HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 555/3/671    most recent jphysiol.2003.060046v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zúñiga, L. Articles by Sepúlveda, F. V. Search for Related Content PubMed PubMed Citation Articles by Zúñiga, L. Articles by Sepúlveda, F. V.

Centro de Estudios Científicos (CECS), Av. Arturo Prat 514, Casilla 1469, Valdivia, Chile

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Functional and structural studies demonstrate that Cl^- channels of the ClC family have a dimeric double-barrelled structure, with each monomer contributing an identical pore. Single protopore gating is a fast process dependent on Cl^- interaction within the selectivity filter and in ClC-0 has a low temperature coefficient over a 10°C range (Q₁₀). A slow gating process closes both protopores simultaneously, has a high Q₁₀, is facilitated by extracellular Zn²⁺ and Cd²⁺ and is abolished or markedly reduced by mutation of a cysteine conserved in ClC-0, -1 and -2. In order to test the hypothesis that similar slow and fast gates exist in the widely expressed ClC-2 Cl^- channel we have investigated the effects of these manoeuvres on ClC-2. We find that the time constants of both components of the double-exponential hyperpolarization-dependent activation (and deactivation) processes have a high temperature dependence, with Q₁₀ values of about 4–5, suggesting important conformational changes of the channel. Mutating C256 (equivalent to C212 in ClC-0) to A, led to a significant fraction of constitutively open channels at all potentials. Activation time constants were not affected but deactivation was slower and significantly less temperature dependent in the C256A mutant. Extracellular Cd²⁺, that inhibits wild-type (WT) channels almost fully, inhibited C256A only by 50%. In the WT, the time constants for opening were not affected by Cd²⁺ but deactivation at positive potentials was accelerated by Cd²⁺. This effect was absent in the C256A mutant. The effect of intracellular Cl^- on channel activation was unchanged in the C256A mutant. Collectively our results strongly support the hypothesis that ClC-2 possesses a common gate and that part of the current increase induced by hyperpolarization represents an opening of the common gate. In contrast to the gating in ClC-0, the protopore gate and the common gate of ClC-2 do not appear to be independent.

(Received 18 December 2003; accepted after revision 9 January 2004; first published online 14 January 2004)
Corresponding author F. Sepúlveda: Centro de Estudios Científicos (CECS), Av. Arturo Prat 514, Casilla 1469, Valdivia, Chile. Email: fsepulveda{at}cecs.cl

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
ClC-2 is a broadly expressed member of the ClC family of Cl^- channels (Thiemann et al. 1992), which includes eight other mammalian ClC channels (Jentsch et al. 2002). The function of ClC-2 has not been elucidated, but studies in ClC-2 knock-out mice have suggested its participation in epithelial transport processes (Bösl et al. 2001). A possible role in epithelial transport is also supported by localization and functional studies in epithelia such as colon (Catalán et al. 2002). In addition to this transport function, a role for ClC-2 in the control of intracellular Cl^- ([Cl^-]_i) has also been proposed for neurones expressing inhibitory GABA receptors (Staley et al. 1996). Recently, mutations in the human ClC-2 gene have been associated with idiopathic generalized epilepsy and attributed to functional effects of the mutations compatible with the proposed neuronal role of ClC-2 (Haug et al. 2003).

Little is known about ClC-2 gating. The better-characterized ClC-0, cloned from Torpedoelectroplax, is thought to be a functional homodimer with subunits containing parallel identical pores. Two gating processes are present: an individual ‘fast’ gate controls each (proto)pore independently and a ‘slow’ or common gate, controls both pores simultaneously. This view was confirmed by mutagenesis and functional assays (Ludewig et al. 1996; Middleton et al. 1996) after the cloning of ClC-0. Functional and biochemical experiments also suggested a homodimeric structure for ClC-1, a closely related mammalian relative of ClC-0 (Fahlke et al. 1997; Saviane et al. 1999). The idea that ClC channels are dimers of the same protein, each containing an identical individual pore, has been demonstrated by the X-ray structure of a bacterial ClC (Dutzler et al. 2002). A possible molecular basis for protopore gating has also been proposed from the crystal structure of E. coli ClC (Dutzler et al. 2003). Mutational work in EcClC and ClC-0 (Dutzler et al. 2003) and -1 (Estévez et al. 2003) is consistent with the view that Cl^- gates the outermost anion coordination site in the selectivity filter. Entry of Cl^- from the extracellular side, promoted by depolarization, would be the basis for the ‘gating by the permeant anion’ (Pusch et al. 1995). This simple model was proposed to account fully for the fast gating process that opens and closes protopores independently. As discussed recently, however, the gating might be more complicated, as both intra- and extracellular Cl^- participate in the gating process of ClC-0 (Chen, 2003) and, as has been argued on the basis of blocker effects, a substantial conformational change might occur upon protopore opening (Accardi & Pusch, 2003).

In ClC-2 it has been shown that gating depends upon intra- but not extracellular Cl^- and that neutralization of E217, homologous to the pore glutamate in EcClC, leads to loss of sensitivity to intracellular Cl^- and, to a great extent, voltage. Experiments testing for transient activation by extracellular protons also demonstrated that E217 was not available for protonation in the closed channel state but became so after opening by hyperpolarization. The results were interpreted to suggest that E217 is a hyperpolarization-dependent protopore gate in ClC-2 and that access of intracellular Cl^- to a site normally occupied by its side-chain in the pore stabilizes the open state. A remaining hyperpolarization-dependent gate was speculated to correspond to a process akin to that closing both pores simultaneously in other ClC channels (Niemeyer et al. 2003).

