ClC chloride channels

Chloride-conducting ion channels of the ClC family are emerging as critical contributors to a host of biological processes. These polytopic membrane proteins form aqueous pathways through which anions are selectively allowed to pass down their concentration gradients. The ClCs are found in nearly all organisms, with members in every mammalian tissue, yet relatively little is known about their mechanism or regulation. It is clear, however, that they are fundamentally different in molecular construction and mechanism from the well-known potassium-, sodium-, and calcium-selective channels. The medical importance of ClC channels - four inherited diseases have been blamed on familial ClC dysfunction to date - highlights their diverse physiological functions and provides strong motivation for further study.     

Bead-like passage of chloride ions through ClC chloride channels

The ClC chloride channels control the ionic composition of the cytoplasm and the volume of cells, and regulate electrical excitability. Recently, it has been proposed that prokaryotic ClC channels are H+-Cl- exchange transporter. Although X-ray and molecular dynamics (MD) studies of bacterial ClC channels have investigated the filter open-close and ion permeation mechanism of channels, details have remained unclear. We performed MD simulations of ClC channels involving H+, Na+, K+, or H3O+ in the intracellular region to elucidate the open-close mechanism, and to clarify the role of H+ ion an H+-Cl- exchange transporter. Our simulations revealed that H+ and Na+ caused channel opening and the passage of Cl- ions. Na+ induced a bead-like string of Cl- -Na+-Cl--Na+-Cl- ions to form and permeate through ClC channels to the intracellular side with the widening of the channel pathway.     

The ClC family of chloride channels and transporters

The ClC proteins are members of a large family of chloride transport proteins, which are involved in a variety of physiological processes. All family members share a conserved molecular architecture consisting of a complex transmembrane transport domain and a soluble regulatory domain. To date, representative structures for the two parts are available, the transmembrane domain from the structure of a bacterial homologue, the soluble domain from a eukaryotic family member. Despite the strong conservation of the structural framework, the family members show an unusually broad variety of functional behaviors, as some members work as gated chloride channels and others as secondary chloride transporters. The conservation in the structure and the functional resemblance in gating and transport mechanism suggest a strong mechanistic relationship between seemingly contradictory transport modes.     

Ion transit pathways and gating in ClC chloride channels

ClC chloride channels possess a homodimeric structure in which each monomer contains an independent chloride ion pathway. ClC channel gating is regulated by chloride ion concentration, pH and voltage. Based on structural and physiological evidence, it has been proposed that a glutamate residue on the extracellular end of the selectivity filter acts as a fast gate. We utilized a new search algorithm that incorporates electrostatic information to explore the ion transit pathways through wild-type and mutant bacterial ClC channels. Examination of the chloride ion permeation pathways supports the importance of the glutamate residue in gating. An external chloride binding site previously postulated in physiological experiments is located near a conserved basic residue adjacent to the gate. In addition, access pathways are found for proton migration to the gate, enabling pH control at hyperpolarized membrane potentials. A chloride ion in the selectivity filter is required for the pH-dependent gating mechanism.     

Intracellular localization of ClC chloride channels and their ability to form hetero-oligomers

ClC chloride channels (ClCs) can be classified into two groups in terms of their cellular localizations: ClCs present in the plasma membranes and those residing in intracellular organelles. Members of the latter group, including ClC-3, ClC-4, ClC-5, ClC-6, and ClC-7, are often co-expressed in a variety of cell types in many organs. Although the localization of individual channels within cells has been investigated, the degree of overlap between the locations of different ClCs in the same cell has not been clarified. To address this question, different combinations of ClCs, engineered to encode specific epitope tags (FLAG or HA), were either transiently or stably transfected into HEK293 cells, and we then compared the intracellular localization of the expressed channel proteins by immunofluorescence microscopy. Immunofluorescence images of the alternatively labeled channels clearly showed significant co-localization between all pair-wise combinations of ClCs. In particular, ClC-3, ClC-4, and ClC-5 showed a high degree of co-localization. As a significant degree of co-localization between ClCs was observed, we used co-immunoprecipitation to evaluate oligomer formation, and found that each ClC tested could form homo-oligomers, and that any pair-wise combination of ClC-3, ClC-4, and ClC-5 could also form hetero-oligomers. Neither ClC-6 nor ClC-7 was co-precipitated with any other channel protein. These results suggest that within cells ClC-3, ClC-4, and ClC-5 may have combinatorial functions, whereas ClC-6 and ClC-7 are more likely to function as homo-oligomers.     

