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.     

The mechanism of fast-gate opening in ClC-0

ClC-0 is a chloride channel whose gating is sensitive to both voltage and chloride. Based on analysis of gating kinetics using single-channel recordings, a five-state model was proposed to describe the dependence of ClC-0 fast-gate opening on voltage and external chloride (Chen, T.-Y., and C. Miller. 1996. J. Gen. Physiol. 108:237-250). We aimed to use this five-state model as a starting point for understanding the structural changes that occur during gating. Using macroscopic patch recordings, we were able to reproduce the effects of voltage and chloride that were reported by Chen and Miller and to fit our opening rate constant data to the five-state model. Upon further analysis of both our data and those of Chen and Miller, we learned that in contrast to their conclusions, (a) the features in the data are not adequate to rule out a simpler four-state model, and (b) the chloride-binding step is voltage dependent. In order to be able to evaluate the effects of mutants on gating (described in the companion paper, see Engh et al. on p. 351 of this issue), we developed a method for determining the error on gating model parameters, and evaluated the sources of this error. To begin to mesh the kinetic model(s) with the known CLC structures, a model of ClC-0 was generated computationally based on the X-ray crystal structure of the prokaryotic homolog ClC-ec1. Analysis of pore electrostatics in this homology model suggests that at least two of the conclusions derived from the gating kinetics analysis are consistent with the known CLC structures: (1) chloride binding is necessary for channel opening, and (2) chloride binding to any of the three known chloride-binding sites must be voltage dependent.     

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.     

Intracellular control of proton extrusion inSaccharomyces cerevisiae

Wild-type and mutant (glucosephosphate isomerase, pyruvate kinase and respiratory deficientrho) strains were used to determine the kinetics of substrate-induced H⁺ efflux in dilute suspensions, glucose-induced production of titratable acidity in intact cells and cell-free extracts, and kinetics of extracellular titratable acidity production (pH-stat). The results indicate that (1) initial phases of H⁺ efflux proceed at the expense of preexisting cell acidity reserves while subsequent efflux is supported by de novo formed acidity, (2) apart from regulation by pH_out the H⁺ efflux is subject to intracellular control, (3) intracellular acidity level is controlled separately from H⁺ efflux. Tentative scheme is proposed for the regulation of H⁺ fluxes inS. cerevisiae.     

Channel-like slippage modes in the human anion/proton exchanger ClC-4

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 × 10⁵ 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.     

Glutamate 268 regulates transport probability of the anion/proton exchanger ClC-5

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.     

Nonequilibrium gating and voltage dependence of the ClC-0 Cl- channel

The gating of ClC-0, the voltage-dependent Cl- channel from Torpedo electric organ, is strongly influenced by Cl- ions in the external solution. Raising external Cl- over the range 1-600 mM favors the fast- gating open state and disfavors the slow-gating inactivated state. Analysis of purified single ClC-0 channels reconstituted into planar lipid bilayers was used to identify the role of Cl- ions in the channel's fast voltage-dependent gating process. External, but not internal, Cl- had a major effect on the channel's opening rate constant. The closing rate was more sensitive to internal Cl- than to external Cl-. Both opening and closing rates varied with voltage. A model was derived that postulates (a) that in the channel's closed state, Cl- is accessible to a site located at the outer end of the conduction pore, where it binds in a voltage-independent fashion, (b) that this closed conformation can open, whether liganded by Cl- or not, in a weakly voltage-dependent fashion, (c) that the Cl(-)-liganded closed channel undergoes a conformational change to a different closed state, such that concomitant with this change, Cl- ion moves inward, conferring voltage-dependence to this step, and (d) that this new Cl(-)- liganded closed state opens with a very high rate. According to this picture, Cl- movement within the pre-open channel is the major source of voltage dependence, and charge movement intrinsic to the channel protein contributes very little to voltage-dependent gating of ClC-0. Moreover, since the Cl- activation site is probably located in the ion conduction pathway, the fast gating of ClC-0 is necessarily coupled to ion conduction, a nonequilibrium process.     

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.     

Crystal structure of the cytoplasmic domain of the chloride channel ClC-0

Ion channels are frequently organized in a modular fashion and consist of a membrane-embedded pore domain and a soluble regulatory domain. A similar organization is found for the ClC family of Cl- channels and transporters. Here, we describe the crystal structure of the cytoplasmic domain of ClC-0, the voltage-dependent Cl- channel from T. marmorata. The structure contains a folded core of two tightly interacting cystathionine beta-synthetase (CBS) subdomains. The two subdomains are connected by a 96 residue mobile linker that is disordered in the crystals. As revealed by analytical ultracentrifugation, the domains form dimers, thereby most likely extending the 2-fold symmetry of the transmembrane pore. The structure provides insight into the organization of the cytoplasmic domains within the ClC family and establishes a framework for guiding future investigations on regulatory mechanisms.     

