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.     

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.     

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.     

The role of protons in fast and slow gating of the Torpedo chloride channel ClC-0

Transmembrane proton transport is of fundamental importance for life. The list of H⁺ transporting proteins has been recently expanded with the discovery that some members of the CLC gene family are stoichiometrically coupled Cl⁻/H⁺ antiporters. Other CLC proteins are instead passive Cl⁻ selective anion channels. The gating of these CLC channels is, however, strongly regulated by pH, likely reflecting the evolutionary relationship with CLC Cl⁻/H⁺ antiporters. The role of protons in the gating of the model Torpedo channel ClC-0 is best understood. ClC-0 is a homodimer with separate pores in each subunit. Each protopore can be opened and closed independently from the other pore by a “fast gate”. A common, slow gate acts on both pores simultaneously. The opening of the fast gate is controlled by a critical glutamate (E166), whose protonation state determines the fast gate’s pH dependence. Extracellular protons likely can arrive directly at E166. In contrast, protonation of E166 from the inside has been proposed to be mediated by the dissociation of an intrapore water molecule. The OH⁻ anion resulting from the water dissociation is stabilized in one of the anion binding sites of the channel, competing with intracellular Cl⁻ ions. The pH dependence of the slow gate is less well understood. It has been shown that proton translocation drives irreversible gating transitions associated with the slow gate. However, the relationship of the fast gate’s pH dependence on the proton translocation and the molecular basis of the slow gate remain to be discovered.     

The Integrity of the TRP Domain Is Pivotal for Correct TRPV1 Channel Gating

Transient receptor potential vanilloid subtype I (TRPV1) is a thermosensory ion channel that is also gated by chemical substances such as vanilloids. Adjacent to the channel gate, this polymodal thermoTRP channel displays a TRP domain, referred to as AD1, that plays a role in subunit association and channel gating. Previous studies have shown that swapping the AD1 in TRPV1 with the cognate from the TRPV2 channel (AD2) reduces protein expression and produces a nonfunctional chimeric channel (TRPV1-AD2). Here, we used a stepwise, sequential, cumulative site-directed mutagenesis approach, based on rebuilding the AD1 domain in the TRPV1-AD2 chimera, to unveil the minimum number of amino acids needed to restore protein expression and polymodal channel activity. Unexpectedly, we found that virtually full restitution of the AD1 sequence is required to reinstate channel expression and responses to capsaicin, temperature, and voltage. This strategy identified E692, R701, and T704 in the TRP domain as important for TRPV1 activity. Even conservative mutagenesis at these sites (E692D/R701K/T704S) impaired channel expression and abolished TRPV1 activity. However, the sole mutation of these positions in the TRPV1-AD2 chimera (D692E/K701R/S704T) was not sufficient to rescue channel gating, implying that other residues in the TRP domain are necessary to endow activity to TRPV1-AD2. A biophysical analysis of a functional chimera suggested that mutations in the TRP domain raised the energetics of channel gating by altering the coupling of stimuli sensing and pore opening. These findings indicate that inter- and/or intrasubunit interactions in the TRP domain are essential for correct TRPV1 gating.     

Common Gating of Both CLC Transporter Subunits Underlies Voltage-dependent Activation of the 2Cl?/1H+ Exchanger ClC-7/Ostm1

