Inhibition of skeletal muscle ClC-1 chloride channels by low intracellular pH and ATP

Skeletal muscle acidosis during exercise has long been thought to be a cause of fatigue, but recent studies have shown that acidosis maintains muscle excitability and opposes fatigue by decreasing the sarcolemmal chloride conductance. ClC-1 is the primary sarcolemmal chloride channel and has a clear role in controlling muscle excitability, but recombinant ClC-1 has been reported to be activated by acidosis. Following our recent finding that intracellular ATP inhibits ClC-1, we investigated here the interaction between pH and ATP regulation of ClC-1. We found that, in the absence of ATP, intracellular acidosis from pH 7.2 to 6.2 inhibited ClC-1 slightly by shifting the voltage dependence of common gating to more positive potentials, similar to the effect of ATP. Importantly, the effects of ATP and acidosis were cooperative, such that ATP greatly potentiated the effect of acidosis. Adenosine had a similar effect to ATP at pH 7.2, but acidosis did not potentiate this effect, indicating that the phosphates of ATP are important for this cooperativity, possibly due to electrostatic interactions with protonatable residues of ClC-1. A protonatable residue identified by molecular modeling, His-847, was found to be critical for both pH and ATP modulation and may be involved in such electrostatic interactions. These findings are now consistent with, and provide a molecular explanation for, acidosis opposing fatigue by decreasing the chloride conductance of skeletal muscle via inhibition of ClC-1. The modulation of ClC-1 by ATP is a key component of this molecular mechanism.     

Mechanism of voltage-dependent gating in skeletal muscle chloride channels.

Voltage-dependent gating was investigated in a recombinant human skeletal muscle Cl- channel, hCIC-1, heterologously expressed in human embryonic kidney (HEK-293) cells. Gating was found to be mediated by two qualitatively distinct processes. One gating step operates on a microsecond time scale and involves the rapid rearrangement of two identical intramembranous voltage sensors, each consisting of a single titratable residue. The second process occurs on a millisecond time scale and is due to a blocking-unblocking reaction mediated by a cytoplasmic gate that interacts with the ion pore of the channel. These results illustrate a rather simple structural basis for voltage sensing that has evolved in skeletal muscle Cl- channels and provides evidence for the existence of a cytoplasmic gating mechanism in an anion channel analogous to the "ball and chain" mechanism of voltage-gated cation channels.     

Sarcolemmal-restricted localization of functional ClC-1 channels in mouse skeletal muscle

Skeletal muscle fibers exhibit a high resting chloride conductance primarily determined by ClC-1 chloride channels that stabilize the resting membrane potential during repetitive stimulation. Although the importance of ClC-1 channel activity in maintaining normal muscle excitability is well appreciated, the subcellular location of this conductance remains highly controversial. Using a three-pronged multidisciplinary approach, we determined the location of functional ClC-1 channels in adult mouse skeletal muscle. First, formamide-induced detubulation of single flexor digitorum brevis (FDB) muscle fibers from 15-16-day-old mice did not significantly alter macroscopic ClC-1 current magnitude (at -140 mV; -39.0 +/- 4.5 and -42.3 +/- 5.0 nA, respectively), deactivation kinetics, or voltage dependence of channel activation (V(1/2) was -61.0 +/- 1.7 and -64.5 +/- 2.8 mV; k was 20.5 ± 0.8 and 22.8 +/- 1.2 mV, respectively), despite a 33% reduction in cell capacitance (from 465 +/- 36 to 312 +/- 23 pF). In paired whole cell voltage clamp experiments, where ClC-1 activity was measured before and after detubulation in the same fiber, no reduction in ClC-1 activity was observed, despite an approximately 40 and 60% reduction in membrane capacitance in FDB fibers from 15-16-day-old and adult mice, respectively. Second, using immunofluorescence and confocal microscopy, native ClC-1 channels in adult mouse FDB fibers were localized within the sarcolemma, 90 degrees out of phase with double rows of dihydropyridine receptor immunostaining of the T-tubule system. Third, adenoviral-mediated expression of green fluorescent protein-tagged ClC-1 channels in adult skeletal muscle of a mouse model of myotonic dystrophy type 1 resulted in a significant reduction in myotonia and localization of channels to the sarcolemma. Collectively, these results demonstrate that the majority of functional ClC-1 channels localize to the sarcolemma and provide essential insight into the basis of myofiber excitability in normal and diseased skeletal muscle.     

