also for the pathophysiology of BS. Our findings show that barttin regulates the subcellular localization not only of kidney ClC-K channels but also of the ClC-5 transporter, and suggest that ClC-5 might potentially play a role not only in kidney proximal tubules but also in tubular kidney segments expressing barttin. In addition, they demonstrate that the spectrum of clinical, genetic and molecular pathophysiology investigation of DD1 should be extended.

Early cell death induced by Clostridium difficile TcdB: Uptake and Rac1-glucosylation kinetics are decisive for cell fate.

Toxin A and Toxin B (TcdA/TcdB) are large glucosyltransferases produced by Clostridium difficile. TcdB but not TcdA induces reactive oxygen species-mediated early cell death (ECD) when applied at high concentrations. We found that nonglucosylated Rac1 is essential for induction of ECD since inhibition of Rac1 impedes this effect. ECD only occurs when TcdB is rapidly endocytosed. This was shown by generation of chimeras using the trunk of TcdB from a hypervirulent strain. TcdB from hypervirulent strain has been described to translocate from endosomes at higher pH values and thus, meaning faster than reference type TcdB. Accordingly, intracellular delivery of the glucosyltransferase domain of reference TcdB by the trunk of TcdB from hypervirulent strain increased ECD. Furthermore, proton transporters such as sodium/proton exchanger (NHE) or the ClC-5 anion/proton exchanger, both of which contribute to endosomal acidification, also affected cytotoxic potency of TcdB: Specific inhibition of NHE reduced cytotoxicity, whereas transfection of cells with the endosomal anion/proton exchanger ClC-5 increased cytotoxicity of TcdB. Our data suggest that both the uptake rate of TcdB into the cytosol and the status of nonglucosylated Rac1 are key determinants that are decisive for whether ECD or delayed apoptosis is triggered.

A novel CLCN5 pathogenic mutation supports Dent disease with normal endosomal acidification.

Dent disease is an X-linked recessive renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, nephrocalcinosis, and progressive renal failure. Inactivating mutations of CLCN5, the gene encoding the 2Cl- /H+ exchanger ClC-5, have been reported in patients with Dent disease 1. In vivo studies in mice harboring an artificial mutation in the "gating glutamate" of ClC-5 (c.632A > C, p.Glu211Ala) and mathematical modeling suggest that endosomal chloride concentration could be an important parameter in endocytosis, rather than acidification as earlier hypothesized. Here, we described a novel pathogenic mutation affecting the "gating glutamate" of ClC-5 (c.632A>G, p.Glu211Gly) and investigated its molecular consequences. In HEK293T cells, the p.Glu211Gly ClC-5 mutant displayed unaltered N-glycosylation and normal plasma membrane and early endosomes localizations. In Xenopus laevis oocytes and HEK293T cells, we found that contrasting with wild-type ClC-5, the mutation abolished the outward rectification, the sensitivity to extracellular H+ and converted ClC-5 into a Cl- channel. Investigation of endosomal acidification in HEK293T cells using the pH-sensitive pHluorin2 probe showed that the luminal pH of cells expressing a wild-type or p.Glu211Gly ClC-5 was not significantly different. Our study further confirms that impaired acidification of endosomes is not the only parameter leading to defective endocytosis in Dent disease 1.

Overexpression of the Endosomal Anion/Proton Exchanger ClC-5 Increases Cell Susceptibility toward Clostridium difficile Toxins TcdA and TcdB.

Virulent C. difficile toxins TcdA and TcdB invade host intestinal epithelia by endocytosis and use the acidic environment of intracellular vesicles for further processing and activation. We investigated the role of ClC-5, a chloride/proton exchanger expressed in the endosomes of gastrointestinal epithelial cells, in the activation and processing of C. difficile toxins. Enhanced intoxication by TcdA and TcdB was observed in cells expressing ClC-5 but not ClC-4, another chloride/proton exchanger with similar function but different localization. In accordance with the established physiological function of ClC-5, its expression lowered the endosomal pH in HEK293T cells by approximately 0.6 units and enhanced approximately 5-fold the internalization of TcdA. In colon HT29 cells, 34% of internalized TcdA localized to ClC-5-containing vesicles defined by colocalization with Rab5, Rab4a, and Rab7 as early and early-to-late of endosomes but not as Rab11-containing recycling endosomes. Impairing the cellular uptake of TcdA by deleting the toxin CROPs domain did not abolish the effects of ClC-5. In addition, the transport-incompetent mutant ClC-5 E268Q similarly enhanced both endosomal acidification and intoxication by TcdA but facilitated the internalization of the toxin to a lower extent. These data suggest that ClC-5 enhances the cytotoxic action of C. difficile toxins by accelerating the acidification and maturation of vesicles of the early and early-to-late endosomal system. The dispensable role of electrogenic ion transport suggests that the voltage-dependent nonlinear capacitances of mammalian CLC transporters serve important physiological functions. Our data shed light on the intersection between the endocytotic cascade of host epithelial cells and the internalization pathway of the large virulence C. difficile toxins. Identifying ClC-5 as a potential specific host ion transporter hijacked by toxins produced by pathogenic bacteria widens the horizon of possibilities for novel therapies of life-threatening gastrointestinal infections.

