Mechanisms of block of muscle type CLC chloride channels (Review)

CLC proteins are a nine-member gene family of Cl - channels that have diverse roles in the plasma membrane and in intracellular organelles. The recent structure determination of bacterial CLC homologues by Dutzler et al. was a break-through for the structure-function analysis of CLC channels. This review describes the mechanisms of inhibition of muscle type CLC channels by two classes of small organic substances: 9-anthracene carboxylic acid (9AC) and p-chlorophenoxy propionic acid (CPP). Both substances block muscle type CLC channels (CLC-0 and CLC-1) from the intracellular side. For CPP, one could show that it inhibits the individual protopores of the double-barrelled channel. A major difference between the two types of blockers is the extremely slow binding- and unbinding-kinetics of 9AC (time scale of min), compared to that of CPP block (time scale of s), while the general mechanism of block seems to be quite similar. In the case of the chiral CPP only the S(-) enantiomer is effective. Both substances exhibit a strongly voltage-dependent block with strong inhibition at negative voltages and relief of block at depolarizing potentials at which the channels tend to open maximally. A quantitative kinetic model was developed for the CPP block of CLC-0 in which the closed state has a much larger affinity for CPP than the open state and opening of drug-bound channels is greatly slowed compared to drug-free channels. First experiments with mutated CLC-0 channels and with derivatives of CPP strongly support the pore localization of the CPP binding site. This work provides the basis for the use of these small organic substances as tools to investigate the pharmacological properties of mammalian CLC channels guided by the crystallographic structure of bacterial CLC homologues. They might also turn out to be useful to obtain information about the intricate coupling of gating and permeation that characterizes CLC channels.     

ATP inhibition of CLC-1 is controlled by oxidation and reduction

The effect of intracellular adenosine triphosphate (ATP) on the “common gating” of the CLC-1 chloride channel has been studied by several laboratories with controversial results. Our previous study on the channel expressed in Xenopus oocytes using excised inside-out patch-clamp methods showed a robust effect of ATP in shifting the open probability curve of the common gate toward more depolarizing voltages (Tseng, P.Y., B. Bennetts, and T.Y. Chen. 2007. J. Gen. Physiol. 130:217–221). The results were consistent with those from studying the channel expressed in mammalian cells using whole cell recording methods (Bennetts, B., M.W. Parker, and B.A. Cromer. 2007. J. Biol. Chem. 282:32780–32791). However, a recent study using excised-patch recording methods for channels expressed in Xenopus oocytes reported that ATP had no direct effect on CLC-1 (Zifarelli, G., and M. Pusch. 2008. J. Gen. Physiol. 131:109–116). Here, we report that oxidation of CLC-1 may be the culprit underlying the controversy. When patches were excised from mammalian cells, the sensitivity to ATP was lost quickly—within 2–3 min. This loss of ATP sensitivity could be prevented or reversed by reducing agents. On the other hand, CLC-1 expressed in Xenopus oocytes lost the ATP sensitivity when patches were treated with oxidizing reagents. These results suggest a novel view in muscle physiology that the mechanisms controlling muscle fatigability may include the oxidation of CLC-1.     

Mechanisms of block of muscle type CLC chloride channels (Review)

CLC proteins are a nine-member gene family of Cl- channels that have diverse roles in the plasma membrane and in intracellular organelles. The recent structure determination of bacterial CLC homologues by Dutzler et al. was a breakthrough for the structure-function analysis of CLC channels. This review describes the mechanisms of inhibition of muscle type CLC channels by two classes of small organic substances: 9-anthracene carboxylic acid (9AC) and p-chlorophenoxy propionic acid (CPP). Both substances block muscle type CLC channels (CLC-0 and CLC-1) from the intracellular side. For CPP, one could show that it inhibits the individual protopores of the double-barrelled channel. A major difference between the two types of blockers is the extremely slow binding- and unbinding-kinetics of 9AC (time scale of min), compared to that of CPP block (time scale of s), while the general mechanism of block seems to be quite similar. In the case of the chiral CPP only the S(-) enantiomer is effective. Both substances exhibit a strongly voltage-dependent block with strong inhibition at negative voltages and relief of block at depolarizing potentials at which the channels tend to open maximally. A quantitative kinetic model was developed for the CPP block of CLC-0 in which the closed state has a much larger affinity for CPP than the open state and opening of drug-bound channels is greatly slowed compared to drug-free channels. First experiments with mutated CLC-0 channels and with derivatives of CPP strongly support the pore localization of the CPP binding site. This work provides the basis for the use of these small organic substances as tools to investigate the pharmacological properties of mammalian CLC channels guided by the crystallographic structure of bacterial CLC homologues. They might also turn out to be useful to obtain information about the intricate coupling of gating and permeation that characterizes CLC channels.     