In addition to its dependence on voltage and intracellular Cl^-, ClC-2 is modulated by extracellular pH (Jordt & Jentsch, 1997). A recent study has examined extracellular pH dependence of ClC-2 in great detail (Arreola et al. 2002). Increasing the extracellular pH from 7.4 to 8.0 decreases the magnitude of the current. The action of alkaline pH was independent of the conformational state of the channel, whereas the contrary was true for low pH. A pH of 5.5 closes channels by preventing channel opening. In contrast, application of pH 5.5 to open channels, at least transiently, activates the currents. The results were interpreted on the basis of two independent protonatable sites, producing activation and inhibition with respective pK values of 7.3 and 6.0, giving a maximum activity near pH 6.5. High pH was proposed to decrease the current by shifting the open probability to more negative membrane potentials, and low pH, which also decreases the current, was thought to act through a proton-dependent stabilization of the channel closed state (Arreola et al. 2002). Our own experiments comparing the effect of pH 5.5 on WT and E217V ClC-2 suggest that the open conformation-dependent activation of ClC-2 by acidification, is due to protonation of the side-chain of the pore E217 residue promoting further opening (Niemeyer et al. 2003).

The presence of a slow (common) gating mechanism in ClC-2 remains to be proven. ClC-2 shows low activity under resting conditions but opens slowly upon hyperpolarization. Deletion experiments demonstrated that ClC-2 channels lacking a cytoplasmic N-terminal domain (ClC-216–61) became constitutively active and independent of voltage (also of cell swelling and extracellular acidification) (Gründer et al. 1992). A potential receptor region, whose deletion also led to loss of voltage dependence, was identified. It was speculated that these data could be explained by a ‘ball-and-chain’ mechanism, where a domain of the N-terminus would constitute a ball that at relatively positive voltages would interact with its receptor keeping the channel closed; hyperpolarization would do the reverse. The ball-and-chain hypothesis therefore could provide a ‘common gating mechanism’ that would account for the opening of ClC-2 by hyperpolarization (Jordt & Jentsch, 1997). An evaluation of the behaviour of the N-terminus-deleted ClC-2 expressed in oocytes and studied in isolated macropatches or in HEK-293 cells by conventional whole-cell patch-clamp revealed that its deletion does not lead to loss of voltage dependence (Pusch et al. 1999; Varela et al. 2002). Varela et al. concluded that the results were incompatible with the ball-and-chain model as previously proposed for ClC-2.

In the present paper we assume, as discussed recently (Estévez et al. 2003), that the general structure is conserved in the ClC family. We seek evidence for a slow gating process in ClC-2, separate from the fast gating of protopores, by examining the temperature dependence, the effect of Cd²⁺, and mutation of a cysteine residue known to alter common gating in other ClC channels. We conclude that the double-exponential activation of ClC-2 by hyperpolarization partially reflects the activation of a common gate that acts on both protopores of the double-barrelled channel. A separate, [Cl^-]_i-dependent fast gate that also responds to hyperpolarization is present in parallel. It is postulated that these two processes are rather strongly coupled in ClC-2.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
The cDNA of rClC-2, originally provided by Dr Thomas J. Jenstch, subcloned into the mammalian expression vector pCR 3.1 (Invitrogen), was used (Varela et al. 2002). The C256A mutation was generated using PCR and confirmed by sequencing analysis.

HEK-293 cells were grown and transiently transfected with expression plasmids for the various ClC-2 constructs and H3-CD8, containing the cDNA of the T-cell antigen CD8 to identify effectively transfected cells as previously described (Cid et al. 2000). Experiments were performed on cells in 35-mm cell-culture plastic Petri dishes mounted directly on the microscope stage. For temperature-controlled experiments a glass-bottomed microchamber was used as described elsewhere (Niemeyer et al. 2000). The bath solution contained (mM): 140 NaCl, 2 CaCl₂, 1 MgCl₂, 22 sucrose, and 10 Hepes (pH 7.4 adjusted with Tris). The pipette solution (35mM Cl^-) contained (mM): 100 Na gluconate, 33 CsCl, 1 MgCl₂, 2 EGTA, and 10 Hepes (pH 7.4 adjusted with Tris). In the 135mM Cl^-, 100mM NaCl replaced the gluconate salt. Low-Cl^- solutions were made by equimolar replacement of 130mM NaCl by the corresponding foreign anion sodium salt. Liquid junction potentials were calculated (Barry, 1994) and appropriate corrections applied.

Standard whole cell patch-clamp recordings were performed as described elsewhere (Díaz & Sepúlveda, 1995) using an Axopatch 200B (Axon Instruments, USA) or an EPC-7 (List, Germany) amplifier. The bath was earthed via an Agar-150mM KCl bridge. Patch-clamp pipettes had resistances of 2–3M. The voltage pulse generator and analysis programs were from Axon Instruments. Unless otherwise stated, when giving trains of pulses, an interval of 60 or 90s between pulses was left, at the holding potential, to allow for complete current deactivation. In some experiments, a ramp taking the voltage from -130 to +30mV in 50ms was given after each pulse to test for selectivity conservation. Experiments where a change in E_rev was seen were discarded. The currents generated by transfection were neither observed in untransfected cells nor in cells transfected with the H3-CD8 plasmid alone.