Mutations associated with Dent's disease affect gating and voltage dependence of the human anion/proton exchanger ClC-5.

Dent's disease is associated with impaired renal endocytosis and endosomal acidification. It is linked to mutations in the membrane chloride/proton exchanger ClC-5; however, a direct link between localization in the protein and functional phenotype of the mutants has not been established until now. Here, two Dent's disease mutations, G212A and E267A, were investigated using heterologous expression in HEK293T cells, patch-clamp measurements and confocal imaging. WT and mutant ClC-5 exhibited mixed cell membrane and vesicular distribution. Reduced ion currents were measured for both mutants and both exhibited reduced capability to support endosomal acidification. Functionally, mutation G212A was capable of mediating anion/proton antiport but dramatically shifted the activation of ClC-5 toward more depolarized potentials. The shift can be explained by impeded movements of the neighboring gating glutamate Gluext, a residue that confers major part of the voltage dependence of ClC-5 and serves as a gate at the extracellular entrance of the anion transport pathway. Cell surface abundance of E267A was reduced by ~50% but also dramatically increased gating currents were detected for this mutant and accordingly reduced probability to undergoing cycles associated with electrogenic ion transport. Structurally, the gating alternations correlate to the proximity of E267A to the proton glutamate Gluin that serves as intracellular gate in the proton transport pathway and regulates the open probability of ClC-5. Remarkably, two other mammalian isoforms, ClC-3 and ClC-4, also differ from ClC-5 in gating characteristics affected by the here investigated disease-causing mutations. This evolutionary specialization, together with the functional defects arising from mutations G212A and E267A, demonstrate that the complex gating behavior exhibited by most of the mammalian CLC transporters is an important determinant of their cellular function.

Multiple discrete transitions underlie voltage-dependent activation in CLC Cl(-)/H(+) antiporters.

Most mammalian chloride channels and transporters in the CLC family display pronounced voltage-dependent gating. Surprisingly, despite the complex nature of the gating process and the large contribution to it by the transport substrates, experimental investigations of the fast gating process usually produce canonical Boltzmann activation curves that correspond to a simple two-state activation. By using nonlinear capacitance measurements of two mutations in the ClC-5 transporter, here we are able to discriminate and visualize discrete transitions along the voltage-dependent activation pathway. The strong and specific dependence of these transitions on internal and external [Cl(-)] suggest that CLC gating involves voltage-dependent conformational changes as well as coordinated movement of transported substrates.

Involvement of ClC-3 chloride/proton exchangers in controlling glutamatergic synaptic strength in cultured hippocampal neurons.

ClC-3 is a member of the CLC family of anion channels and transporters that localizes to early and late endosomes as well as to synaptic vesicles (SV). Its genetic disruption in mouse models results in pronounced hippocampal and retinal neurodegeneration, suggesting that ClC-3 might be important for normal excitatory and/or inhibitory neurotransmission in central neurons. To characterize the role of ClC-3 in glutamate accumulation in SV we compared glutamatergic synaptic transmission in cultured hippocampal neurons from WT and Clcn3-/- mice. In Clcn3-/- neurons the amplitude and frequency of miniature as well as the amplitudes of action-potential evoked EPSCs were significantly increased as compared to WT neurons. The low-affinity competitive AMPA receptor antagonist γ-DGG reduced the quantal size of synaptic events more effectively in WT than in Clcn3-/- neurons, whereas no difference was observed for the high-affinity competitive non-NMDA antagonist NBQX. Paired pulse ratios of evoked EPSCs were significantly reduced, whereas the size of the readily releasable pool was not affected by the genetic ablation of ClC-3. Electron microscopy revealed increased volumes of SV in hippocampi of Clcn3-/- mice. Our findings demonstrate that ClC-3 controls fast excitatory synaptic transmission by regulating the amount of neurotransmitter as well as the release probability of SV. These results provide novel insights into the role of ClC-3 in synaptic transmission and identify excessive glutamate release as a likely basis of neurodegeneration in Clcn3-/-.