Structural basis for ion conduction and gating in ClC chloride channels

Members of the ClC family of voltage-gated chloride channels are found from bacteria to mammals with a considerable degree of conservation in the membrane-inserted, pore-forming region. The crystal structures of the ClC channels of Escherichia coli and Salmonella typhimurium provide a structural framework for the entire family. The ClC channels are homodimeric proteins with an overall rhombus-like shape. Each ClC dimer has two pores each contained within a single subunit. The ClC subunit consists of two roughly repeated halves that span the membrane with opposite orientations. This antiparallel architecture defines a chloride selectivity filter within the 15-A neck of a hourglass-shaped pore. Three Cl(-) binding sites within the selectivity filter stabilize ions by interactions with alpha-helix dipoles and by chemical interactions with nitrogen atoms and hydroxyl groups of residues in the protein. 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.     

Cysteine modification of a putative pore residue in ClC-0: implication for the pore stoichiometry of ClC chloride channels

The ClC channel family consists of chloride channels important for various physiological functions. Two members in this family, ClC-0 and ClC-1, share approximately 50-60% amino acid identity and show similar gating behaviors. Although they both contain two subunits, the number of pores present in the homodimeric channel is controversial. The double-barrel model proposed for ClC-0 was recently challenged by a one-pore model partly based on experiments with ClC-1 exploiting cysteine mutagenesis followed by modification with methanethiosulfonate (MTS) reagents. To investigate the pore stoichiometry of ClC-0 more rigorously, we applied a similar strategy of MTS modification in an inactivation-suppressed mutant (C212S) of ClC-0. Mutation of lysine 165 to cysteine (K165C) rendered the channel nonfunctional, but modification of the introduced cysteine by 2-aminoethyl MTS (MTSEA) recovered functional channels with altered properties of gating-permeation coupling. The fast gate of the MTSEA-modified K165C homodimer responded to external Cl(-) less effectively, so the P(o)-V curve was shifted to a more depolarized potential by approximately 45 mV. The K165C-K165 heterodimer showed double-barrel-like channel activity after MTSEA modification, with the fast-gating behaviors mimicking a combination of those of the mutant and the wild-type pore, as expected for the two-pore model. Without MTSEA modification, the heterodimer showed only one pore, and was easier to inactivate than the two-pore channel. These results showed that K165 is important for both the fast and slow gating of ClC-0. Therefore, the effects of MTS reagents on channel gating need to be carefully considered when interpreting the apparent modification rate.     

Ion-binding properties of the ClC chloride selectivity filter

The ClC channels are members of a large protein family of chloride (Cl-) channels and secondary active Cl- transporters. Despite their diverse functions, the transmembrane architecture within the family is conserved. Here we present a crystallographic study on the ion-binding properties of the ClC selectivity filter in the close homolog from Escherichia coli (EcClC). The ClC selectivity filter contains three ion-binding sites that bridge the extra- and intracellular solutions. The sites bind Cl- ions with mM affinity. Despite their close proximity within the filter, the three sites can be occupied simultaneously. The ion-binding properties are found conserved from the bacterial transporter EcClC to the human Cl- channel ClC-1, suggesting a close functional link between ion permeation in the channels and active transport in the transporters. In resemblance to K+ channels, ions permeate the ClC channel in a single file, with mutual repulsion between the ions fostering rapid conduction.     

Different fast-gate regulation by external Cl(-) and H(+) of the muscle-type ClC chloride channels.

The fast gate of the muscle-type ClC channels (ClC-0 and ClC-1) opens in response to the change of membrane potential (V). This gating process is intimately associated with the binding of external Cl(-) to the channel pore in a way that the occupancy of Cl(-) on the binding site increases the channel's open probability (P(o)). External H(+) also enhances the fast-gate opening in these channels, prompting a hypothesis that protonation of the binding site may increase the Cl(-) binding affinity, and this is possibly the underlying mechanism for the H(+) modulation. However, Cl(-) and H(+), modulate the fast-gate P(o)-V curve in different ways. Varying the external Cl(-) concentrations ([Cl(-)](o)) shifts the P(o)-V curve in parallel along the voltage axis, whereas reducing external pH mainly increases the minimal P(o) of the curve. Furthermore, H(+) modulations at saturating and nonsaturating [Cl(-)](o) are similar. Thus, the H(+) effect on the fast gating appears not to be a consequence of an increase in the Cl(-) binding affinity. We previously found that a hyperpolarization-favored opening process is important to determine the fast-gate P(o) of ClC-0 at very negative voltages. This [Cl(-)](o)-independent mechanism attracted little attention, but it appears to be the opening process that is modulated by external H(+).     

