Alexi K. Alekov

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

CLCN2 variants in idiopathic generalized epilepsy

To the Editor: In 2003, we reported that mutations in CLCN2, the gene encoding the voltage-gated chloride channel ClC-2, are associated with the four major subtypes of idiopathic generalized epilepsy in three pedigrees with an apparent autosomal dominant mode of inheritance. We concluded that these mutations confer a major gene effect. The observed heterogeneity of the epileptic phenotypes prompted us to suggest that other, unknown genes might also be involved, in agreement with complex inheritance1. Re-examination of the families and the molecular genetic data by a neurologist and a geneticist who were not involved in the original study has now revealed major differences from the published data in two of the three published pedigrees (presented in Figs. 1a and 1b of the aforementioned publication1), which are described in detail below. All studies were approved by the Ethics Committee of the University of Bonn and informed consent was obtained from all subjects. In the previously published pedigree of Family 1 (ref. 1), the identified mutation cosegregates in an autosomal dominant manner in five affected family members. Three of these were indicated to be suffering from juvenile myoclonic epilepsy (JME) and one from epilepsy with grand mal (also known as generalized tonic-clonic) seizures on awakening (EGMA), and one was reported as showing generalized spike and wave discharges on an EEG without being clinically affected (Fig. 1a). The family was re-contacted and re-analyzed. This second evaluation revealed that only the index case had JME. She reported classical bilateral myoclonic jerks on awakening since the age of 17 and one generalized tonicclonic seizure in the morning that occurred when she was 18 years of age. She has been treated with valproate and has since remained seizure free for more than 15 years. EEG recordings were available only from the period when she was receiving valproate, and they did not show epileptic discharges. No other family members were reported as suffering from epileptic seizures, and the pedigree structure was found to be different from that initially described (Fig. 1b). We re-collected blood samples from nine available family members (Fig. 1b). The family structure was re-evaluated through molecular fingerprinting with 15 highly polymorphic STR markers and one sex-specific marker (Powerplex 16, Promega). The family structure obtained through the second recruitment proved to be correct. Re-sequencing of the CLCN2 gene revealed the published mutation c.597insG (p.M200fsX231) in three family members (the index patient, her unaffected sister and her unaffected father). Furthermore, three of the original DNA samples with the mutation proved to stem from one individual. The previously published pedigree of Family 2 (ref. 1) indicates an autosomal dominant form of epilepsy with eight affected members. The index case was reported as suffering from childhood absence epilepsy (CAE), his sister as showing generalized spike and wave discharges on the EEG without being clinically affected, five other family members as suffering from EGMA, and the deceased great-grandfather of the index case was reported to have had unclassifiable epileptic seizures (Fig. 1c). The family was re-contacted and the clinical phenotypes were re-evaluated using information obtained from the parents of the index I

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-/-.

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.

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.

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.

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

Dents 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 Dents 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.

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.