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J Physiol (2003), 553.3, pp. 873-879
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.055988
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ABSTRACT |
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ClC-2 is a ubiquitously expressed, two-pore homodimeric Cl- channel opened by hyperpolarisation. Little is known about its gating mechanisms. Crystallographic and functional studies in other ClC channels suggest that a conserved glutamate residue carboxylate side-chain can close protopores by interacting with a Cl--binding site in the pore. Competition for this site is thought to provide the molecular basis for gating by extracellular Cl-. We now show that ClC-2 gating depends upon intra- but not extracellular Cl- and that neutralisation of E217, the homologous pore glutamate, leads to loss of sensitivity to intracellular Cl- and voltage. Experiments testing for transient activation by extracellular protons demonstrate that E217 is not available for protonation in the closed channel state but becomes so after opening by hyperpolarisation. The results suggest that E217 is a hyperpolarisation-dependent protopore gate in ClC-2 and that access of intracellular Cl- to a site normally occupied by its side-chain in the pore stabilises the open state. A remaining hyperpolarisation-dependent gate might correspond to that closing both pores simultaneously in other ClC channels.
(Resubmitted 27 September 2003; accepted after revision 11 November 2003; first published online 14 November 2003)
Corresponding author F. V. Sepúlveda: Centro de Estudios Científicos (CECS), Av. Arturo Prat 514, Casilla 1469, Valdivia, Chile. Email: fsepulveda{at}cecs.cl
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INTRODUCTION |
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Chloride channels of the ClC family play a variety of physiological roles and a number of human inherited diseases are caused by mutations in the genes encoding them (Jentsch et al. 2002). ClC-2 is a ubiquitously expressed member of the family that is gated by voltage and intracellular Cl-. Recently three different mutations in the ClC-2 gene have been proposed to be associated with idiopathic generalised epilepsy (Haug et al. 2003). One of these decreases drastically the sensitivity of the channel to intracellular Cl-, an effect that, it is argued, leads to hyperexcitability. It is, therefore, of great theoretical and practical interest to understand ClC-2 gating, in particular the mechanism of its coupling to Cl- ion movements.
The best-studied ClC channel, ClC-0 from Torpedo, was proposed to be a double-barrelled functional homodimer (Miller, 1982; Miller & White, 1984). Two gating processes were distinguished in ClC-0, a 'fast' gate operating on each pore independently and a 'slow' or common gate, capable of closing the two pores simultaneously. The contention that ClC channels are homodimers, with each monomer contributing with an individual pore to the structure, received definitive confirmation with the 3.0 Å resolution of a bacterial ClC structure (Dutzler et al. 2002). The selectivity filter was shown as highly conserved sequences occurring at N-termini of 
-helices with a contribution by nitrogen atoms and hydroxyl groups.
Many of the ClC channels are voltage gated. The fast gate of ClC-0 is opened by depolarisation, but the voltage required for this process depends strongly on extracellular Cl-. It is postulated that voltage dependence is conferred by the movement of the anion within the pore (Pusch et al. 1995; Chen & Miller, 1996). A molecular basis for this phenomenon has recently been proposed based on a combination of structural and mutagenesis analysis of Escherichia coli ClC (EcClC) (Dutzler et al. 2003). This study shows two sites, internal and central, for Cl- coordination within the selectivity filter. The carboxyl group of E148 obstructs the opening to the external side of the pore. When E148 is mutated to a neutral amino acid, a Cl- ion takes the place of the E148 side-chain. This configuration, with a third, external site occupied by Cl-, is taken to be the open state of the protopores (Dutzler et al. 2003).
ClC-2 is closely related to ClC-0 yet it opens in response to hyperpolarisation and is gated by intracellular Cl- (Jordt & Jentsch, 1997; Pusch et al. 1999; Varela et al. 2002; Haug et al. 2003). The molecular mechanisms for these effects have not been elucidated. Given the high homology between the pore segments identified in EcClC and the equivalent positions in ClC-2, it seems reasonable to assume that they play a similar role in this mammalian channel. In the present paper we confirm the strong coupling of ClC-2 gating to intracellular Cl- and go on to show that it is independent of the extracellular anion. We also show that neutralising E217, the conserved glutamate residue in ClC-2, makes the channel independent of intracellular Cl- and decreases its voltage sensitivity. We propose that E217 is a hyperpolarisation-dependent gate of ClC-2 protopores and that intracellular Cl- stabilises the open state.