Intracellular calcium regulation of connexin43

The mechanism by which intracellular Ca(2+) concentration ([Ca(2+)](i)) regulates the permeability of gap junctions composed of connexin43 (Cx43) was investigated in HeLa cells stably transfected with this connexin. Extracellular addition of Ca(2+) in the presence of the Ca(2+) ionophore ionomycin produced a sustained elevation in [Ca(2+)](i) that resulted in an inhibition of the cell-to-cell transfer of the fluorescent dye Alexa fluor 594 (IC(50) of 360 nM Ca(2+)). The Ca(2+) dependency of this inhibition of Cx43 gap junctional permeability is very similar to that described in sheep lens epithelial cell cultures that express the three sheep lens connexins (Cx43, Cx44, and Cx49). The intracellular Ca(2+)-mediated decrease in cell-to-cell dye transfer was prevented by an inhibitor of calmodulin action but not by inhibitors of Ca(2+)/calmodulin-dependent protein kinase II or protein kinase C. In experiments that used HeLa cells transfected with a Cx43 COOH-terminus truncation mutant (Cx43(Delta257)), cell-to-cell coupling was similarly decreased by an elevation of [Ca(2+)](i) (IC(50) of 310 nM Ca(2+)) and similarly prevented by the addition of an inhibitor of calmodulin. These data indicate that physiological concentrations of [Ca(2+)](i) regulate the permeability of Cx43 in a calmodulin-dependent manner that does not require the major portion of the COOH terminus of Cx43.     

Cysteine accessibility in ClC-0 supports conservation of the ClC intracellular vestibule

ClC chloride channels, which are ubiquitously expressed in mammals, have a unique double-barreled structure, in which each monomer forms its own pore. Identification of pore-lining elements is important for understanding the conduction properties and unusual gating mechanisms of these channels. Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels. In this study we used cysteine-scanning mutagenesis and modification (SCAM) to screen >50 residues in the intracellular vestibule of ClC-0. We identified 14 positions sensitive to the negatively charged thiol-modifying reagents sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) or sodium 4-acetamido-4'-maleimidylstilbene-2'2-disulfonic acid (AMS) and show that 11 of these alter pore properties when modified. In addition, two MTSES-sensitive residues, on different helices and in close proximity in the prokaryotic structures, can form a disulfide bond in ClC-0. When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004). These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity.     

Myotonia-related mutations in the distal C-terminus of ClC-1 and ClC-0 chloride channels affect the structure of a poly-proline helix

Apoptosis of VSMCs (vascular smooth-muscle cells) leads to features of atherosclerotic plaque instability. We have demonstrated previously that plaque-derived VSMCs have reduced IGF1 (insulin-like growth factor 1) signalling, resulting from a decrease in the expression of IGF1R (IGF1 receptor) compared with normal aortic VSMCs [Patel, Zhang, Siddle, Soos, Goddard, Weissberg and Bennett (2001) Circ. Res. 88, 895-902]. In the present study, we show that apoptosis induced by oxidative stress is inhibited by ectopic expression of IGF1R. Oxidative stress repressed IGF1R expression at multiple levels, and this was also blocked by mutant p53. Oxidative stress also induced p53 phosphorylation and apoptosis in VSMCs. p53 negatively regulated IGF1R promoter activity and expression and, consistent with this, p53-/- VSMCs demonstrated increased IGF1R expression, both in vitro and in advanced atherosclerotic plaques in vivo. Oxidative-stress-induced interaction of endogenous p53 with TBP (TATA-box-binding protein) was dependent on p53 phosphorylation. Oxidative stress also increased the association of p53 with HDAC1 (histone deacetylase 1). Trichostatin A, a specific HDAC inhibitor, or p300 overexpression relieved the repression of IGF1R following oxidative stress. Furthermore, acetylated histone-4 association with the IGF1R promoter was reduced in cells subjected to oxidative stress. These results suggest that oxidative-stress-induced repression of IGF1R is mediated by the association of phosphorylated p53 with the IGF1R promoter via TBP, and by the subsequent recruitment of chromatin-modifying proteins, such as HDAC1, to the IGF1R promoter-TBP-p53 complex.     

Intracellular redox state regulation by parthenolide

In the present paper, we report a strong intracellular antioxidant activity of the sesquiterpene lactone parthenolide in the hippocampal HT22 cells. This effect is mediated by an increase of total glutathione at both, low (5 microM) and high (10 microM), concentrations. Parthenolide also increases the activation of the antioxidant/electrophile response element. This effect is the possible mediator of glutathione increase, since the limiting enzyme on its synthesis possesses this response element on its promoter. Finally, we demonstrate that its antioxidant properties do not mediate its antiproliferative effect nor its inhibition of NF-kappaB.     