Time courses for current activation and deactivation were described by fitting a double-exponential plus a constant term equation (Cid et al. 2000) of the form:

(1)

Where I(t) is current as a function of time and I is current at steady state, for activation, or at time zero, for deactivation. To obtain an estimate of apparent open probability, tail currents as a function of voltage were adjusted by a Boltzmann distribution of the form:

(2)

Where G, G₀, and G_max are conductance as a function of voltage, residual conductance independent of voltage, and maximal conductance at full activation (extrapolated), respectively. V_0.5 is the voltage at which 50% activation occurs, and k is the slope factor. Relative permeabilities of foreign anions were calculated from the changes in reversal potential after partial extracellular replacement (Díaz et al. 1993). The expression used was:

(3)

Where ¹[Cl]_o is the original extracellular Cl^- concentration, and ²[Cl]_o and [X]_o are the extracellular concentrations of Cl^- and the foreign anion, respectively, after the change of solution. E_rev is the new reversal potential minus the original, and R, T and F have their usual meanings.

Significance of differences between means was determined by unpaired t test.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Gating of protopores in ClC-0 has been shown to be little dependent on temperature, with a Q₁₀ of about 2. The common gating process, on the other hand, has the highest temperature dependence described so far for ion channel gating (Pusch et al. 1997), suggesting it is mediated by a major conformational change of the protein. We have looked at the effect of temperature on ClC-2 gating to try to gain evidence for a process compatible with a common gate in its opening. Figure 1A and B shows currents evoked by voltage pulses in cells transfected with ClC-2 and recorded at 22 (A) or 37°C (B). Both current development during hyperpolarizing pulses and channel closure during the postpulse to 30mV were clearly faster at 37°C. Quantification of the changes was done by fitting eqn (1) to the current relaxations (Cid et al. 2000). Figure 1C and D shows the time constants for current activation. They were voltage dependent, becoming faster with hyperpolarization as previously described (Varela et al. 2002). Both constants were markedly decreased at 37°C. No major changes in the steady-state activation curve occurred by increasing temperature (Fig. 1E). The V_0.5 and slope factor obtained were -102 ± 5mV and -24 ± 1.5mV (n= 3), respectively. The corresponding values at 22°C (see below) were -117 ± 2mV and -22 ± 1mV, n= 4. The channel closure at a positive potential also appeared faster at 37 than at 22°C. Time constants for deactivation at 30mV after an activating pulse at -130mV, at 22°C (37°C), were 771 ± 69ms (78 ± 8ms) and 137 ± 23ms (20 ± 1.7ms), n= 11(3). The fractional amplitudes were similar and deactivation was complete at the two temperatures. To evaluate channel gating over a range of temperatures, an activating pulse to -130mV followed by a deactivation pulse to 30mV were used. As the temperature was changed, the length of the pulse was varied to allow for measurement of the kinetic parameters. Figure 2 shows Arrhenius plots for time constants for activation (A) and deactivation (B) of ClC-2-mediated currents. The effect of temperature on each time constant could be described by single activation energy (E_a) values in the range of temperatures examined. Similar results were obtained in separate experiments. E_a values were 101 ± 9 and 112 ± 7kJmol^-1 (means ±S.E.M., n= 5) for the processes described by _s and _f, respectively, for activation at -130mV. The corresponding Q₁₀ values at 293K are 4.1 ± 0.5 and 4.6 ± 0.4. The time constants for deactivation at 30mV were also temperature dependent, with E_a values of 115 ± 10 and 109 ± 18kJmol^-1 (means ±S.E.M., n= 5), giving Q₁₀ values of 4.9 ± 0.6 and 4.8 ± 1.2.

View larger version (20K): [in this window] [in a new window]   Figure 1.  Temperature dependence of rClC-2 chloride currents A and B, representative current traces, elicited from a Vh of -10mV in response to test pulses, delivered every 60 s, ranging from -10 to -190mV in 20mV steps. These were followed by a pulse to 30mV. The duration of the main pulses was increased at more positive voltages in order to approximate full activation of the conductance. For illustration proposes, the beginnings of the tail currents at 30mV were set at the same time. C and D, temperature dependence of the activation. Equation (1) was fitted to the time course and the slow (s) and fast (f) time constants are plotted against voltage at 22°C () and 37°C (•). E, steady-state activation of the channel at 37°C (•) is plotted as apparent Popen as a function of voltage and a Boltzmann distribution adjusted as per eqn (2). , equivalent data obtained at 22°C.

View larger version (16K): [in this window] [in a new window]   Figure 2.  Temperature dependence of the time constants for ClC-2 opening and closing A, Arrhenius plots for s and f for opening during a pulse to -130mV given from a holding of -10mV. B shows data obtained in the same cell as in A, but during the closure at 30mV after the activating pulse of -130mV.

View larger version (32K): [in this window] [in a new window]   Figure 3.  Gating properties of the C256A mutant A, representative whole cell recording obtained from the mutant. B and C, results of fitting eqn (1) to the activation process. As, Af and A0 are fractional amplitudes for the slow, fast and instant term, respectively. Voltage dependence of the slow (s) and fast time constant (f) are shown in C. •, WT; , C256A. Time constants (D) and fractional amplitudes (E) for the deactivation process during a pulse to 30mV. A0 is the fraction of the current that does not deactivate. As and Af are the fractional amplitudes of the time-dependent portion of the deactivation. In D, the left- and right-hand axes apply to s and f, respectively. open bars, WT ClC-2; hatched bars, C256A. Means ±S.E.M. of 7 separate experiments for both WT and C256A. Temperature 22°C.