Structural insights into chloride and proton-mediated gating of CLC chloride channels

CLC Cl(-) channels fulfill numerous physiological functions as demonstrated by their involvement in several human genetic diseases. They have an unusual homodimeric architecture in which each subunit forms an individual pore whose open probability is regulated by various physicochemical factors, including voltage, Cl(-) concentration, and pH. The voltage dependence of Torpedo channel CLC-0 is derived probably indirectly from the translocation of a Cl(-) ion through the pore during the opening step. Recent structure determinations of bacterial CLC homologues marked a breakthrough for the structure-function analysis of CLC channels. The structures revealed a complex fold with 18 alpha-helices and two Cl(-) ions per subunit bound in the center of the protein. The side chain of a highly conserved glutamate residue that resides in the putative permeation pathway appears to be a major component of the channel gate. First studies have begun to exploit the bacterial structures as guides for a rational structure-function analysis. These studies confirm that the overall structure seems to be conserved from bacteria to humans. A full understanding of the mechanisms of gating of eukaryotic CLC channels is, however, still lacking.     

Action potential duration and activation of ATP-sensitive potassium current in isolated guinea-pig ventricular myocytes

It is difficult to associate the ATP-sensitive potassium (K-ATP) channel of cardiac muscle with hypoxia/ischemia induced action potential shortening because this occurs before intracellular ATP falls to levels associated in vitro with channel opening. This leaves the cardiac K-ATP channel without any obvious physiological function. We have quantitatively examined the relationship between action potential duration and K-ATP channel activity in enzymatically isolated ventricular myocytes of the guinea-pig. In whole-cell voltage-clamp recording experiments when the K-ATP channel opener SR 44866 (2-10 microM) stimulated an outward membrane current greater than 50 pA at 0 mV membrane potential (the equivalent of 30 open K-ATP channels or 1% of the cell K-ATP channel population) action potential duration was reduced by more than 50%. In the majority of cell-attached membrane patch recordings metabolic inhibition stimulated K-ATP channel open probability of 1-2% which continued for long periods (7-25 min) before cell contracture and coincident major K-ATP channel activation (open probability 65%). Our quantitative analysis thus shows that physiologically relevant activity of K-ATP channels in cardiac muscle is confined to a very small percentage of the possible cell K-ATP current and thus intracellular ATP would not have to fall very far before the opening of K-ATP channels would influence cardiac excitability.     

pH effects on high conductance Ca2+-activated K+ channels (BK(Ca)) in human internal mammary artery smooth muscle cells

INTRODUCTION: In vascular smooth muscle cells, different types of K+ channels participate in the regulation of membrane potential and consequently in the contractile behavior of the vessel. There is little information about the properties and role of K+ channels in human internal mammary artery (HIMA), the vessel of choice for coronary revascularization. METHODS: Patch-clamp technique on isolated HIMA smooth muscle cells was used. RESULTS: This work presents for the first time single-channel properties of the high conductance Ca2+-activated K+ channel (BK(Ca)) of HIMA. It presents a single-channel conductance of 228+/-4 pS (n=44, 8 cells), is sensitive to 100 nM iberiotoxin, and its open probability is Ca2+- and voltage-dependent. Inside-out results show that BK(Ca) channels in HIMA are directly activated by increasing the pH of intracellular media (NPo=0.096+/-0.032 at pH 7.4 and NPo=0.459+/-0.111 at pH 7.6, n=12 cells, p<0.05) and inhibited by lowering this pH (NPo=0.175+/-0.067 at pH 7.4 and NPo=0.051+/-0.019 at pH 6.8, n=13 cells, p<0.05). CONCLUSIONS: The evidences presented about single-channel properties and intracellular pH sensitivity of BK(Ca) from HIMA smooth muscle cells provide useful information to elucidate physiological or pathological mechanisms in this vessel, as well as for future studies where drugs could have BK(Ca) channels as targets for pharmacological therapies.     