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METHODS |
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The ClC-2 cDNA was gpClC-2
77-86 from guinea-pig (Cid et al. 2000). Numbering corresponds to GenBank sequence no. AF113529. Mutants were generated using PCR and confirmed by sequencing.
HEK-293 cells were grown and transiently transfected with expression plasmids for the ClC-2 constructs as described previously (Cid et al. 2000). The pipette solution (35 mM Cl-) contained (mM): 100 sodium gluconate, 33 CsCl, 1 MgCl2, 2 EGTA and 10 Hepes pH 7.4 adjusted with Tris. The bath solution contained (mM): 140 NaCl, 2 CaCl2, 1 MgCl2, 22 sucrose and 10 Hepes pH 7.4 (7.0 or 8.0). Low extracellular Cl- (16 mM) solution was made by equimolar replacement of 130 mM NaCl with the sodium gluconate salt. At pH 5.5, Hepes was replaced by Mes. Cl- concentration in the pipette was varied by equimolar replacement of sodium gluconate by NaCl and, when necessary, by partial replacement of CsCl with sodium gluconate. No interference with cationic currents was ever detected in these experiments. Liquid junction potentials were calculated (Barry, 1994) and corrected for. Standard whole-cell patch-clamp recordings were performed as described elsewhere (Cid et al. 2000).
To obtain an estimate of apparent open probabilities, steady-state relative conductance was plotted as a function of voltage and adjusted to a Boltzmann distribution of the form: G = G0 + Gmax/(1 + exp{(V - V0.5)/k}, where G, G0 and Gmax are conductance, voltage-independent residual conductance and maximal conductance (extrapolated), respectively. V0.5 is the voltage for 50 % activation and k is the slope factor. In some cases maximal value was not reached but the general shape of the curve allowed its extrapolation. The validity of this approach has some support from the calculation of similar open probability values from analysis of variance at least at one voltage (not shown).
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RESULTS |
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Effect of intra- and extracellular chloride on ClC-2 gating
Native ClC-2 channels are notable for opening upon hyperpolarisation. This behaviour is illustrated in Fig. 1A where a functional assay of ClC-2 was conducted by whole-cell patch-clamp examination of acutely transfected HEK-293 cells. The currents, elicited by voltages between -190 and 10 mV, were small at positive or moderately negative potentials, but activated slowly with strong hyperpolarisation. The data were obtained with 135 mM intracellular Cl- ([Cl-]i) and with 146 mM extracellular Cl- ([Cl-]o). Traces at 35 and 10 mM [Cl-]i are shown in Fig. 1B and C. In high [Cl-]i large currents were elicited with potentials between -55 and -125 mV, whilst hyperpolarising pulses greater than -150 mV were required to elicit sizeable currents at low [Cl-]i. The main effect was a shift in V0.5, which was linearly related to [Cl-]i with a slope of 52 mV per decade increase in [Cl-]i. These results confirm quantitatively with the construct used here the marked [Cl-]i dependence of human ClC-2 (Haug et al. 2003) and qualitatively those of an N-terminal-deleted rat ClC-2 (Pusch et al. 1999). Decreasing [Cl-]o down to 6 mM (Fig. 1D) had no marked effect on the currents elicited by a negative voltage. In Fig. 1E and F, the voltage dependence at low [Cl-]o is shown. The apparent open probability as a function of voltage is plotted for 16 and 146 mM [Cl-]o (Fig. 1F), confirming the lack of effect of extracellular anions on gating. Therefore, the voltage required for half-maximal activation (V0.5) of ClC-2 channels was strongly dependent upon [Cl-]i but was not altered by changing [Cl-]o.