View larger version (23K): [in this window] [in a new window]   Figure 4.  Temperature dependence of C256A ClC-2 chloride currents A, representative current traces, elicited from a Vh of -10mV in response to test pulses, delivered every 60 s, ranging from 10 to -170mV. These were followed by a pulse to 30mV. The duration of the main pulses was increased at more positive voltages in order to approximate full activation of the conductance. For illustration proposes, the beginnings of the tail currents at 30mV were set at the same time. B, steady-state activation of the channel at 37°C is plotted as apparent Popen as a function of voltage, with a fit of eqn (2). C and D, temperature dependence of the activation. Equation (1) was fitted to the time course, and the slow (s) and fast (f) time constants obtained are plotted against voltage at 22°C () and 37°C (•). E, Arrhenius plots for s and f for opening during a pulse to -130mV given from a holding of -10mV. F, data obtained in the same cell as in E, but during the closure at 30mV after the activating pulse of -130mV.

View larger version (16K): [in this window] [in a new window]   Figure 5.  Steady-state activation for WT ClC-2 and its C256A mutant Apparent open probabilities, calculated as described in the text, are shown for WT (•) and the C256A mutant (). Results in A and B were obtained using 35 and 135mM[Cl-]i, respectively. Temperature was 22°C.

View larger version (23K): [in this window] [in a new window]   Figure 6.  Effect of extracellular Cd2+ on ClC-2 and C256A-mediated currents A and B, representative Cl- currents elicited by a voltage step from -10 to -130mV and then returning to 30mV, for WT (A) and C256A (B). Currents before, during and after extracellular application of 100µM Cd2+ are shown. C, dose–response curve. The IC50 values were 48 and 50µM for WT and mutant, respectively. D, effect of Cd2+ on the deactivation. Time constants for the fit of eqn (1) to the deactivation at 30mV are shown for WT (•) and C256A (). Data are means ±S.E.M. of 4 experiments each. Temperature was 22°C.

	Discussion

Top Abstract Introduction Methods Results Discussion References

The model for channel gating in ClC-0, the prototype ClC channel, invokes two independent gates. A fast gate controls the individual pores of the single subunits, and a slow gate controls both of these protopores simultaneously. If the ‘gating by the permeant anion’ hypothesis of Dutzler et al. (2003) were correct, this model could account fully for the fast gating process that opens and closes protopores independently. Further refinement, however, might be required, since pore opening is now thought to involve more than the simple movement of the pore glutamate side-chain (Chen, 2003; Accardi & Pusch, 2003). A gating mechanism has been proposed for protopore gating of ClC-2, where hyperpolarization is required to remove a pore carboxylate group from an external Cl^- binding site within the pore (Niemeyer et al. 2003). In the present work we seek to determine whether a slow gating mechanism is present in ClC-2 equivalent to that described as a common gate in ClC-0.

Work with concatameric forms of ClC-2 has shown that it forms homodimers (Weinreich & Jentsch, 2001). It seems reasonable to assume that each monomer harbours a protopore, but no evidence for a common (slow) gating process exists. An attractive ball-and-chain hypothesis for gating was proposed, which would give rise to the inactivation process expected for a common gate (Gründer et al. 1992). This view has recently been challenged, as normal gating persists in ball-ablated channels (Varela et al. 2002), a fact that had already been noticed in oocyte macropatch experiments (Pusch et al. 1999).

Voltage dependence of ClC-2 opening

Opening of ClC-2 channels is a function of voltage and can be described by a bi-exponential function with time constants that differ by around 10-fold. It is tempting to speculate that they might correspond to the equivalent fast and slow gating transitions in ClC-0 and, consequently, pertain to the gating of protopores and a common gate, respectively. Recently we have shown that a mutant in which a pore-lining glutamic residue is neutralized loses intracellular Cl^- dependence and becomes largely voltage insensitive (Niemeyer et al. 2003). Some voltage dependence remains in the mutant and, if we accept that the pore-lining glutamic residue controls protopore fast gating, this might correspond to a slow common gate.

At a qualitative level, the voltage dependence of ClC-2 opening can be described by slow and fast processes. The opening rate for both processes is voltage dependent, with a similar pattern, becoming faster with hyperpolarization. This is exactly the opposite to the behaviour of ClC-1, in which case both gates are opened by depolarization (Accardi & Pusch, 2000). Fast gating of ClC-0 is also increased by depolarization, but the slow gate opens in response to hyperpolarization (Chen & Miller, 1996), as does the slow component of ClC-2. The fast gate could correspond to a mechanism controlling the individual pores, whilst the slow process might correspond to the so-called common gate in ClC-0. These speculations can only be substantiated on the basis of single-channel studies. These are hampered by the low single-channel conductance of ClC-2, although they are not impossible (Weinreich & Jentsch, 2001). Mutations enhancing the conductance would be most useful in overcoming these problems.