CLC-3 channels modulate excitatory synaptic transmission in hippocampal neurons

It is well established that ligand-gated chloride flux across the plasma membrane modulates neuronal excitability. We find that a voltage-dependent Cl(-) conductance increases neuronal excitability in immature rodents as well, enhancing the time course of NMDA receptor-mediated miniature excitatory postsynaptic potentials (mEPSPs). This Cl(-) conductance is activated by CaMKII, is electrophysiologically identical to the CaMKII-activated CLC-3 conductance in nonneuronal cells, and is absent in clc-3(-/-) mice. Systematically decreasing [Cl(-)](i) to mimic postnatal [Cl(-)](i) regulation progressively decreases the amplitude and decay time constant of spontaneous mEPSPs. This Cl(-)-dependent change in synaptic strength is absent in clc-3(-/-) mice. Using surface biotinylation, immunohistochemistry, electron microscopy, and coimmunoprecipitation studies, we find that CLC-3 channels are localized on the plasma membrane, at postsynaptic sites, and in association with NMDA receptors. This is the first demonstration that a voltage-dependent chloride conductance modulates neuronal excitability. By increasing postsynaptic potentials in a Cl(-) dependent fashion, CLC-3 channels regulate neuronal excitability postsynaptically in immature neurons.     

Trek-like Potassium Channels in Rat Cardiac Ventricular Myocytes Are Activated by Intracellular ATP

Large (111 +/- 3.0 pS) K+ channels were recorded in membrane patches from adult rat ventricular myocytes using patch-clamp techniques. The channels were not blocked by 4-AP (5 mM), intracellular TEA (5 mM) or glybenclamide (100 mM). Applying stretch to the membrane (as pipette suction) increased channel open probability (Po) in both cell-attached and isolated patches (typically, Po approximately equals 0.005 with no pressure; approximately equals 0.328 with 90 cm H2O: Vm = 40 mV, pHi = 7.2). The channels were activated by a decrease in intracellular pH; decreasing pHi to 5.5 from 7.2 increased Po to 0.16 from approx. 0.005 (no suction, Vm held at 40 mV). These properties are consistent with those demonstrated for TREK-1, a member of the recently cloned tandem pore family. We confirmed, using RT-PCR, that TREK-1 is expressed in rat ventricle, suggesting that the channel being recorded is indeed TREK-1. However, we show also that the channels are activated by millimolar concentrations of intracellular ATP. At a pH of 6 with no ATP at the intracellular membrane face, Po was 0.048 +/-0.023, whereas Po increased to 0.22 +/- 0.1 with 1 mM ATP, and to 0.348 +/- 0.13 with 3 mM (n = 5; no membrane stretch applied). The rapid time course of the response and the fact that we see the effect in isolated patches appear to preclude phosphorylation. We conclude that intracellular ATP directly activates TREK-like channels, a property not previously described.     

Expression of the voltage-gated sodium channel NaV1.5 in the macrophage late endosome regulates endosomal acidification

Voltage-gated sodium channels expressed on the plasma membrane activate rapidly in response to changes in membrane potential in cells with excitable membranes such as muscle and neurons. Macrophages also require rapid signaling mechanisms as the first line of defense against invasion by microorganisms. In this study, our goal was to examine the role of intracellular voltage-gated sodium channels in macrophage function. We demonstrate that the cardiac voltage-gated sodium channel, NaV1.5, is expressed on the late endosome, but not the plasma membrane, in a human monocytic cell line, THP-1, and primary human monocyte-derived macrophages. Although the neuronal channel, NaV1.6, is also expressed intracellularly, it has a distinct subcellular localization. In primed cells, NaV1.5 regulates phagocytosis and endosomal pH during LPS-mediated endosomal acidification. Activation of the endosomal channel causes sodium efflux and decreased intraendosomal pH. These results demonstrate a functionally relevant intracellular voltage-gated sodium channel and reveal a novel mechanism to regulate macrophage endosomal acidification.     