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Figure 1. Effect of intra- and extracellular chloride on ClC-2 A- E, representative current traces, elicited from a VH of -30 mV (0 mV in A and D) in response to test pulses to the indicated potential (in mV). These were followed by a pulse to 30 mV (-35 mV in E, 100 mV in D). The duration of the main pulses was increased at more positive voltages in order to approximate full activation of the conductance and was kept as short as possible at more negative potentials to avoid [Cl-]i depletion (Varela et al. 2002). For illustration purposes, the beginning of tail currents at 30 mV was set at the same time. The intra- and extracellular Cl- concentrations used are indicated. An apparent open probability, calculated as described in the text, is plotted against voltage in F (means ± S.E.M.); | ||
Effect of E217V mutation upon ClC-2 chloride channel
The effect of neutralising E217 of ClC-2 was tested. Figure 2A shows currents in a cell transfected with the E217V mutant of ClC-2, recorded with 35 mM [Cl-]i and with an initial [Cl-]o of 146 mM. Hyperpolarising to -110 mV elicited an inward current with little time dependence. Giving a post-pulse to 30 mV elicited a sustained outward current. The selectivity to Cl- was maintained in this mutant as shown by the current-voltage relationships obtained from the ramps (Fig. 2B). The current reversed sign at -36 mV (the chloride equilibrium potential, ECl) for ramps at 146 mM [Cl-]o. Lowering [Cl-]o reversibly decreased outward current with little effect on inward current (Fig. 2A and B). A very similar result was obtained with the E217A mutant of ClC-2 (Fig. 2C and D).
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Figure 2. Effect of mutations E217V and E217A on ClC-2 A, representative current traces, elicited from a VH of -30 mV in response to a test pulse to -110 mV followed by a tail current pulse to 30 mV in a cell transfected with the E217V mutant of ClC-2. Towards the end of the main pulse, a ramp taking the potential to 30 mV was given. Traces before, during and after decreasing the extracellular concentration of Cl- from 146 to 16 mM are shown. Intracellular Cl- concentration was 35 mM. B, current-voltage relationships, from the ramps in A, before ( | ||
The voltage dependence of the current mediated by the E217V mutant was investigated next. Figure 3A shows a family of currents evoked by potentials between -230 and 30 mV at 35 mM [Cl-]i. The currents were time independent between -90 and 30 mV with further channel opening obvious at potentials more negative than -110 mV. A very similar result was obtained with 135 mM [Cl-]i (Fig. 3B). An attempt to quantify this is shown in the activation curves of Fig. 3C. About 50 % of the conductance was voltage independent and [Cl-]i had no effect. These data are radically different from those obtained with WT ClC-2 and suggest that the mutant has lost its sensitivity to [Cl-]i and is found open at all potentials, although further opening can be evoked by strong hyperpolarisation.
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Figure 3. Effect of intracellular chloride on the E217V mutant of ClC-2 A and B, representative current traces, elicited from a VH of -30 mV (0 mV in B) in response to test pulses to the indicated potential (in mV), given in 20 mV increasing steps. These were followed by pulses to 30 mV. The intra- and extracellular Cl- concentrations used are indicated in the panels. An apparent open probability, calculated as described in Methods, is plotted against voltage in C. Results are means ± S.E.M. from 7 experiments with 135 mM [Cl-]i ( | ||
Effect of pH on gating of ClC-2 and the E217V mutant
Extracellular pH affects ClC-2 gating in a complex manner. Acidification to pH 6.5 activates ClC-2, but further acidification produces a strong inhibition (Jordt & Jentsch, 1997; Arreola et al. 2002). We have observed a similar inhibition at pH 5.5 for both WT and E217V ClC-2 (see Supplementary Material). Paradoxically, extracellular acidification to pH 5.5 transiently activates ClC-2 if applied during an opening by hyperpolarisation (Arreola et al. 2002). We have tested the hypothesis that the transient activation by extracellular acidification might be due to protonation of E217. Figure 4A shows that ClC-2 was indeed activated by pHo 5.5 administered during a hyperpolarising pulse. This activation led to a transient further opening that was followed by the expected steady-state inhibition caused by acidification, which occurred at a slower rate. The pulse of acidification was not able to evoke any increase in current at a voltage at which channels are closed, e.g. 0 mV (results not shown). In contrast to the response in WT channels, acidification during an activating pulse produced only the inhibition of the E217V mutant of ClC-2 without any evidence for further transient activation (Fig. 4B). A summary of the main results is given in Fig. 4C. The acute activating effect of acidification was significant in WT channels but only the slower onset inhibition was present with E217V ClC-2.