Effect of temperature on ClC-2 opening

Increasing the temperature from 22 to 37°C greatly accelerated the activation of ClC-2. Both time constants for opening were affected similarly by this increase in temperature without much variation in the fractional amplitudes. The steady-state activation curve did not change markedly either. Analyses of time constants on Arrhenius plots gave single slope activation energies with Q₁₀ values at 293K of 4–5 for the processes described by _s and _f, respectively (at -130mV). The time constants for deactivation at 30mV were also markedly temperature dependent (Q₁₀ values of 4.9 and 4.8). If the opening of ClC-2 involved a process equivalent to the common gating of ClC-0, it might be assumed that at least one of these constants could be related to such a slow opening. The figures for Q₁₀ estimated here are one order of magnitude smaller than that (40) for the common gating process of ClC-0. Both are also higher than that for the fast gate of ClC-0, which was found to be 2.2 (Pusch et al. 1997). A Q₁₀ value of 4 has been measured for the common gating of ClC-1 (Bennetts et al. 2001). In fact, Q₁₀ values for the rates of inactivation processes in ion channels have been compared and found to lie in the range 1.7–7.2 (DeCoursey & Cherny, 1998). A high Q₁₀ value of 5 is observed for inactivation of the ShB K⁺ channel by the ball peptide. This involves interaction between two peptide moieties, with the ball peptide having a low probability of adopting a conformation suitable for binding (Murrell-Lagnado & Aldrich, 1993). The strong temperature effect on the opening process of ClC-2 is therefore likely to be due to a significant conformational change(s) of the channel protein. Although there is a huge difference in the temperature dependence of the slow gate of ClC-0 and the temperature dependencies of ClC-2 gating, this does no necessarily suggest that they are fundamentally different in nature. The slow component of gating of ClC-2 is affected by temperature in a similar way to the slow gate of ClC-1, suggesting a conformational change of similar magnitude in both channels and in clear contrast with the equivalent process in ClC-0.

Building upon structural data on a bacterial ClC and mutational analysis of ClC-0 (Dutzler et al. 2003), protopore gating in ClC-2 has been postulated to involve the removal of a glutamic side-chain from an externally located Cl^- binding site at the mouth of the pore (Niemeyer et al. 2003). As discussed by Niemeyer et al. hyperpolarization would be required to promote the removal of this side-chain from its binding site. This could be accomplished through a conformational change that would destabilize the side-chain interaction with the Cl^- binding site, and thus the high temperature dependence of this process. For ClC-0 and 1 the fast gate has a lower temperature dependence, characterized by Q₁₀ values of 2.2 and 3, respectively (Pusch et al. 1997; Bennetts et al. 2001). This might imply that, if fast gating in those channels is related to the movement of an equivalent residue side-chain, this would involve a conformational change of smaller magnitude than in ClC-2. One question that arises from the temperature dependence of ClC-2 is the similarity in Q₁₀ values for _s and _f for both opening and closure. This could be coincidental, but might also suggest that the same energy barrier is rate-determining for all these parameters (DeCoursey & Cherny, 1998).

Effect of mutation C256A on ClC-2 channel opening

Mutation C256A in ClC-2 alters its gating in such a way that a sizable instantaneous current appears at the expense of time- and voltage-dependent components of activation, without altering the time constants. Mutating the equivalent residue in ClC-0, C212, completely removed slow gating without affecting fast gating (Lin et al. 1999). So, does the mutation throw light upon the presence in ClC-2 of a slow gate of similar nature to that in ClC-0?

The effect of a C256A mutation is to increase the residual, voltage-independent open state of the channel. The appearance of this instant component occurs at the expense of both the contribution of the fast and slow components to the opening process. A simplistic interpretation of the results would be that the mutation has affected the equilibrium of both the slow and fast gating processes by favouring the open state. The opening rates are not strongly affected by the mutation. These effects seem quite different from those caused by the equivalent mutation in ClC-0. They are reminiscent, however, of the effects of mutation C277S in ClC-1, which strongly reduces the slow component of macroscopic gating (by 50%) without affecting the time constants for the slow current relaxations, and at the same time also altering fast gating (Accardi et al. 2001). The authors of this work point out that ‘the coupling between the fast and slow process is different and stronger in ClC-1 than in ClC-0’. If our interpretation is correct, a C256A mutation on ClC-2 would affect both fast and slow opening processes and might imply that, if separate, they are rather strongly coupled.

One may ask at this point: what is the possible meaning of ‘coupling’ between these two putative gating mechanisms? As argued above, we attribute the clear alterations in gating of ClC-2 by mutation C256A to a reduction in slow and fast components of gating which, it is speculated here, might correspond to the common and protopore gating mechanisms seen in ClC-0 and -1. We have previously proposed that the fast gating mechanism in ClC-2 involves the hyperpolarization-dependent removal of a glutamic side-chain from a Cl^- binding site, with the open state being stabilized by competition with intracellular Cl^-. If the slow gating process of ClC-2 were related to a conformational change that decreased the stability of the side-chain interaction, this might explain the coupling between the two processes.

In the mutant C256A, activation occurred with a V_0.5 20–30mV more positive than in the WT. In addition a voltage-independent component, which is absent in the WT, appeared in the mutant C256A. The V_0.5 for activation of ClC-2 increases as a function of [Cl^-]_i (Pusch et al. 1999; Niemeyer et al. 2003; Haug et al. 2003). An increase of a comparable magnitude was seen with WT and C256A ClC-2 channels. These results imply that the effect of [Cl^-]_i on fast (protopore?) gating of ClC-2 has remained after mutation.

ClC-2 deactivation at positive potentials also has slow and fast components. The slow time constant describing the rate of channel closure at positive potentials is slower in the mutation C256A. If the opening rate has not changed significantly, then we might deduce that the closing rate has been slowed down in the mutant.