ClC-3 is an intracellular chloride/proton exchanger with large voltage-dependent nonlinear capacitance.

The chloride/proton exchangers ClC-3, ClC-4 and ClC-5 are localized in distinct intracellular compartments and regulate their luminal acidity. We used electrophysiology combined with fluorescence pH measurements to compare the functions of these three transporters. Since the expression of WT ClC-3 in the surface membrane was negligible, we removed an N-terminal retention signal for standard electrophysiological characterization of this isoform. This construct (ClC-313-19A) mediated outwardly rectifying coupled Cl(-)/H(+) antiport resembling the properties of ClC-4 and ClC-5. In addition, ClC-3 exhibited large electric capacitance, exceeding the nonlinear capacitances of ClC-4 and ClC-5. Mutations of the proton glutamate, a conserved residue at the internal side of the protein, decreased ion transport but increased nonlinear capacitances in all three isoforms. This suggests that nonlinear capacitances in mammalian ClC transporters are regulated in a similar manner. However, the voltage dependence and the amplitudes of these capacitances differed strongly between the investigated isoforms. Our results indicate that ClC-3 is specialized in mainly performing incomplete capacitive nontransporting cycles, that ClC-4 is an effective coupled transporter, and that ClC-5 displays an intermediate phenotype. Mathematical modeling showed that such functional differences would allow differential regulation of luminal acidification and chloride concentration in intracellular compartments.

ClC1 chloride channel in myotonic dystrophy type 2 and ClC1 splicing in vitro.

Myotonic dystrophy type 2 (DM2) is caused by CCTG-repeat expansions. Occurrence of splicing and mutations in the muscle chloride channel gene CLCN1 have been reported to contribute to the phenotype. To examine the effect of CLCN1 in DM2 in Germany, we determined the frequency of a representative ClC1 mutation, R894X, and its effect on DM2 clinical features. Then, we examined CLCN1 mRNA splice variants in patient muscle functionally expressed the most abundant variant, and determined its subcellular localization. Finally, we established a cellular system for studying mouse clcn1 pre-mRNA splicing and tested effects of expression of (CCUG)18, (CUG)24 and (AAG)24 RNAs. The R894X mutation was present in 7.7% of DM2 families. DM2 R894X-carriers had more myotonia and myalgia than non-carriers. The most abundant CLCN1 splice variant in DM2 (80% of all transcripts) excluded exons 6-7 and lead to a truncated ClC1(236X) protein. Heterologous ClC1(236X) expression did not yield functional channels. Co-expression with ClC1 did not show a dominant negative effect, but a slightly suppressive effect. In C2C12 cells, the clc1 splice variants generated by (CCUG)18-RNA resembled those in DM2 muscle and differed from those generated by (CUG)24 and (AAG)24. We conclude that ClC1 mutations exert gene dose effects and enhance myotonia and pain in DM2 in Germany. Additionally, the ClC1(236X) splice variant may contribute to myotonia in DM2. Since splice variants depend on the types of repeats expressed in the cellular C2C12 model, similar cell models of other tissues may be useful for studying repeatdependent pathogenetic mechanisms more easily than in transgenic animals.

Glutamate 268 regulates transport probability of the anion/proton exchanger ClC-5.

The Cl(-)/H(+) exchange mediated by ClC transporters can be uncoupled by external SCN(-) and mutations of the proton glutamate, a conserved residue at the internal side of the protein. We show here for the mammalian ClC transporter ClC-5 that acidic internal pH led to a greater increase in currents upon exchanging extracellular Cl(-) for SCN(-). However, transport uncoupling, unitary current amplitudes, and the voltage dependence of the depolarization-induced activation were not altered by low pH values. Therefore, it is likely that an additional gating process regulates ClC-5 transport. Higher internal [H(+)] and the proton glutamate mutant E268H altered the ratio between ClC-5 transport and nonlinear capacitance, indicating that the gating charge movements in ClC-5 arise from incomplete transport cycles and that internal protons increase the transport probability of ClC-5. This was substantiated by site-directed sulfhydryl modification of the proton glutamate mutant E268C. The mutation exhibited small transport currents together with prominent gating charge movements. The charge restoration using a negatively charged sulfhydryl reagent reinstated also the WT phenotype. Neutralization of the charge of the gating glutamate 211 by the E211C mutation abolished the effect of internal protons, showing that the increased transport probability of ClC-5 results from protonation of this residue. S168P (a mutation that decreases the anion affinity of the central binding site) reduced also the internal pH dependence of ClC-5. These results support the idea that protonation of the gating glutamate 211 at the central anion-binding site of ClC-5 is mediated by the proton glutamate 268.