CLC anion transporters form dimers that function either as Cl⁻ channels or as electrogenic Cl⁻/H⁺ exchangers. CLC channels display two different types of “gates,” “protopore” gates that open and close the two pores of a CLC dimer independently of each other and common gates that act on both pores simultaneously. ClC-7/Ostm1 is a lysosomal 2Cl⁻/1H⁺ exchanger that is slowly activated by depolarization. This gating process is drastically accelerated by many CLCN7 mutations underlying human osteopetrosis. Making use of some of these mutants, we now investigate whether slow voltage activation of plasma membrane-targeted ClC-7/Ostm1 involves protopore or common gates. Voltage activation of wild-type ClC-7 subunits was accelerated by co-expressing an excess of ClC-7 subunits carrying an accelerating mutation together with a point mutation rendering these subunits transport-deficient. Conversely, voltage activation of a fast ClC-7 mutant could be slowed by co-expressing an excess of a transport-deficient mutant. These effects did not depend on whether the accelerating mutation localized to the transmembrane part or to cytoplasmic cystathionine-β-synthase (CBS) domains of ClC-7. Combining accelerating mutations in the same subunit did not speed up gating further. No currents were observed when ClC-7 was truncated after the last intramembrane helix. Currents and slow gating were restored when the C terminus was co-expressed by itself or fused to the C terminus of the β-subunit Ostm1. We conclude that common gating underlies the slow voltage activation of ClC-7. It depends on the CBS domain-containing C terminus that does not require covalent binding to the membrane domain of ClC-7.     

Large movement in the C terminus of CLC-0 chloride channel during slow gating

Chloride channels and transporters of the CLC gene family are expressed in virtually all cell types and are crucial in the regulation of membrane potential, chloride homeostasis and intravesicular pH. There are two gating processes that open CLC channels-fast and slow. The fast gating process in CLC channels has recently been linked to a small movement of a glutamate side chain. However, the molecular mechanism underlying the slow gating process is still elusive. Using spectroscopic microscopy, we observed a large backbone movement in the C terminus of the CLC-0 chloride channel that was functionally linked to slow gating. We further showed that the C-terminal movement had a time course similar to slow gating. In addition, a mutation known to lock the slow gate open prevented movement of the C terminus. When combined with recent structural information on the CLC C terminus, our findings provide a structural model for understanding the conformational changes linked to slow gating in CLC transport proteins.     

Regions in the carboxy terminus of alpha-bENaC involved in gating and functional effects of actin

Gating differences occur between the alpha-subunits of the bovine and rat clones of an amiloride-sensitive epithelial Na+ channel (ENaC). Deletion of the carboxy terminus of bovine alpha-ENaC (alpha-bENaC) at R567 converted the gating properties to that of rat alpha-ENaC (alpha-rENaC). The equivalent truncation in alpha-rENaC was without effect on the gating of the rat homologue. The addition of actin to ENaC channels composed of either alpha-rENaC or alpha-bENaC alone produced a twofold reduction in conductance and an increase in open probability. Neither alpha-rENaC (R613X) nor alpha-bENaC (R567X) was responsive to actin. Using a chimera consisting of alpha-rENaC1-615 and alpha-bENaC570-650, we examined several different carboxy-terminal truncation mutants plus and minus actin. When incorporated into planar bilayers, the gating pattern of this construct was identical to wild-type (wt) alpha-bENaC. Premature stop mutations proximal to E685X produced channels with gating patterns like alpha-rENaC. Actin had no effect on the E631X truncation, whereas more distal truncations all interacted with actin, as did wt alpha-bENaC. Key findings were confirmed using channels expressed in Xenopus oocytes and studied by cell-attached patch-clamp recording. Our results suggest that the site of actin regulation at the carboxy terminus of the chimera is located between residues 631 and 644.     

The Carboxyl Terminal Residues 220–283 Are Not Required for Voltage Gating of a Chimeric Connexin32 Hemichannel

Connexin hemichannels display two distinct forms of voltage-dependent gating, corresponding to the operation of V_j- or fast gates and loop- or slow gates. The carboxyl terminus (CT) of connexin 32 has been reported to be required for the operation of the V_j (fast) gates, but this conclusion was inferred from the loss of a fast kinetic component in macroscopic currents of CT-truncated intercellular channels elicited by transjunctional voltage. Such inferences are complicated by presence of both fast and slow gates in each hemichannel and the serial head-to-head arrangement of these gates in the intercellular channel. Examination of voltage gating in undocked hemichannels and V_j gate polarity reversal by a negative charge substitution (N2E) in the amino terminal domain allow unequivocal separation of the two gating processes in a Cx32 chimera (Cx32^∗43E1). This chimera expresses currents as an undocked hemichannel in Xenopus oocytes and provides a model system to study the molecular determinants and mechanisms of Cx32 voltage gating. Here, we demonstrate that both V_j- and loop gates are operational in a truncation mutation that removes all but the first four CT residues (ACAR²¹⁹) of the Cx32^∗43E1 hemichannel. We conclude that an operational Cx32 V_j (fast) gate does not require CT residues 220–283, as reported previously by others.     