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Figure 4. Effect of extracellular protons on open ClC-2 channels A and B, representative current records taken from cells expressing ClC-2 (A) or E217V ClC-2 (B). The records shown were taken with 60 s gaps between them under continuous superfusion with solutions of the pH values indicated. VH was -30 mV; a pulse was given to -70 mV, followed by a post-pulse to 30 mV. A ramp to 30 mV towards the end of the main pulse was also delivered to check the reversal potential (Vrev). The voltage protocol is shown under the last stimulation. Acidification was effected during the activating pulse to ascertain the effect of protons on the open channel. C, summary results giving the effect of acidification on the tail currents of the pulses identified as a and b. Means ± S.E.M. of 6 and 5 experiments for WT and E217V channels, respectively. * Statistically significant (P < 0.05 by paired t test) difference from control (first pulse in the sequence). | ||
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DISCUSSION |
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The gating of ClC-2 is poorly understood. Work with concatameric forms of ClC-2 has shown that it forms homodimers, with each subunit contributing a separate pore to the channel (Weinreich & Jentsch, 2001). A ball-and-chain hypothesis was proposed to explain voltage-dependent ClC-2 gating (Gründer et al. 1992), but it was recently demonstrated to be incorrect (Varela et al. 2002) and the voltage dependence of ClC-2 opening has remained without explanation.
The model for channel gating in ClC-0, the prototype and best studied of ClC channels, involves a fast gate controlling the individual pores of the homodimer, and a slow gate that controls the two protopores simultaneously. A plausible molecular basis for the fast gating has recently been proposed from examination of the crystal structure of bacterial ClC channels (Dutzler et al. 2002, 2003). The selectivity filter of EcClC channels consists of three Cl--binding sites, termed Sint, Scent and Sext, lining the pore from the internal to the external aspect of each protopore of the channel. Parallel mutational analysis in EcClC and ClC-0 (Dutzler et al. 2003) is consistent with the view that Cl- and the side-chain of E148 of EcClC (E166 in ClC-0) compete for the outermost transported anion coordination site (Sext) in the selectivity filter. Entry of Cl- from the extracellular side, promoted by depolarisation, would be the basis for the 'gating by the permeant anion'. Residue E148 in EcClC is conserved in ClC-2, at position 217. Indeed there is a high degree of sequence conservation in the regions of the selectivity filter and it may be productive to assume a certain conservation of structure to try to understand the gating mechanism of other ClC channels. As shown here, ClC-2 is unaffected by extracellular Cl- but is activated by hyperpolarisation and by Cl-i. This observation raises the question of whether E217 plays a role in this process.
Neutralisation of E217 in ClC-2 leads to the appearance of a sizeable voltage-independent current, and therefore channel opening, of a wide range of voltages. Some considerable hyperpolarisation-activated current remains at negative potentials but all trace of intracellular Cl- dependence is lost. It is tempting to speculate that neutralisation of E217 has abolished protopore gating, with the remaining voltage-dependent component being due to a different type of gate, perhaps corresponding to the common gate of ClC-0. Corroboration of this hypothesis would require single-channel analysis, which is difficult due to the low single-channel conductance (Weinreich & Jentsch, 2001). If the interpretation were, however, correct, and all Cl--dependent gating relied on E217 side-chain movement, one might ask why it is intra- and not extracellular Cl- that gates the selectivity filter in ClC-2. If the closed protopore state indeed corresponds to the picture provided by the structure of EcClC, perhaps the configuration with the E217 carboxylate side-chain sitting at Sext is much more stable in ClC-2 than in ClC-0. Hyperpolarisation would therefore be required to dislodge the gate from its closed-state position. Under these conditions Cl- entry into the channel would occur from the intracellular side and therefore [Cl-]o would have no effect in stabilising the open gate. Effects of extracellular anions should be expected, however, on open channels, particularly at positive potentials. Indeed such effects have been reported as replacement of Cl-o with Br- accelerates channel closure at positive potentials in WT ClC-2 and replacement with Br- or I- decreases the activity of a partially open ClC-2 mutant (K566Q in the rat channel) (Pusch et al. 1999).