Electrostatic control and chloride regulation of the fast gating of ClC-0 chloride channels

The opening and closing of chloride (Cl-) channels in the ClC family are thought to tightly couple to ion permeation through the channel pore. In the prototype channel of the family, the ClC-0 channel from the Torpedo electric organ, the opening-closing of the pore in the millisecond time range known as "fast gating" is regulated by both external and internal Cl- ions. Although the external Cl- effect on the fast-gate opening has been extensively studied at a quantitative level, the internal Cl- regulation remains to be characterized. In this study, we examine the internal Cl- effects and the electrostatic controls of the fast-gating mechanism. While having little effect on the opening rate, raising [Cl-]i reduces the closing rate (or increases the open time) of the fast gate, with an apparent affinity of >1 M, a value very different from the one observed in the external Cl- regulation on the opening rate. Mutating charged residues in the pore also changes the fast-gating properties-the effects are more prominent on the closing rate than on the opening rate, a phenomenon similar to the effect of [Cl-]i on the fast gating. Thus, the alteration of fast-gate closing by charge mutations may come from a combination of two effects: a direct electrostatic interaction between the manipulated charge and the negatively charged glutamate gate and a repulsive force on the gate mediated by the permeant ion. Likewise, the regulations of internal Cl- on the fast gating may also be due to the competition of Cl- with the glutamate gate as well as the overall more negative potential brought to the pore by the binding of Cl-. In contrast, the opening rate of the fast gate is only minimally affected by manipulations of [Cl-]i and charges in the inner pore region. The very different nature of external and internal Cl- regulations on the fast gating thus may suggest that the opening and the closing of the fast gate are not microscopically reversible processes, but form a nonequilibrium cycle in the ClC-0 fast-gating mechanism.     

Oxidation and reduction control of the inactivation gating of Torpedo ClC-0 chloride channels

Oxidation and reduction (redox) are known to modulate the function of a variety of ion channels. Here, we report a redox regulation of the function of ClC-0, a chloride (Cl(-)) channel from the Torpedo electric organ. The study was motivated by the occasional observation of oocytes with hyperpolarization-activated Cl(-) current when these oocytes expressed ClC-0. We find that these atypical recording traces can be turned into typical ClC-0 current by incubating the oocyte in millimolar concentrations of reducing agents, suggesting that the channel function is regulated by oxidation and reduction. The redox control apparently results from an effect of oxidation on the slow (inactivation) gating: oxidation renders it more difficult for the channel to recover from the inactivated states. Introducing the point mutation C212S in ClC-0 suppresses the inactivation state, and this inactivation-suppressed mutant is no longer sensitive to the inhibition by oxidizing reagents. However, C212 is probably not the target for the redox reaction because the regulation of the inactivation gating by oxidation is still present in a pore mutant (K165C/K165 heterodimer) in which the C212S mutation is present. Taking advantage of the K165C/K165 heterodimer, we further explore the oxidation effect in ClC-0 by methane thiosulfonate (MTS) modifications. We found that trimethylethylammonium MTS modification of the introduced cysteine can induce current in the K165C/K165 heterodimer, an effect attributed to the recovery of the channel from the inactivation state. The current induction by MTS reagents is subjected to redox controls, and thus the extent of this current induction can serve as an indicator to report the oxidation state of the channel. These results together suggest that the inactivation gating of ClC-0 is affected by redox regulation. The finding also provides a convenient method to "cure" those atypical recording traces of ClC-0 expressed in Xenopus oocytes.     

Low single channel conductance of the major skeletal muscle chloride channel, ClC-1.

We expressed the skeletal muscle chloride channel, ClC-1, in HEK293 cells and investigated it with the patch-clamp technique. Macroscopic properties are similar to those obtained after expression in Xenopus oocytes, except that faster gating kinetics are observed in mammalian cells. Nonstationary noise analysis revealed that both rat and human ClC-1 have a low single channel conductance of about 1 pS. This finding may explain the lack of single-channel data for chloride channels from skeletal muscle despite its high macroscopic chloride conductance.     