Anion- and proton-dependent gating of ClC-4 anion/proton transporter under uncoupling conditions.

ClC-4 is a secondary active transporter that exchanges Cl(-) ions and H(+) with a 2:1 stoichiometry. In external SCN(-), ClC-4 becomes uncoupled and transports anions with high unitary transport rate. Upon voltage steps, the number of active transporters varies in a time-dependent manner, resembling voltage-dependent gating of ion channels. We here investigated modification of the voltage dependence of uncoupled ClC-4 by protons and anions to quantify association of substrates with the transporter. External acidification shifts voltage dependence of ClC-4 transport to more positive potentials and leads to reduced transport currents. Internal pH changes had less pronounced effects. Uncoupled ClC-4 transport is facilitated by elevated external [SCN(-)] but impaired by internal Cl(-) and I(-). Block by internal anions indicates the existence of an internal anion-binding site with high affinity that is not present in ClC channels. The voltage dependence of ClC-4 coupled transport is modulated by external protons and internal Cl(-) in a manner similar to what is observed under uncoupling conditions. Our data illustrate functional differences but also similarities between ClC channels and transporters.

Channel-like slippage modes in the human anion/proton exchanger ClC-4.

The ClC family encompasses two classes of proteins with distinct transport functions: anion channels and transporters. ClC-type transporters usually mediate secondary active anion-proton exchange. However, under certain conditions they assume slippage mode behavior in which proton and anion transport are uncoupled, resulting in passive anion fluxes without associated proton movements. Here, we use patch clamp and intracellular pH recordings on transfected mammalian cells to characterize exchanger and slippage modes of human ClC-4, a member of the ClC transporter branch. We found that the two transport modes differ in transport mechanisms and transport rates. Nonstationary noise analysis revealed a unitary transport rate of 5 x 10(5) s(-1) at +150 mV for the slippage mode, indicating that ClC-4 functions as channel in this mode. In the exchanger mode, unitary transport rates were 10-fold lower. Both ClC-4 transport modes exhibit voltage-dependent gating, indicating that there are active and non-active states for the exchanger as well as for the slippage mode. ClC-4 can assume both transport modes under all tested conditions, with exchanger/channel ratios determined by the external anion. We propose that binding of transported anions to non-active states causes transition from slippage into exchanger mode. Binding and unbinding of anions is very rapid, and slower transitions of liganded and non-liganded states into active conformations result in a stable distribution between the two transport modes. The proposed mechanism results in anion-dependent conversion of ClC-type exchanger into an anion channel with typical attributes of ClC anion channels.

Anion channels: regulation of ClC-3 by an orphan second messenger.

ClC-3 is a ubiquitously expressed chloride channel isoform whose biological function has been a matter of debate for many years. A recent study reporting its regulation by Ins(3,4,5,6)P(4) assigns novel transport functions and cellular roles to ClC-3 and identifies a regulatory pathway that affects epithelial transport and endosomal pH regulation.

Gating of human ClC-2 chloride channels and regulation by carboxy-terminal domains.

Eukaryotic ClC channels are dimeric proteins with each subunit forming an individual protopore. Single protopores are gated by a fast gate, whereas the slow gate is assumed to control both protopores through a cooperative movement of the two carboxy-terminal domains. We here study the role of the carboxy-terminal domain in modulating fast and slow gating of human ClC-2 channels, a ubiquitously expressed ClC-type chloride channel involved in transepithelial solute transport and in neuronal chloride homeostasis. Partial truncation of the carboxy-terminus abolishes function of ClC-2 by locking the channel in a closed position. However, unlike other isoforms, its complete removal preserves function of ClC-2. ClC-2 channels without the carboxy-terminus exhibit fast and slow gates that activate and deactivate significantly faster than in WT channels. In contrast to the prevalent view, a single carboxy-terminus suffices for normal slow gating, whereas both domains regulate fast gating of individual protopores. Our findings demonstrate that the carboxy-terminus is not strictly required for slow gating and that the cooperative gating resides in other regions of the channel protein. ClC-2 is expressed in neurons and believed to open at negative potentials and increased internal chloride concentrations after intense synaptic activity. We propose that the function of the ClC-2 carboxy-terminus is to slow down the time course of channel activation in order to stabilize neuronal excitability.