Extracellular Zinc Ion Inhibits ClC-0 Chloride Channels by Facilitating Slow Gating

Extracellular Zn²⁺ was found to reversibly inhibit the ClC-0 Cl⁻ channel. The apparent on and off rates of the inhibition were highly temperature sensitive, suggesting an effect of Zn²⁺ on the slow gating (or inactivation) of ClC-0. In the absence of Zn²⁺, the rate of the slow-gating relaxation increased with temperature, with a Q₁₀ of ∼37. Extracellular Zn²⁺ facilitated the slow-gating process at all temperatures, but the Q₁₀ did not change. Further analysis of the rate constants of the slow-gating process indicates that the effect of Zn²⁺ is mostly on the forward rate (the rate of inactivation) rather than the backward rate (the rate of recovery from inactivation) of the slow gating. When ClC-0 is bound with Zn²⁺, the equilibrium constant of the slow-gating process is increased by ∼30-fold, reflecting a 30-fold higher Zn²⁺ affinity in the inactivated channel than in the open-state channel. As examined through a wide range of membrane potentials, Zn²⁺ inhibits the opening of the slow gate with equal potency at all voltages, suggesting that a two-state model is inadequate to describe the slow-gating transition. Following a model originally proposed by Pusch and co-workers (Pusch, M., U. Ludewig, and T.J. Jentsch. 1997. J. Gen. Physiol. 109:105–116), the effect of Zn²⁺ on the activation curve of the slow gate can be well described by adding two constraints: (a) the dissociation constant for Zn²⁺ binding to the open channel is 30 μM, and (b) the difference in entropy between the open state and the transition state of the slow-gating process is increased by 27 J/ mol/°K for the Zn²⁺-bound channel. These results together indicate that extracellular Zn²⁺ inhibits ClC-0 by facilitating the slow-gating process.     

P-糖蛋白的细胞内转运及表达调控

肿瘤多药耐药(multidrug resistance,MDR)的发生多与P-糖蛋白(P-glycoprotein,P-gp)过度表达相关。作为一种糖蛋白,P-糖蛋白在内质网中合成、折叠,然后转运到高尔基体进行加工、修饰,最终定位于细胞膜,且只有定位于细胞膜的P-糖蛋白才与肿瘤多药耐药的产生相关。P-糖蛋白的表达与多种信号通路如MAPK、Wnt/β-catenin、PKC、NF-κB有关。研究证实,还有多种miRNA与肿瘤多药耐药的发生相关。本文综述了P-糖蛋白的细胞内转运过程及P-糖蛋白表达相关信号通路的研究进展,为以P-糖蛋白为靶标的肿瘤多药耐药逆转剂提供新的研究策略。     

A Three-State Multi-Ion Kinetic Model for Conduction Properties of ClC-0 Chloride Channel

A three-state, multiion kinetic model was proposed to enable the conduction properties of the mammalian channel ClC-0 to be well characterized. Using this rate-theory based model, the current-voltage and conductance-concentration relations were obtained. The five parameters needed were determined by fitting the data of conduction experiments of the wild-type ClC-0 and its K519C mutant. The model was then tested against available calculation and simulation data, and the energy differences between distinct chloride-occupancy states computed agreed with an independent calculation on the binding free energies solved by using the Poisson-Boltzmann equation. The average ion number of conduction and the ion passing duration calculated closely resembled the values obtained from Brownian dynamics simulations. According to the model, the decrease of conductance caused by mutating residue K519 to C519 can be attributed to the effect of K519C mutation on translocation rate constants. Our study sets up a theoretical model for ion permeation and conductance in ClC-0. It provides a starting point for experimentalists to test the three-state model, and would help in understanding the conduction mechanism of ClC-0.     

Intracellular regulation of neuronal nicotinic cholinergic receptors

Effects of substances affecting intracellular secondary messengers on the membrane currents evoked by ionophoretic application of acetylcholine (ACh currents) and on the excitatory postsynaptic currents (EPSC) evoked by single stimuli applied to preganglionic nerve fibres, were studied in neurones of the rat isolated superior cervical ganglion. Forskolin, the protein kinase A activator, and isobutyl-methyxanthine, the phosphodiesterase inhibitor, decreased the ACh currents. Neither forskolin nor isobutyl-methylxanthine affected the EPSC amplitude or the EPSC decay time constant. Phorbol ester, the protein kinase C activator, decreased the ACh current but did not affect either EPSC amplitude or the EPSC decay time constant. Thapsigargin, the intracellular calcium releaser, decreased the ACh current and the EPSC amplitude but did not affect the EPSC decay time constant. The data obtained suggest that nicotinic acetylcholine receptors (nAChRs) of ganglion neurones are not modulated through the pathways involving protein kinase A or protein kinase C. The nAChRs sensitivity to both exogenous and nerve-released acetylcholine is reduced by intracellular calcium without affecting kinetics of their ionic channels.