Localization of mouse CLC-6 and CLC-7 mRNA and their functional complementation of yeast CLC gene mutant

CLC-6 and CLC-7 belong to the family of voltage-dependent chloride channels. To learn more about the in vivo roles of CLC-6 and CLC-7, we performed in situ hybridization of these CLC channels in various mouse organs. Mouse CLC-6 (mCLC-6) was expressed in the peripheral region of seminiferous tubules in the testis, tracheal epithelium, epithelium of bronchioles, alveolar cells in the lung, acinar cells in the pancreas, and intestinal epithelium, but we could not detect signals from pancreatic islets. Mouse CLC-7 (mCLC-7) was expressed in neurons in the medulla oblongata, Purkinje cells in the cerebellum, proximal tubules in the kidney, and hepatocytes in the liver. The distribution of mCLC-6 and mCLC-7 were similar in the lung, pancreas, and testis. mCLC-6 functionally complemented the gef1 phenotype of a yeast strain in which a single CLC channel (GEF1) had been disrupted by homologous recombination. In contrast, mCLC-7 did not complement this gef1 phenotype. This study identified the cell types that express mCLC-6 and mCLC-7 in the mouse tissues, and the complementation assay suggested that mCLC-6 functions as an intracellular chloride channel.     

Molecular modeling of p-chlorophenoxyacetic acid binding to the CLC-0 channel

Molecular simulation techniques were applied to predict the interaction of the CLC-0 Cl(-) channel and the channel-blocking molecule p-chlorophenoxyacetic acid (CPA). A three-dimensional model of the CLC-0 channel was constructed on the basis of the homology with the bacterial Cl(-) channel StCLC, the structure of which has been solved by X-ray crystallography. Docking of the CPA molecule was obtained by using a geometric recognition algorithm, yielding 5000 possible conformations. By restraining the simulation to those conformations in which CPA is near the intracellular mouth of the channel, the CPA-protein complex models were reduced to three sets of conformations, which are interconvertible within 2 ns when molecular dynamics is applied to the system. Point mutations of CLC-0 at three different positions predicted to interact with CPA in these configurations did, however, not greatly alter CPA inhibition, suggesting a deeper final binding location. In the model, binding of CPA to a more internal position in the ionic pathway was obtained by applying a constant force vector to CPA, pushing it toward the center of the channel. This technique allowed us to outline the possible intrachannel pathway of CPA and to describe qualitatively the binding sites and energy barriers of this pathway. The consistency of the obtained models and the experimental data indicates that the CLC-0-CPA complex model is reasonable and can be used in further biological studies, such as rational design of blocking agents of and mutagenesis of CLC Cl(-) channels.     

Increased Activity of Calcium Leak Channels Caused by Proteolysis Near Sarcolemmal Ruptures

Dystrophin, a 427 kD membrane-associated structural protein in muscle cells, is thought to confer strength to the myofiber sarcolemma and protect the membrane from rupture during the stresses of contraction. Dystrophin is absent in muscle cells from Duchenne muscular dystrophy (DMD) patients and mdx mice, a DMD model. Dystrophic muscle membranes undergo more frequent transient, nonlethal tears than normal cell membranes, especially during exercise. In addition, the mean open probability of a background (``leak') calcium channel is higher in dystrophic muscle cells, which leads to higher intracellular free calcium levels. Because elevated calcium levels may contribute to the eventual necrosis of muscle cells in DMD, we examined the possibility that the history of sarcolemmal rupture at a specific location on the membrane affects the open probability of nearby calcium leak channels. Membrane ruptures left by the excision of cell-attached patch-clamp electrodes were used to mimic natural tears. Patches made within 5 microns of excision sites contained channels with a fourfold greater mean open probability than channels in patches 50 μm away from ruptures. The increased leak channel activity near ruptures was seen continuously through the duration of the recordings and was not seen if the rupture was made in the presence of the protease inhibitor leupeptin. Calcium background channels proteolytically activated near ruptures, perhaps in a calcium-dependent manner, may thus be the lasting consequence of the weaker dystrophic sarcolemma, leading to chronically raised intracellular free calcium, increased calcium-dependent proteolysis and, eventually, necrosis. Received: 29 November 1999/Revised: 13 April 2000     