The effect of temperature on the opening rate of ClC-2 is not affected in the mutant C256A. Closing rates at positive potential, however, become only slightly temperature dependent in the C256A mutant. We do not have a straightforward interpretation for this, but we speculate that closure in the mutant depends simply on a diffusion-limited event that might correspond to flipping the fast (glutamic side-chain) gate into the closed position in the absence of hyperpolarization. We postulate that there are two conformations for the channel: one of stable interaction of the pore glutamic side-chain with an externally located Cl^- coordination site in the selectivity filter (S_ext in the original nomenclature of Dutzler et al. 2003) and one of weak interaction, which is reached by hyperpolarization. This conformational change, leading to a less stable interaction of the glutamic side-chain and S_ext, might be reached by opening the slow gate. Fast gating has to do with removal of the pore glutamic side-chain from S_ext, is also dependent upon hyperpolarization and can be promoted by intracellular Cl^- occupancy of S_ext. This fast gating process might determine closing rates. If closure of the fast gate requires the slow gate to be in the open conformation, and if mutation shifted the equilibrium of the slow gate to the open conformation, this might explain the observed low temperature dependence.

Interaction of Cd²⁺ with the ClC-2 Cl^- channel

Divalent cations Cd²⁺ and Zn²⁺ have been shown to inhibit ClC channels, including ClC-2. The effect has been explored in some detail for ClC-0, where it occurs by a facilitation of the slow gating process (Chen, 1998). The effect of Cd²⁺ on ClC-2 occurred with an IC₅₀ of 48µM, was nearly complete and took place without affecting the rate of activation, but accelerating that of deactivation. These results agree with Cd²⁺ favouring closure of what we have tentatively identified as slow and fast gates of ClC-2. Consistent with this, the effect of Cd²⁺ is much reduced in the C256A mutant of ClC-2. Indeed the rate constants for activation or deactivation were not altered by the divalent cation in the mutated channel. The residual inhibition could be due to a mechanism different from interference with gating. A similar partial effect is observed for the equivalent mutant of ClC-0 (Lin et al. 1999).

In conclusion, we have shown that processes that differ in their time course by about one order of magnitude can describe opening of ClC-2 by hyperpolarization. Both of these are highly dependent on temperature. The Q₁₀ values observed suggest that gating of ClC-2 is mediated by conformational change(s) probably less radical than the common gating process of ClC-0, with its very high Q₁₀, but more complex than its protopore gating. Channel closure is facilitated by extracellular Cd²⁺, leading to inhibition. Mutation of a conserved cysteine known to abolish, or decrease, slow gating in other ClC channels, favours the open state in ClC-2. We postulate that this is brought about by a weakening the interaction of a pore-lining glutamic side-chain with the outermost binding site for Cl^- in the selectivity filter. Closure in the C256A mutant has a diminished Cd²⁺ and temperature sensitivity. The results are interpreted in terms of the presence in ClC-2 of fast and slow gating mechanisms. Whether these two gates correspond to the common and protopore gating of ClC-0 remains an open question. Unlike what is seen with ClC-0, however, these two gating mechanism are highly coupled in ClC-2. We conclude that a conformational change leading to the opening of a slow gate in ClC-2 is also capable of facilitating the fast opening mechanism.

	References

Top Abstract Introduction Methods Results Discussion References

 
Accardi A, Ferrera L & Pusch M (2001). Drastic reduction of the slow gate of human muscle chloride channel (ClC-1) by mutation C277S. J Physiol (London) 534, 745–752.[Abstract/Free Full Text]

Accardi A & Pusch M (2000). Fast and slow gating relaxations in the muscle chloride channel CLC-1. J General Physiol 116, 433–444.[Abstract/Free Full Text]

Accardi A & Pusch M (2003). Conformational changes in the pore of CLC-0. J General Physiol 122, 277–294.[Abstract/Free Full Text]

Arreola J, Begenisich T & Melvin JE (2002). Conformation-dependent regulation of inward rectifier chloride channel gating by extracellular protons J Physiol 541, 103–112.[Abstract/Free Full Text]

Barry PH (1994). JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements. J Neurosci Meth 51, 107–116.[CrossRef][Medline]

Bennetts B, Roberts ML, Bretag AH & Rychkov GY (2001). Temperature dependence of human muscle ClC-1 chloride channel. J Physiol 535, 83–93.[Abstract/Free Full Text]

Bösl MR, Stein V, Hubner C, Zdebik AA, Jordt SE, Mukhopadhyay AK, Davidoff MS, Holstein AF & Jentsch TJ (2001). Male germ cells and photoreceptors, both dependent on close cell–cell interactions, degenerate upon ClC-2 Cl^- channel disruption. EMBO J 20, 1289–1299.[CrossRef][Medline]

Catalán M, Cornejo I, Figueroa C, Niemeyer MI, Sepúlveda FV & Cid LP (2002). Expression of ClC-2 chloride channels in surface epithelium of guinea pig colon: mRNA, protein and functional evidence. Am J Physiol 283, G1004–G1013.

Chen TY (1998). Extracellular zinc ion inhibits ClC-0 chloride channels by facilitating slow gating. J General Physiol 112, 715–726.[Abstract/Free Full Text]

Chen TY (2003). Coupling gating with ion permeation in ClC channels. Sci. STKE 2003, pe23.[Abstract/Free Full Text]

Chen TY & Miller C (1996). Nonequilibrium gating and voltage dependence of the ClC-0 Cl^- channel. J General Physiol 108, 237–250.[Abstract/Free Full Text]

Cid LP, Niemeyer MI, Ramírez A & Sepúlveda FV (2000). Splice variants of a ClC-2 chloride channel with differing functional characteristics. Am J Physiol 279, C1198–C1210.