Is the chemical gate of connexins voltage sensitive? Behavior of Cx32 wild-type and mutant channels

Connexinchannels are gated by transjunctional voltage(V_j)or CO₂ via distinct mechanisms.The cytoplasmic loop (CL) and arginines of a COOH-terminal domain(CT₁) of connexin32 (Cx32) wereshown to determine CO₂sensitivity, and a gating mechanism involvingCL-CT₁ association-dissociationwas proposed. This study reports that Cx32 mutants, tandem, 5R/E, and5R/N, designed to weaken CL-CT₁interactions, display atypicalV_jand CO₂ sensitivities when testedheterotypically with Cx32 wild-type channels inXenopus oocytes. In tandems, two Cx32monomers are linked NH₂-to-COOH terminus. In 5R/E and 5R/N mutants, glutamates or asparagines replaceCT₁ arginines. On the basis of theintriguing sensitivity of the mutant-32 channel toV_jpolarity, the existence of a "slow gate" distinct from theconventionalV_jgate is proposed. To a lesser extent the slow gatemanifests itself also in homotypic Cx32 channels. Mutant-32 channelsare more CO₂ sensitive than homotypic Cx32 channels, andCO₂-induced chemical gating isreversed with relative depolarization of the mutant oocyte, suggesting V_jsensitivity of chemical gating. A hypothetical pore-plugging modelinvolving an acidic cytosolic protein (possibly calmodulin) is discussed.

     

Inward Rectification in ClC-0 Chloride Channels Caused by Mutations in Several Protein Regions

Several cloned ClC-type Cl⁻ channels open and close in a voltage-dependent manner. The Torpedo electric organ Cl⁻ channel, ClC-0, is the best studied member of this gene family. ClC-0 is gated by a fast and a slow gating mechanism of opposite voltage direction. Fast gating is dependent on voltage and on the external and internal Cl⁻ concentration, and it has been proposed that the permeant anion serves as the gating charge in ClC-0 (Pusch, M., U. Ludewig, A. Rehfeldt, and T.J. Jentsch. 1995. Nature (Lond.). 373:527–531). The deactivation at negative voltages of the muscular ClC-1 channel is similar but not identical to ClC-0. Different from the extrinsic voltage dependence suggested for ClC-0, an intrinsic voltage sensor had been proposed to underlie the voltage dependence in ClC-1 (Fahlke, C., R. Rüdel, N. Mitrovic, M. Zhou, and A.L. George. 1995. Neuron. 15:463–472; Fahlke, C., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rüdel. 1996. Biophys. J. 71:695–706). The gating model for ClC-1 was partially based on the properties of a point-mutation found in recessice myotonia (D136G). Here we investigate the functional effects of mutating the corresponding residue in ClC-0 (D70). Both the corresponding charge neutralization (D70G) and a charge conserving mutation (D70E) led to an inwardly rectifying phenotype resembling that of ClC-1 (D136G). Several other mutations at very different positions in ClC-0 (K165R, H472K, S475T, E482D, T484S, T484Q), however, also led to a similar phenotype. In one of these mutants (T484S) the typical wild-type gating, characterized by a deactivation at negative voltages, can be partially restored by using external perchlorate (ClO₄ ⁻) solutions. We conclude that gating in ClC-0 and ClC-1 is due to similar mechanisms. The negative charge at position 70 in ClC-0 does not specifically confer the voltage sensitivity in ClC-channels, and there is no need to postulate an intrinsic voltage sensor in ClC-channels.     