Ca2+-activated K+ channels from cultured renal medullary thick ascending limb cells: Effects of pH

Summary Ca²⁺-activated K⁺ channels were studied in cultured medullary thick ascending limb cells (MTAL) using the patch-clamp technique. The purpose was to determine the effect of acidic pH on channel properties in excised patches of apical cell membrane. At pH 7.4, increasing Ca²⁺ on the intracellular side or applying positive voltages increases channel open probability. Reducing pH to 5.8 on the intracellular face of the channel decreases channel open probability at each voltage and Ca²⁺ concentration. Channel mean open times display two distributions and mean closed times display three distributions. Increasing Ca²⁺ or applying depolarizing voltages lengthens each of the mean open times and shortens each of the closed times. Lowering pH to 5.8 decreases the mean open times and increases mean closed times at each Ca²⁺ and voltage with the greatest effect on the mean closed times. In contrast, both single-channel conductance and channel kinetics are unaffected when pH is reduced to 5.8 on the extracellular face of the membrane. We conclude that protons interfere with Ca²⁺ binding to the gate of Ca²⁺-activated K⁺ channels reducing the probability of channel opening.     

The muscle chloride channel ClC-1 is not directly regulated by intracellular ATP

ClC-1 belongs to the gene family of CLC Cl(-) channels and Cl(-)/H(+) antiporters. It is the major skeletal muscle chloride channel and is mutated in dominant and recessive myotonia. In addition to the membrane-embedded part, all mammalian CLC proteins possess a large cytoplasmic C-terminal domain that bears two so-called CBS (from cystathionine-beta-synthase) domains. Several studies indicate that these domains might be involved in nucleotide binding and regulation. In particular, Bennetts et al. (J. Biol. Chem. 2005. 280:32452-32458) reported that the voltage dependence of hClC-1 expressed in HEK cells is regulated by intracellular ATP and other nucleotides. Moreover, very recently, Bennetts et al. (J. Biol. Chem. 2007. 282:32780-32791) and Tseng et al. (J. Gen. Physiol. 2007. 130:217-221) reported that the ATP effect was enhanced by intracellular acidification. Here, we show that in striking contrast with these findings, human ClC-1, expressed in Xenopus oocytes and studied with the inside-out configuration of the patch-clamp technique, is completely insensitive to intracellular ATP at concentrations up to 10 mM, at neutral pH (pH 7.3) as well as at slightly acidic pH (pH 6.2). These results have implications for a general understanding of nucleotide regulation of CLC proteins and for the physiological role of ClC-1 in muscle excitation.     

Effect of [Cl(-)]i on ENaC activity from mouse cortical collecting duct cells

Na(+) transport via epithelial Na(+) channel (ENaC) occurs across many epithelial surfaces and plays a key role in regulating salt and water absorption. In this study, we have examined the effects of cytosolic Na(+) and Cl(-) on ENaC activity by patch clamping single channel recording method in mouse cortical collecting duct cells (M1). Cytosolic Na(+) exerts its effect in change of ENaC open probability (Po). High cytosolic Na(+) significantly reduces ENaC Po. No change in channel conductance by cytosolic Na(+) is observed. However, decrease of cytosolic Cl(-) concentration significantly increases channel conductance and ENaC Po. This effect is due to the right shift of ENaC I-V curve to positive membrane potential. The virtue of ENaC conductance remains the same. Cl(-) channels like CFTR and VRAC are unlikely to be involved in this regulation. The results suggest that cytosolic Cl(-) could serve as a mediator to regulate ENaC activity, in accordance with the activities of Cl(-) channels.     