Clark S, Jordt SE, Jentsch TJ & Mathie A (1998). Characterization of the hyperpolarization-activated chloride current in dissociated rat sympathetic neurons. J Physiol 506, 665–678.[Abstract/Free Full Text]

DeCoursey TE & Cherny VV (1998). Temperature dependence of voltage-gated H⁺ currents in human neutrophils, rat alveolar epithelial cells, and mammalian phagocytes. J General Physiol 112, 503–522.[Abstract/Free Full Text]

Díaz M & Sepúlveda FV (1995). Characterisation of Ca²⁺-dependent inwardly rectifying K⁺ currents in HeLa cells. Pflügers Arch 430, 168–180.[CrossRef][Medline]

Díaz M, Valverde MA, Higgins CF, Rucareanu C & Sepúlveda FV (1993). Volume-activated chloride channels in HeLa cells are blocked by verapamil and dideoxyforskolin. Pflügers Arch 422, 347–353.[CrossRef][Medline]

Dutzler R, Campbell EB, Cadene M, Chait BT & MacKinnon R (2002). X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity. Nature 415, 287–294.[CrossRef][Medline]

Dutzler R, Campbell EB & MacKinnon R (2003). Gating the selectivity filter in ClC chloride channels. Science 300, 108–112.[Abstract/Free Full Text]

Estévez R, Schroeder BC, Accardi A, Jentsch TJ & Pusch M (2003). Conservation of chloride channel structure revealed by an inhibitor binding site in ClC-1. Neuron 38, 47–59.[CrossRef][Medline]

Fahlke C, Knittle T, Gurnett CA, Campbell KP & George ALJ (1997). Subunit stoichiometry of human muscle chloride channels. J General Physiol 109, 93–104.[Abstract/Free Full Text]

Gründer S, Thiemann A, Pusch M & Jentsch TJ (1992). Regions involved in the opening of ClC-2 chloride channel by voltage and cell volume. Nature 360, 759–762.[CrossRef][Medline]

Haug K, Warnstedt M, Alekov AK, Sander T, Ramirez A, Poser B et al.. (2003). Mutations in CLCN2 encoding a voltage- gated chloride channel are associated with idiopathic generalized epilepsies. Nat Genet 33, 527–532.[CrossRef][Medline]

Jentsch TJ, Stein V, Weinreich F & Zdebik AA (2002). Molecular structure and physiological function of chloride channels. Physiol Rev 82, 503–568.[Abstract/Free Full Text]

Jordt SE & Jentsch TJ (1997). Molecular dissection of gating in the ClC-2 chloride channel. EMBO J 16, 1582–1592.[CrossRef][Medline]

Kürz L, Wagner S, George AL Jr & Rüdel R (1997). Probing the major skeletal muscle chloride channel with Zn²⁺ and other sulfhydryl-reactive compounds. Pflügers Arch 433, 357–363.[CrossRef][Medline]

Lin YW, Lin CW & Chen TY (1999). Elimination of the slow gating of ClC-0 chloride channel by a point mutation. J General Physiol 114, 1–12.

Ludewig U, Pusch M & Jentsch TJ (1996). Two physically distinct pores in the dimeric ClC-0 chloride channel. Nature 383, 340–343.[CrossRef][Medline]

Middleton RE, Pheasant DJ & Miller C (1996). Homodimeric architecture of a ClC-type chloride ion channel. Nature 383, 337–340.[CrossRef][Medline]

Miller C (1982). Open-state substructure of single chloride channels from Torpedo electroplax. Phil Trans Roy Soc London B 299, 401–411.[Medline]

Murrell-Lagnado RD & Aldrich RW (1993). Energetics of ShakerK channels block by inactivation peptides. J General Physiol 102, 977–1003.[Abstract/Free Full Text]

Niemeyer MI, Cid LP, Zúñiga L, Catalán M & Sepúlveda FV (2003). A conserved pore-lining glutamate as a voltage- and chloride-dependent gate in the ClC-2 chloride channel. J Physiol 553, 873–879.[Abstract/Free Full Text]

Niemeyer MI, Hougaard C, Hoffmann EK, Jørgensen F, Stutzin A & Sepúlveda FV (2000). Characterisation of a cell swelling-activated K⁺-selective conductance of Ehrlich mouse ascites tumour cells. J Physiol 524, 757–767.[Abstract/Free Full Text]

Pusch M, Jordt SE, Stein V & Jentsch TJ (1999). Chloride dependence of hyperpolarization-activated chloride channel gates. J Physiol 515, 341–353.[Abstract/Free Full Text]

Pusch M, Ludewig U & Jentsch TJ (1997). Temperature dependence of fast and slow gating relaxations of ClC-0 chloride channels. J General Physiol 109, 105–116.[Abstract/Free Full Text]

Pusch M, Ludewig U, Rehfeldt A & Jentsch TJ (1995). Gating of the voltage-dependent chloride channel ClC-0 by the permeant anion. Nature 373, 527–531.[CrossRef][Medline]

Rychkov GY, Pusch M, Roberts ML, Jentsch TJ & Bretag AH (1998). Permeation and block of the skeletal muscle chloride channel, ClC-1, by foreign anions. J General Physiol 111, 653–665.[Abstract/Free Full Text]