Identification of the pH sensor and activation by chemical modification of the ClC-2G Cl- channel

Rabbit and human ClC-2GCl channels are voltagesensitive and activated by protein kinase A and low extracellular pH.The objective of the present study was to investigate the mechanism involved in acid activation of the ClC-2GCl channel and to determinewhich amino acid residues play a role in this acid activation. Channelopen probability(P_o) at ±80 mV holding potentials increased fourfold in a concentration-dependent manner with extracellular H⁺concentration (that is, extracellular pH,pH_trans), with anapparent acidic dissociation constant of pH 4.95 ± 0.27. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide-catalyzed amidation of the channel with glycine methyl ester increasedP_o threefold atpH_trans 7.4, at which the channelnormally exhibits lowP_o. Withextracellular pH reduction (protonation) or amidation, increasedP_o was due to asignificant increase in open time constants and a significant decreasein closed time constants of the channel gating, and this effect wasinsensitive to applied voltage. With the use of site-directedmutagenesis, the extracellular region EELE (amino acids416-419) was identified as the pH sensor and amino acid Glu-419was found to play the key or predominant role in activation of theClC-2G Cl channel byextracellular acid.

     

细胞外氯离子浓度对大鼠ClC-1通道去激活动力学特性的影响

为探讨C1C－1通道的门控机制,实验应用爪蟾母细胞异源性表达大鼠野生型C1C－1（WT RC1C-1)通道基因,并使用双电极电压钳法记录通道电流。通过改变细胞外氯离子浓度,采用双指数拟合的方法分析通道去激活电流,对其去激活门控动力学特性进行了研究。结果表明,降低细胞外氯离子浓度可增加快速去激活电流成分,减少慢速去激活成分;同时,慢速去激活和快速去激活电流的时间常数都显著减小,说明细胞外氯离子浓度的改变可影响通道去激活动力学参数,从而改变通道的门控过程。     

The Carboxyl Terminal Residues 220–283 Are Not Required for Voltage Gating of a Chimeric Connexin32 Hemichannel

Connexin hemichannels display two distinct forms of voltage-dependent gating, corresponding to the operation of V_j- or fast gates and loop- or slow gates. The carboxyl terminus (CT) of connexin 32 has been reported to be required for the operation of the V_j (fast) gates, but this conclusion was inferred from the loss of a fast kinetic component in macroscopic currents of CT-truncated intercellular channels elicited by transjunctional voltage. Such inferences are complicated by presence of both fast and slow gates in each hemichannel and the serial head-to-head arrangement of these gates in the intercellular channel. Examination of voltage gating in undocked hemichannels and V_j gate polarity reversal by a negative charge substitution (N2E) in the amino terminal domain allow unequivocal separation of the two gating processes in a Cx32 chimera (Cx32^∗43E1). This chimera expresses currents as an undocked hemichannel in Xenopus oocytes and provides a model system to study the molecular determinants and mechanisms of Cx32 voltage gating. Here, we demonstrate that both V_j- and loop gates are operational in a truncation mutation that removes all but the first four CT residues (ACAR²¹⁹) of the Cx32^∗43E1 hemichannel. We conclude that an operational Cx32 V_j (fast) gate does not require CT residues 220–283, as reported previously by others.     

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.     

Mechanism of block of single protopores of the Torpedo chloride channel ClC-0 by 2-(p-chlorophenoxy)butyric acid (CPB).