The idea of the inaccessibility of the E217 side-chain in the closed state was further explored by looking at the pH sensitivity of ClC-2. Extracellular acidification to pH 5.5 opens ClC-0 channels, abolishing the voltage gating of protopores, an effect attributed to protonation of the E166 side-chain as it is mimicked by replacement with neutral residues (Dutzler et al. 2003). This degree of extracellular acidification completely blocks ClC-2 (Arreola et al. 2002). We speculated that perhaps access of extracellular protons to the carboxylate side-chain of E217 was prevented in the closed-state configuration and that this might also prevent access of external Cl- ions to the equivalent of Sext. Although extracellular acidification, e.g. to pHo 5.5, inhibits ClC-2 channels, this degree of acidification is capable of further activating open ClC-2 transiently in fast application experiments (Arreola et al. 2002). We found that extracellular protons were capable of activating WT ClC-2 transiently only when applied on hyperpolarisation-opened channels. In contrast, the E217V mutant of ClC-2 was inhibited by pHo 5.5 but did not show any transient activation. This suggests that the transient acidification-dependent activation of ClC-2 channels requires the presence of E217 and could be ascribed to protonation of its side-chain. As transient acidification-dependent activation requires the channels to be open, we assume that in the closed protopore configuration the carboxylate side-chain of E217 is stably bound, presumably to the equivalent of the Sext coordination site, and not available to react with protons. Hyperpolarisation is required to remove the side-chain of E217, which is then protonated, this being reflected in the transient opening. We assume that the subsequent inhibition of the channel must be due to the interaction of protons with other parts of the protein yet to be explored. It is interesting that in a model developed recently (Arreola et al. 2002), two sites for H+ modulation of ClC-2 are demonstrated. In addition, we propose the E217 side-chain as the site responsible for the transient, state-dependent activation. A similar reasoning would explain the inability of [Cl-]o to gate the selectivity filter of ClC-2. This time extracellular Cl- cannot access the Sext site due to the proposed stability of its interaction with the E217 side-chain. It must be stressed that alternative explanations are possible, including an allosteric effect of mutating E217. On the other hand, it has been pointed out that the control of protopores in ClC-0 might be more complicated than a simple local movement of an extracellular facing glutamate (Chen, 2003) and that a more substantial conformational change must occur upon channel opening (Accardi & Pusch, 2003; Traverso et al. 2003). The same might be true for ClC-2, although in the absence of further evidence we tentatively favour the simpler model presented here.
In conclusion, we hypothesise that the conserved E217 pore residue acts as a protopore gate in the ClC-2 channel, as proposed previously from structural and mutational work on EcClC and ClC-0 (Dutzler et al. 2003). At variance with other ClC channels, opening of ClC-2 would require hyperpolarisation to remove the carboxylate group from an external Cl--binding site within the pore. Intracellular Cl- would favour the open state of the channel by competition for this site, but hyperpolarisation would be needed to maintain the gate open. We also propose that voltage-dependent gating remaining after neutralisation of E217 is due to a gating mechanism akin to the common gate of ClC-0 and 1. These ideas should provide a working hypothesis for future experiments probing the gating mechanisms of ClC-2.
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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.
Supplementary material
The online version of this paper can be found at:
DOI: 10.1113/jphysiol.2003.055988
and contains material entitled:
Effect of pH on gating of ClC-2 and E217V mutant
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 M. I. Niemeyer, Y. R. Yusef, I. Cornejo, C. A. Flores, F. V. Sepulveda, and L. P. Cid Functional evaluation of human ClC-2 chloride channel mutations associated with idiopathic generalized epilepsies Physiol Genomics, September 16, 2004; 19(1): 74 - 83. [Abstract] [Full Text] [PDF]
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L. Zuniga, M. I. Niemeyer, D. Varela, M. Catalan, L. P. Cid, and F. V. Sepulveda The voltage-dependent ClC-2 chloride channel has a dual gating mechanism J. Physiol., March 15, 2004; 555(3): 671 - 682. [Abstract] [Full Text] [PDF]
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, 146 mM [Cl-]o (n = 9); 
, 16 mM [Cl-]o (n = 6).
) the decrease in [Cl-]o. ECl at 146 mM [Cl-]o is indicated. C and D, a similar experiment to that in A and B but carried out with the E217A mutant of ClC-2.