Barttin modulates trafficking and function of ClC-K channels.

Barttin is an accessory subunit of a subgroup of ClC-type chloride channels expressed in renal and inner ear epithelia. In this study, we examined the effects of barttin on two ClC-K channel isoforms, rat ClC-K1 and human ClC-Kb, using heterologous expression, patch clamping, confocal imaging, and flow cytometry. In the absence of barttin, only a small percentage of rClC-K1 and hClC-Kb channels are inserted into the plasma membrane. Coexpression of barttin enhances surface membrane insertion and furthermore modifies permeation and gating of ClC-K channels. hClC-Kb channels are nonfunctional without barttin and require the coexpressed accessory subunit to become anion conducting. In contrast, rClC-K1 channels are active without barttin, but at the cost of reduced unitary conductance as well as altered voltage dependence of activation. We mapped the separate functions of barttin to structural domains by a deletion analysis. Whereas the transmembrane core is necessary and sufficient to promote ClC-K channel exit from the endoplasmic reticulum, a short cytoplasmic segment following the second transmembrane helix modifies the unitary conductance. The entire cytoplasmic carboxyl terminus affects the open probability of ClC-K channels. The multiple functions of barttin might be necessary for a tight adjustment of epithelial Cl(-) conductances to ensure a precise regulation of body salt content and endocochlear potential.

Modeling of single noninactivating Na+ channels: evidence for two open and several fast inactivated states.

Voltage-gated Na(+) channels play a fundamental role in the excitability of nerve and muscle cells. Defects in fast Na(+) channel inactivation can cause hereditary muscle diseases with hyper- or hypoexcitability of the sarcolemma. To explore the kinetics and gating mechanisms of noninactivating muscle Na(+) channels on a molecular level, we analyzed single channel currents from wild-type and five mutant Na(+) channels. The mutations were localized in different protein regions which have been previously shown to be important for fast inactivation (D3-D4-linker, D3/S4-S5, D4/S4-S5, D4/S6) and exhibited distinct grades of defective fast inactivation with varying levels of persistent Na(+) currents caused by late channel reopenings. Different gating schemes were fitted to the data using hidden Markov models with a correction for time interval omission and compared statistically. For all investigated channels including the wild-type, two open states were necessary to describe our data. Whereas one inactivated state was sufficient to fit the single channel behavior of wild-type channels, modeling the mutants with impaired fast inactivation revealed evidence for several inactivated states. We propose a single gating scheme with two open and three inactivated states to describe the behavior of all five examined mutants. This scheme provides a biological interpretation of the collected data, based on previous investigations in voltage-gated Na(+) and K(+) channels.

Preferred mexiletine block of human sodium channels with IVS4 mutations and its pH-dependence.

The effects of extracellular pH (6.2, 7.4 and 8.2) and 0.1 mM mexiletine, a channel blocker of the lidocaine type, are studied on two mutations of the fourth voltage sensor of the Nav1.4 sodium channel, R1448H/C. The fast inactivated channel state to which mexiletine preferentially binds is destabilized by the mutations. By contrast to the expected low response of R1448H/C carriers, mexiletine is particularly effective in preventing exercise-induced stiffness and paralysis from which these patients suffer. Our measurements performed in the whole-cell mode on stably transfected HEK cells show for the first time that the mutations strikingly accelerate closed-state inactivation and, as steady-state fast inactivation is shifted to more negative potentials, stabilize the fast inactivated channel state in the potential range around the resting potential. At pH 7.4 and 8.2, the phasic mexiletine block is larger for R1448C (55%) and R1448H (47%) than for wild-type channels (31%) due to slowed recovery from block (tau is approximately 520 ms for R1448C versus 270 ms for wild-type at pH 7.4) although the recovery from inactivation is slightly faster for the mutants (tau is approximately 1.9 ms for R1448C versus 3.8 ms for wild-type at pH 7.4). At pH 6.2, recovery from block is relatively fast (tau is approximately 35 ms for R1448H/C and 14 ms for wild-type) and thus shows no use-dependence. We conclude that enhanced closed-state inactivation expands the concept of a mutation-induced uncoupling of channel inactivation from activation to a new potential range and that the higher mexiletine efficacy in R1448H/C carriers compared to other myotonic patients offers a pharmacogenetic strategy for mutation-specific treatment.