We investigated in detail the mechanism of inhibition by the S(-) enantiomer of 2-(p-chlorophenoxy)butyric acid (CPB) of the Torpedo Cl(-)channel, ClC-0. The substance has been previously shown to inhibit the homologous skeletal muscle channel, CLC-1. ClC-0 is a homodimer with probably two independently gated protopores that are conductive only if an additional common gate is open. As a simplification, we used a mutant of ClC-0 (C212S) that has the common gate "locked open" (Lin, Y.W., C.W. Lin, and T.Y. Chen. 1999. J. Gen. Physiol. 114:1-12). CPB inhibits C212S currents only when applied to the cytoplasmic side, and single-channel recordings at voltages (V) between -120 and -80 mV demonstrate that it acts independently on individual protopores by introducing a long-lived nonconductive state with no effect on the conductance and little effect on the lifetime of the open state. Steady-state macroscopic currents at -140 mV are half-inhibited by approximately 0.5 mM CPB, but the inhibition decreases with V and vanishes for V > or = 40 mV. Relaxations of CPB inhibition after voltage steps are seen in the current responses as an additional exponential component that is much slower than the gating of drug-free protopores. For V = 60 mV) with an IC50 of approximately 30-40 mM. Altogether, these findings support a model for the mechanism of CPB inhibition in which the drug competes with Cl(-) for binding to a site of the pore where it blocks permeation. CPB binds preferentially to closed channels, and thereby also strongly alters the gating of the single protopore. Since the affinity of CPB for open WT pores is extremely low, we cannot decide in this case if it acts also as an open pore blocker. However, the experiments with the mutant K519E strongly support this interpretation. CPB block may become a useful tool to study the pore of ClC channels. As a first application, our results provide additional evidence for a double-barreled structure of ClC-0 and ClC-1.     

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.     

Pores formed by single subunits in mixed dimers of different CLC chloride channels

CLC chloride channels comprise a gene family with nine mammalian members. Probably all CLC channels form homodimers, and some CLC proteins may also associate to heterodimers. ClC-0 and ClC-1, the only CLC channels investigated at the single-channel level, display two conductances of equal size which are thought to result from two separate pores, formed individually by the two monomers. We generated concatemeric channels containing one subunit of ClC-0 together with one subunit of ClC-1 or ClC-2. They should display two different conductances if one monomer were sufficient to form one pore. Indeed, we found a 8-picosiemens (pS) conductance (corresponding to ClC-0) that was associated with either a 1.8-pS (ClC-1) or a 2.8-pS (ClC-2) conductance. These conductances retained their typical gating, but the slow gating of ClC-0 that affects both pores simultaneously was lost. ClC-2 and ClC-0 current components were modified by point mutations in the corresponding subunit. The ClC-2 single pore of the mixed dimer was compared with the pores in the ClC-2 homodimer and found to be unaltered. We conclude that each monomer individually forms a gated pore. CLC dimers in general must be imagined as having two pores, as shown previously for ClC-0.     

Isolation and Characterization of a High Affinity Peptide Inhibitor of ClC-2 Chloride Channels