TREK-1 regulation by nitric oxide and cGMP-dependent protein kinase. An essential role in smooth muscle inhibitory neurotransmission

Potassium channels activated by membrane stretch may contribute to maintenance of relaxation of smooth muscle cells in visceral hollow organs. Previous work has identified K(+) channels in murine colon that are activated by stretch and further regulated by NO-dependent mechanisms. We have screened murine gastrointestinal, vascular, bladder, and uterine smooth muscles for the expression of TREK and TRAAK mRNA. Although TREK-1 was expressed in many of these smooth muscles, TREK-2 was expressed only in murine antrum and pulmonary artery. TRAAK was not expressed in any smooth muscle cells tested. Whole cell currents from TREK-1 expressed in mammalian COS cells were activated by stretch, and single channel recordings showed that the stretch-dependent conductance was due to 90 pS channels. Sodium nitroprusside (10(-6) or 10(-5) m) and 8-Br-cGMP (10(-4) or 10(-3) m) increased TREK-1 currents in perforated whole cell and single channel recordings. Mutation of the PKG consensus sequence at serine 351 blocked the stimulatory effects of sodium nitroprusside and 8-Br-cGMP on open probability without affecting the inhibitory effects of 8-Br-cAMP. TREK-1 encodes a component of the stretch-activated K(+) conductance in smooth muscles and may contribute to nitrergic inhibition of gastrointestinal muscles.     

The relationship between cAMP, Ca(2)+, and transport of CFTR to the plasma membrane

The mechanism whereby cAMP stimulates Cl(-) flux through CFTR ion channels in secretory epithelia remains controversial. It is generally accepted that phosphorylation by cAMP-dependent protein kinase increases the open probability of the CFTR channel. A more controversial hypothesis is that cAMP triggers the translocation of CFTR from an intracellular pool to the cell surface. We have monitored membrane turnover in Calu-3 cells, a cell line derived from human airway submucosal glands that expresses high levels of CFTR using membrane capacitance and FM1-43 fluorescence measurements. Using a conventional capacitance measurement technique, we observe an apparent increase in membrane capacitance in most cells that exhibit an increase in Cl(-) current. However, after we carefully correct our recordings for changes in membrane conductance, the apparent changes in capacitance are eliminated. Measurements using the fluorescent membrane marker FM1-43 also indicate that no changes in membrane turnover accompany the activation of CFTR. Robust membrane insertion can be triggered with photorelease of caged Ca(2)+ in Calu-3 cells. However, no increase in Cl(-) current accompanies Ca(2)+-evoked membrane fusion. We conclude that neither increases in cAMP or Ca(2)+ lead to transport of CFTR to the plasma membrane in Calu-3 cells. In addition, we conclude that membrane capacitance measurements must be interpreted with caution when large changes in membrane conductance occur.     

Molecular cloning of CLC chloride channels in Oreochromis mossambicus and their functional complementation of yeast CLC gene mutant

We have cloned two members of the CLC chloride channel family (OmCLC-3 and OmCLC-5) from gill cDNA libraries of the euryhaline tilapia Oreochromis mosammbicus. At the amino acid level, OmCLC-3 is 90.5% identical to rat CLC-3 and OmCLC-5 is 79.2% identical to rat CLC-5. Ribonuclease protection assay revealed that OmCLC-5 was mainly expressed in the gill, kidney, and intestine in both freshwater- (FW) and seawater- (SW) adapted tilapia. Although the mRNA of OmCLC-3 was broadly expressed in tissues of FW- and SW-adapted tilapia, the most intense signals were observed in the gill, kidney, intestine, and brain. Injection of OmCLC-3 and OmCLC-5 cRNAs into Xenopus oocytes did not elicit chloride currents, but these clones did functionally complement the gef1 phenotype of YPH250(gef), a yeast strain in which a single CLC channel (GEF1) has been disrupted by homologous recombination. These results clearly indicated that CLC channels closely related to the mammalian CLC-3, -4, and -5 subfamily exist also in tilapia and that OmCLC-3 and OmCLC-5 function as intracellular chloride channels.