The fast gating mechanism in ClC-0 channels

We investigate and then modify the hypothesis that a glutamate side chain acts as the fast gate in ClC-0 channels. We first create a putative open-state configuration of the prokaryotic ClC Cl- channel using its crystallographic structure as a basis. Then, retaining the same pore shape, the prokaryotic ClC channel is converted to ClC-0 by replacing all the nonconserved polar and charged residues. Using this open-state channel model, we carry out molecular dynamics simulations to study how the glutamate side chain can move between open and closed configurations. When the side chain extends toward the extracellular end of the channel, it presents an electrostatic barrier to Cl- conduction. However, external Cl- ions can push the side chain into a more central position where, pressed against the channel wall, it does not impede the motion of Cl- ions. Additionally, a proton from a low-pH external solution can neutralize the extended glutamate side chain, which also removes the barrier to conduction. Finally, we use Brownian dynamics simulations to demonstrate the influence of membrane potential and external Cl- concentration on channel open probability.     

Cloning and characterisation of amphibian ClC-3 and ClC-5 chloride channels

Amphibians have provided important model systems to study transepithelial transport, acid-base balance and cell volume regulation. Several families of chloride channels and transporters are involved in these functions. The purpose of this review is to report briefly on some of the characteristics of the chloride channels so far reported in amphibian epithelia, and to focus on recently cloned members of the ClC family and their possible physiological roles. The electrophysiological characterisation, distribution, localisation and possible functions are reviewed and compared to their mammalian orthologs.     

ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation

We investigated the involvement of ClC-3 chloride channels in endosomal acidification by measurement of endosomal pH and chloride concentration [Cl-] in control versus ClC-3-deficient hepatocytes and in control versus ClC-3-transfected Chinese hamster ovary cells. Endosomes were labeled with pH or [Cl-]-sensing fluorescent transferrin (Tf), which targets to early/recycling endosomes, or alpha2-macroglobulin (alpha2M), which targets to late endosomes. In pulse label-chase experiments, [Cl-] was 19 mM just after internalization in alpha2M-labeled endosomes in primary cultures of hepatocytes from wild-type mice, increasing to 58 mM over 45 min, whereas pH decreased from 7.1 to 5.4. Endosomal acidification and [Cl-] accumulation were significantly impaired in hepatocytes from ClC-3 knock-out mice, with [Cl-] increasing from 16 to 43 mM and pH decreasing from 7.1 to 6.0. Acidification and Cl- accumulation were blocked by bafilomycin. In Tf-labeled endosomes, [Cl-] was 46 mM in wild-type versus 35 mM in ClC-3-deficient hepatocytes at 15 min after internalization, with corresponding pH of 6.1 versus 6.5. Approximately 4-fold increased Cl- conductance was found in alpha2M-labeled endosomes isolated from hepatocytes of wild-type versus ClC-3 null mice. In contrast, Golgi acidification was not impaired in ClC-3-deficient hepatocytes. In transfected Chinese hamster ovary cells expressing ClC-3A, endosomal acidification and [Cl-] accumulation were enhanced. [Cl-] in alpha2M-labeled endosomes was 42 mM (control) versus 53 mM (ClC-3A) at 45 min, with corresponding pH 5.8 versus 5.2; [Cl-] in Tf-labeled endosomes at 15 min was 37 mM (control) versus 49 mM (ClC-3A) with pH 6.3 versus 5.9. Our results provide direct evidence for involvement of ClC-3 in endosomal acidification by Cl- shunting of the interior-positive membrane potential created by the vacuolar H+ pump.     

Aging-associated down-regulation of ClC-1 expression in skeletal muscle: phenotypic-independent relation to the decrease of chloride conductance

In order to clarify the mechanism underlying the reduction of resting membrane chloride conductance (gCl) during aging, the levels of mRNA encoding the principal skeletal muscle chloride channel, ClC-1, were measured. Total RNA samples isolated from tibialis anterior muscles of aged (24-29 months old) and adult (3-4 months old) rats were examined for ClC-1 expression using Northern blot analysis, and macroscopic gCl was recorded from extensor digitorum longus muscle fibers from each adult and aged rat in vitro using a two intracellular microelectrode technique. Although interindividual variability was observed, aged rats exhibited a parallel reduction of both gCl and ClC-1 mRNA expression as compared to adult rats. A linear correlation exists between individual values of ClC-1 mRNA and gCl. These results provide evidence that ClC-1 is the main determinant of sarcolemmal gCl and demonstrate that the decrease of gCl observed during aging is associated with a down-regulation of ClC-1 expression in muscle.     

Molecular dissection of gating in the ClC-2 chloride channel.