Exploring the gating mechanism in the ClC chloride channel via metadynamics

Computer simulations have been used to probe the gating mechanism in the Salmonella serovar typhimurium chloride channel (st-ClC). Specifically, the recently developed metadynamics methodology has been exploited to construct free energy surfaces as a function of the positions of either one or two chloride ions inside the pore, the position and protonation state of the key E148 residue, and the number of water molecules coordinating the translocating ions. The present calculations confirm the multi-ion mechanism in which an ion-push-ion effect lowers the main barriers to chloride ion translocation. When a second anion is taken into account, the barrier for chloride passage through the E148 narrow region is computed to be 6 kcal/mol in the wild-type channel, irrespective of the protonation state of the E148 residue, which is shown to only affect the entrance barrier. In the E148A mutant, this barrier is much lower, amounting to 3 kcal/mol. The metadynamics calculations reported herein also demonstrate that before reaching the periplasmic solution, chloride ions have to overcome an additional barrier arising from two different effects, namely the rearrangement of their solvation shell and a flip in the backbone angles of the residues E148 and G149, which reside at the end of the alphaF helix.     

ClC channels: leaving the dark ages on the verge of a new millennium.

The field of molecular physiology of ClC chloride channels has witnessed a tremendous surge in knowledge over the past few years; however, fundamental issues such as the stoichiometry of ClC channels and the identification of pore-lining sequences have only recently begun to be addressed. New studies have also provided important insights into the role of ClC channels in cell volume regulation and their function in intracellular organelles.     

The role of the carboxyl terminus in ClC chloride channel function

The human muscle chloride channel ClC-1 has a 398-amino acid carboxyl-terminal domain that resides in the cytoplasm and contains two CBS (cystathionine-beta-synthase) domains. To examine the role of this region, we studied various carboxyl-terminal truncations by heterologous expression in mammalian cells, whole-cell patch clamp recording, and confocal imaging. Channel constructs lacking parts of the distal CBS domain, CBS2, did not produce functional channels, whereas deletion of CBS1 was tolerated. ClC channels are dimeric proteins with two ion conduction pathways (protopores). In heterodimeric channels consisting of one wild type subunit and one subunit in which the carboxyl terminus was completely deleted, only the wild type protopore was functional, indicating that the carboxyl terminus supports the function of the protopore. All carboxyl-terminal-truncated mutant channels fused to yellow fluorescent protein were translated and the majority inserted into the plasma membrane as revealed by confocal microscopy. Fusion proteins of cyan fluorescent protein linked to various fragments of the carboxyl terminus formed soluble proteins that could be redistributed to the surface membrane through binding to certain truncated channel subunits. Stable binding only occurs between carboxyl-terminal fragments of a single subunit, not between carboxyl termini of different subunits and not between carboxyl-terminal and transmembrane domains. However, an interaction with transmembrane domains can modify the binding properties of particular carboxyl-terminal proteins. Our results demonstrate that the carboxyl terminus of ClC-1 is not necessary for intracellular trafficking but is critical for channel function. Carboxyl termini fold independently and modify individual protopores of the double-barreled channel.     

From 'omes to biology

Technologies that have emerged from the genome project have dramatically increased our ability to generate data on the way in which organisms respond to their environments, how they execute their programmes of development and growth, and how these are altered in the development of disease states. However, our ability to analyse these large datasets has not kept pace with our ability to generate them and consequently new strategies must be developed to address the issues associated with their analysis. One approach that we have employed quite successfully is to look at data from microarrays (or proteomics or metabolomics experiments) not as independent datasets, but rather as elements of a much larger body of biological information across various scales that must be integrated with, and interpreted within, the context of such ancillary data. Here we outline the general approach and provide three examples from published studies of the way in which we have applied this strategy.     

From 'omes to biology

Technologies that have emerged from the genome project have dramatically increased our ability to generate data on the way in which organisms respond to their environments, how they execute their programmes of development and growth, and how these are altered in the development of disease states. However, our ability to analyse these large datasets has not kept pace with our ability to generate them and consequently new strategies must be developed to address the issues associated with their analysis. One approach that we have employed quite successfully is to look at data from microarrays (or proteomics or metabolomics experiments) not as independent datasets, but rather as elements of a much larger body of biological information across various scales that must be integrated with, and interpreted within, the context of such ancillary data. Here we outline the general approach and provide three examples from published studies of the way in which we have applied this strategy.     

Localization of a putative ClC chloride channel in spinach chloroplasts

Seven genes seem to encode for putative ClC chloride channels (AtClC-a to AtClC-g) in Arabidopsis thaliana. Their function and localization is still largely unknown. AtClC-f shares considerable sequence similarity with putative ClC channel proteins from Synechocystis, considered to represent the precursor of chloroplasts. We show by biochemical and mass spectrometry analysis that ClC-f is located in the outer envelope membrane of spinach chloroplasts. Consistent with the plastidial localization of ClC-f, p-chlorophenoxy-acetic acid (CPA) reduces photosynthetic activity and the protein is expressed in etioplasts and chloroplasts but not in root tissue. These findings may represent a step toward the molecular identification of ion channel activities in chloroplast membranes.