The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent K_D of ∼20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function.ClC proteins form a family of voltage-gated Cl⁻ channels and Cl⁻/H⁺ exchangers that are found in animals, plants, and bacteria (1). These proteins are expressed on the plasma membrane and some intracellular membranes in both excitable and nonexcitable cells (1, 2). There are nine mammalian members of the ClC family that perform functions as varied as maintenance of membrane potential in neuronal cells (ClC-2) (3), Cl⁻ transport across plasma membranes of epithelial and skeletal muscle cells (ClC-1, ClC-2, and ClC-Ka/b) (1, 4), and participation in lysosomal acidification (ClC-5 and ClC-6) (2). Defects in the genes encoding ClC proteins are linked to a number of diseases including myotonia, epilepsy, Dent''s disease, and Bartter''s syndrome (1–3). It has been suggested recently that ClC-2 may play a role in constipation-associated irritable bowel disease as well as in atherosclerosis (5, 6). Most ClC channels show localized tissue expression; ClC-1, for example, is expressed solely in skeletal muscle, whereas ClC-Ka/b is localized to the kidney. ClC-2, on the other hand, is expressed nearly ubiquitously, suggesting that this channel plays an important, yet largely undefined, physiological role (1, 2).ClC proteins are structurally unrelated to cation channels, with the functional unit being a homodimer (1). ClC channels display two equidistant conductance levels for a single channel opening. In 2002, the crystal structure of a bacterial ClC protein from Salmonella typhimurium was solved, revealing a very complicated membrane topology consisting of 18 α-helical units/subunit in the homodimer, only some of which fully traverse the membrane (7). Examination of the crystal structure revealed no obvious pore, such as is evident in K⁺ channel structures, even though bound Cl⁻ ions were present near the proposed selectivity filter (7, 8). Shortly after the crystal structure was solved, it was shown that the bacterial ClC protein was actually a Cl⁻/H⁺ exchanger and not a channel (9). Comparison of the amino acid sequence of the bacterial ClC protein with that of the eukaryotic ClC channels ClC-0, -1, and -2 revealed only 22, 16, and 19% overall identity, respectively (data not shown). The divergence is largely in the cytoplasmic domains, which are absent in bacterial ClC proteins; sequence identity is much higher in the transmembrane domains.Single-channel gating in ClC proteins is complicated, involving both fast and slow gating processes, which are thought to involve separate regions of the protein (1). Fast gating controls the opening and closing of both protopores independently, operating on the millisecond time scale or faster. Through examination of the crystal structure and subsequent electrophysiological analysis, the fast gating process was revealed to involve a conserved glutamate residue deep within each pore (10). This acidic residue lies near a Cl⁻-binding site and moves slightly to open the pathway in response to changes in membrane voltage and subsequent changes in occupancy of that site, thus providing the link between permeation and gating observed in ClC channels (4). In contrast, slow gating controls both pores simultaneously, operating on the hundreds of milliseconds to seconds time scale. Unlike with fast gating, the regions of the ClC protein involved in slow gating are still unknown, despite the availability of the bacterial ClC crystal structure. It is believed that the dimer interface contributes to slow gating, as well as the long cytoplasmic C-terminal domain, an isolated version of which was recently crystallized (11–13). However, the conformational changes involved in the fast and slow gating processes are still largely unknown. Also, in both ClC-1 and -2, fast and slow gating are linked through an undetermined mechanism (14, 15).Despite the availability of the bacterial ClC protein crystal structure, our understanding of gating mechanisms and structural rearrangements of ClC proteins has lagged behind that of their cation channel counterparts. This is due in large part to a lack of useful pharmacological agents, such as peptide toxins, that may be used as tools. Toxins from venomous animals such as scorpions, snakes, and cone snails have been used for a number of years to define the permeation pathways and gating processes of cation channels (16). However, no peptide toxins have been isolated that inhibit a ClC channel, and only one toxin has been isolated that inhibits any Cl⁻ channel of known molecular identity (17). We recently showed that venom from the scorpion Leiurus quinquestriatus hebraeus contains a peptide component that inhibits the ClC-2 chloride channel (18). Here, we report the isolation of this peptide toxin, its proteomic properties, and primary characteristics of the biophysical mechanism of inhibition.     

Electrophysiological Characteristics of Six Mutations in hClC-1 of Korean Patients with Myotonia Congenita

ClC-1 is a member of a large family of voltage-gated chloride channels, abundantly expressed in human skeletal muscle. Mutations in ClC-1 are associated with myotonia congenita (MC) and result in loss of regulation of membrane excitability in skeletal muscle. We studied the electrophysiological characteristics of six mutants found among Korean MC patients, using patch clamp methods in HEK293 cells. Here, we found that the autosomal dominant mutants S189C and P480S displayed reduced chloride conductances compared to WT. Autosomal recessive mutant M128I did not show a typical rapid deactivation of Cl⁻ currents. While sporadic mutant G523D displayed sustained activation of Cl⁻ currents in the whole cell traces, the other sporadic mutants, M373L and M609K, demonstrated rapid deactivations. V_1/2 of these mutants was shifted to more depolarizing potentials. In order to identify potential effects on gating processes, slow and fast gating was analyzed for each mutant. We show that slow gating of the mutants tends to be shifted toward more positive potentials in comparison to WT. Collectively, these six mutants found among Korean patients demonstrated modifications of channel gating behaviors and reduced chloride conductances that likely contribute to the physiologic changes of MC.