The ClC-2 chloride channel is probably involved in the regulation of cell volume and of neuronal excitability. Site-directed mutagenesis was used to understand ClC-2 activation in response to cell swelling, hyperpolarization and acidic extracellular pH. Similar to equivalent mutations in ClC-0, neutralizing Lys566 at the end of the transmembrane domains results in outward rectification and a shift in voltage dependence, but leaves the basic gating mechanism, including swelling activation, intact. In contrast, mutations in the cytoplasmic loop between transmembrane domains D7 and D8 abolish all three modes of activation by constitutively opening the channel without changing its pore properties. These effects resemble those observed with deletions of an amino-terminal inactivation domain, and suggest that it may act as its receptor. Such a 'ball-and-chain' type mechanism may act as a final pathway in the activation of ClC-2 elicited by several stimuli.     

MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1

The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families—muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins—are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5′ end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.     

Serum and glucocorticoid inducible kinases functionally regulate ClC-2 channels

ClC-2 participates in the regulation of neuronal excitability, chloride secretion, and cell volume. The ClC-2 sequence contains a consensus site (Ser82) for phosphorylation by the serum and glucocorticoid inducible kinase isoforms SGK1-3. Thus, the present study explored whether ClC-2 is regulated by those kinases. ClC-2 expression in Xenopus oocytes induced inwardly rectifying currents that increased upon coexpression of SGK1-3 and the related kinase PKB. The stimulatory effect was still present upon disruption of the SGK phosphorylation site. SGKs can phosphorylate the ubiquitin ligase Nedd4-2 and prevent Nedd4-2 from binding to its target. Therefore, the role of Nedd4-2 in ClC-2 modulation was investigated. ClC-2 activity decreased upon Nedd4-2 coexpression, an effect reversed by the kinases. According to chemiluminescence ClC-2 membrane abundance was enhanced by SGKs and diminished by Nedd4-2. These observations suggest that SGK1-3 and Nedd4-2 regulate ClC-2 at least in part by modulating ClC-2 abundance at the plasma membrane.     

Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy

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.     

Review. Proton-coupled gating in chloride channels

The physiologically indispensable chloride channel (CLC) family is split into two classes of membrane proteins: chloride channels and chloride/proton antiporters. In this article we focus on the relationship between these two groups and specifically review the role of protons in chloride-channel gating. Moreover, we discuss the evidence for proton transport through the chloride channels and explore the possible pathways that the protons could take through the chloride channels. We present results of a mutagenesis study, suggesting the feasibility of one of the pathways, which is closely related to the proton pathway proposed previously for the chloride/proton antiporters. We conclude that the two groups of CLC proteins, although in principle very different, employ similar mechanisms and pathways for ion transport.     

Quantitative analysis of the voltage-dependent gating of mouse parotid ClC-2 chloride channel

Various ClC-type voltage-gated chloride channel isoforms display a double barrel topology, and their gating mechanisms are thought to be similar. However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1. To delineate the gating of ClC-2 in quantitative terms, we have determined the voltage (V(m)) and time dependence of the protopore (P(f)) and common (P(s)) gates that control the opening and closing of the double barrel. mClC-2 was cloned from mouse salivary glands, expressed in HEK 293 cells, and the resulting chloride currents (I(Cl)) were measured using whole cell patch clamp. WT channels had I(Cl) that showed inward rectification and biexponential time course. Time constants of fast and slow components were approximately 10-fold different at negative V(m) and corresponded to P(f) and P(s), respectively. P(f) and P(s) were approximately 1 at -200 mV, while at V(m) > or = 0 mV, P(f) approximately 0 and P(s) approximately 0.6. Hence, P(f) dominated open kinetics at moderately negative V(m), while at very negative V(m) both gates contributed to gating. At V(m) > or = 0 mV, mClC-2 closes by shutting off P(f). Three- and two-state models described the open-to-closed transitions of P(f) and P(s), respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter P(f) or P(s) in other ClC channels. Based on the time and V(m) dependence of the two gates in WT and mutant channels, we constructed a model to explain the gating of mClC-2. In this model the E213 residue contributes to P(f), the dominant regulator of gating, while the C258 residue alters the V(m) dependence of P(f), probably by interacting with residue E213. These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.     

ClC-2 chloride channels contribute to HTC cell volume homeostasis

Membrane Cl(-) channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl(-) currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl(-) currents when non-ClC-2 currents were blocked by DIDS (100 microM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl(-) currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl(-) permeability and cell volume homeostasis.