RESUMO
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.
Assuntos
Canais de Cloreto , Prótons , Animais , Ânions/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Concentração de Íons de Hidrogênio , Transporte de ÍonsRESUMO
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.
Assuntos
Antiporters/metabolismo , Canais de Cloreto/metabolismo , Prótons , Antiporters/genética , Canais de Cloreto/genética , Células HEK293 , Humanos , Ativação do Canal Iônico , Mutação PuntualRESUMO
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.
Assuntos
Canais de Cloreto/metabolismo , Ácido Glutâmico/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Canais de Cloreto/química , Canais de Cloreto/genética , Ácido Glutâmico/química , Ácido Glutâmico/genética , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Mutação de Sentido Incorreto , Prótons , Compostos de Sulfidrila/farmacologiaRESUMO
Idiopathic generalized epilepsy (IGE) is an inherited neurological disorder affecting about 0.4% of the world's population. Mutations in ten genes causing distinct forms of idiopathic epilepsy have been identified so far, but the genetic basis of many IGE subtypes is still unknown. Here we report a gene associated with the four most common IGE subtypes: childhood and juvenile absence epilepsy (CAE and JAE), juvenile myoclonic epilepsy (JME), and epilepsy with grand mal seizures on awakening (EGMA; ref. 8). We identified three different heterozygous mutations in the chloride-channel gene CLCN2 in three unrelated families with IGE. These mutations result in (i) a premature stop codon (M200fsX231), (ii) an atypical splicing (del74-117) and (iii) a single amino-acid substitution (G715E). All mutations produce functional alterations that provide distinct explanations for their pathogenic phenotypes. M200fsX231 and del74-117 cause a loss of function of ClC-2 channels and are expected to lower the transmembrane chloride gradient essential for GABAergic inhibition. G715E alters voltage-dependent gating, which may cause membrane depolarization and hyperexcitability.
Assuntos
Canais de Cloreto/genética , Epilepsia Generalizada/genética , Mutação , Adolescente , Adulto , Sequência de Bases , Membrana Celular/metabolismo , Códon de Terminação , Análise Mutacional de DNA , DNA Complementar/metabolismo , Eletrofisiologia , Saúde da Família , Feminino , Heterozigoto , Humanos , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Modelos Biológicos , Dados de Sequência Molecular , Linhagem , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
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.
Assuntos
Canais de Cloreto/química , Canais de Cloreto/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Prótons , Ânions/metabolismo , Ânions/farmacologia , Eletricidade , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismoRESUMO
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.
Assuntos
Canais de Cloreto/fisiologia , Fosfatos de Inositol/fisiologia , Transdução de Sinais , Transporte Biológico , Linhagem Celular , Canais de Cloreto/antagonistas & inibidores , Humanos , Fosfatos de Inositol/metabolismo , Isoformas de Proteínas/fisiologiaRESUMO
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.
RESUMO
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.
RESUMO
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-/-.
RESUMO
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.
Assuntos
Canais de Cloreto/fisiologia , Capacitância Elétrica , Membranas Intracelulares/fisiologia , Proteínas Musculares/fisiologia , Dinâmica não Linear , Animais , Canais de Cloreto/química , Cobaias , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/química , Ativação do Canal Iônico/fisiologia , Proteínas Musculares/químicaRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular , Canais de Cloreto/genética , Humanos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Transporte de Íons , Cinética , Potenciais da Membrana , Modelos Biológicos , Técnicas de Patch-Clamp , Tiocianatos/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Ativação do Canal Iônico/fisiologia , Rim/fisiologia , Modelos Biológicos , Modelos Químicos , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Canais de Sódio/química , Canais de Sódio/metabolismo , Linhagem Celular , Membrana Celular/fisiologia , Simulação por Computador , Humanos , Canal de Sódio Disparado por Voltagem NAV1.4 , Sódio/química , Sódio/metabolismoRESUMO
Coexpression of KCNQ2 and KCNQ3 channels results in a 10-fold increased current amplitude compared to that of KCNQ2 alone, suggesting the formation of heteromultimeric channels. There is no interaction of either channel with KCNQ1. We evaluated the C-terminus as a potential interaction domain by construction of chimeras with interchanged C-termini of KCNQ1, KCNQ2 and KCNQ3 and functional expression in Xenopus oocytes. The chimera of KCNQ1 with a KCNQ2 C-terminus (Q1ctQ2) showed an 8-fold increase in current amplitude, and Q1ctQ3 a 3-fold increase when coexpressed with KCNQ3 and KCNQ2, respectively, indicating that the C-terminus contains an interaction domain. To characterize this interacting region, we studied further chimeras of KCNQ1 containing different parts of the KCNQ3 C-terminus for interaction with KCNQ2. We also evaluated short sequences of the KCNQ2 C-terminus for a dominant-negative effect on Q1ctQ3. According to the results of these experiments, functional interaction of KCNQ2 and KCNQ3 requires a highly conserved region of about 80 amino acids, previously called the A-domain, plus either 40 residues downstream of the A-domain (B-domain) or the proximal C-terminus between S6 and the A-domain. Furthermore, the chimeras Q1ctQ3 and Q2ctQ3 showed > 10-fold increased current amplitudes compared to KCNQ1 or KCNQ2 alone and a strong depolarizing shift of voltage-dependent activation. The proximal part of the KCNQ3 C-terminus was necessary to produce these effects. Our results indicate that specific parts of the C-terminus enable the interaction between KCNQ2 and KCNQ3 channels and that different parts of the KCNQ3 C-terminus are important for regulating current amplitude.
Assuntos
Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Eletrofisiologia , Feminino , Humanos , Ativação do Canal Iônico/fisiologia , Canal de Potássio KCNQ2 , Canal de Potássio KCNQ3 , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/biossíntese , Canais de Potássio/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Xenopus laevisRESUMO
Cytoplasmic S4-S5 loops have been shown to be involved in fast inactivation of voltage-gated ion channels. We studied mutations in these loops and their potential cooperative effects in domains D3 (N1151C, A1152C, I1160C/A) and D4 (F1473C, L1482C/A) of the human skeletal muscle Na(+) channel alpha-subunit (hNa(v)1.4) using expression in tsA201 cells and the whole cell patch-clamp technique. All cysteine mutations were accessible to intracellularly applied sulfhydryl reagents which considerably destabilized fast inactivation. For different combinations of corresponding D3/D4 double mutations, fast inactivation could be almost completely removed. Thermodynamic cycle analysis indicated an additive effect for N1151C/F1473C and a significant cooperative effect for I1160/L1482 double mutations. Application of oxidizing reagents such as Cu-phenanthroline to link two cysteines via a disulfide bridge did not reveal evidence for a direct physical interaction of cysteines in D3 and D4. In addition to the pronounced alterations of fast inactivation, mutations of I1160 shifted steady-state activation in the hyperpolarizing direction and slowed the kinetics of both activation and deactivation. Sulfhydryl reagents had charge-dependent effects on I1160C suggesting interaction with negative charges in another protein region. We conclude that fast inactivation of the Na(+) channel involves both S4-S5 loops in D3 and D4 in a cooperative manner. D3/S4-S5 also plays an important role in activation and deactivation.