Saviane C, Conti F & Pusch M (1999). The muscle chloride channel ClC-1 has a double-barrelled appearance that is differentially affected in dominant and recessive myotonia. J General Physiol 113, 457–467.[Abstract/Free Full Text]

Staley K, Smith R, Schaack J, Wilcox C & Jentsch TJ (1996). Alteration of GABA_A receptor function following gene transfer of the ClC-2 chloride channel. Neuron 17, 543–551.[CrossRef][Medline]

Thiemann A, Gründer S, Pusch M & Jentsch TJ (1992). A chloride channel widely expressed in epithelial and non- epithelial cells. Nature 356, 57–60.[CrossRef][Medline]

Varela D, Niemeyer MI, Cid LP & Sepúlveda FV (2002). Effect of an N-terminus deletion on voltage-dependent gating of ClC-2 chloride channel. J Physiol 544, 363–372.[Abstract/Free Full Text]

Weinreich F & Jentsch TJ (2001). Pores formed by single subunits in mixed dimers of different CLC chloride channels. J Biol Chem 276, 2347–2353.[Abstract/Free Full Text]

	Acknowledgements

 
This work was supported by Fondecyt Grant 1030627. CECS is a Millennium Science Institute and is funded in part by grants from Fundación Andes, the Tinker Foundation and Empresas CMPC.

Author's present address
D. Varela: ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Casilla 70058, Chile.

This article has been cited by other articles:

				H. F. Bao, L. Liu, J. Self, B. J. Duke, R. Ueno, and D. C. Eaton A synthetic prostone activates apical chloride channels in A6 epithelial cells Am J Physiol Gastrointest Liver Physiol, August 1, 2008; 295(2): G234 - G251. [Abstract] [Full Text] [PDF]

				T.-Y. Chen and T.-C. Hwang CLC-0 and CFTR: Chloride Channels Evolved From Transporters Physiol Rev, April 1, 2008; 88(2): 351 - 387. [Abstract] [Full Text] [PDF]

				G. Bouyer, S. Egee, and S. L. Y. Thomas Toward a unifying model of malaria-induced channel activity PNAS, June 26, 2007; 104(26): 11044 - 11049. [Abstract] [Full Text] [PDF]

				J. Blanz, M. Schweizer, M. Auberson, H. Maier, A. Muenscher, C. A. Hubner, and T. J. Jentsch Leukoencephalopathy upon Disruption of the Chloride Channel ClC-2 J. Neurosci., June 13, 2007; 27(24): 6581 - 6589. [Abstract] [Full Text] [PDF]

				A. Hinzpeter, J. Fritsch, F. Borot, S. Trudel, D.-L. Vieu, F. Brouillard, M. Baudouin-Legros, J. Clain, A. Edelman, and M. Ollero Membrane Cholesterol Content Modulates ClC-2 Gating and Sensitivity to Oxidative Stress J. Biol. Chem., January 26, 2007; 282(4): 2423 - 2432. [Abstract] [Full Text] [PDF]

				Y. R. Yusef, L. Zuniga, M. Catalan, M. I. Niemeyer, L. P. Cid, and F. V. Sepulveda Removal of gating in voltage-dependent ClC-2 chloride channel by point mutations affecting the pore and C-terminus CBS-2 domain J. Physiol., April 1, 2006; 572(1): 173 - 181. [Abstract] [Full Text] [PDF]

				J. Denton, K. Nehrke, X. Yin, A. M. Beld, and K. Strange Altered gating and regulation of a carboxy-terminal ClC channel mutant expressed in the Caenorhabditis elegans oocyte Am J Physiol Cell Physiol, April 1, 2006; 290(4): C1109 - C1118. [Abstract] [Full Text] [PDF]

				L. P. Cid, M. I. Niemeyer, and F. V. Sepulveda ClC-2 channels get new partners. Focus on "Association between Hsp90 and the ClC-2 chloride channel upregulates channel function" Am J Physiol Cell Physiol, January 1, 2006; 290(1): C42 - C44. [Full Text] [PDF]

				A. Hinzpeter, J. Lipecka, F. Brouillard, M. Baudoin-Legros, M. Dadlez, A. Edelman, and J. Fritsch Association between Hsp90 and the ClC-2 chloride channel upregulates channel function Am J Physiol Cell Physiol, January 1, 2006; 290(1): C45 - C56. [Abstract] [Full Text] [PDF]

				J. A. de Santiago, K. Nehrke, and J. Arreola Quantitative Analysis of the Voltage-dependent Gating of Mouse Parotid ClC-2 Chloride Channel J. Gen. Physiol., November 28, 2005; 126(6): 591 - 603. [Abstract] [Full Text] [PDF]

				N. J. Ernest, A. K. Weaver, L. B. Van Duyn, and H. W. Sontheimer Relative contribution of chloride channels and transporters to regulatory volume decrease in human glioma cells Am J Physiol Cell Physiol, June 1, 2005; 288(6): C1451 - C1460. [Abstract] [Full Text] [PDF]

				J. Denton, K. Nehrke, X. Yin, R. Morrison, and K. Strange GCK-3, a Newly Identified Ste20 Kinase, Binds To and Regulates the Activity of a Cell Cycle-dependent ClC Anion Channel J. Gen. Physiol., January 31, 2005; 125(2): 113 - 125. [Abstract] [Full Text] [PDF]