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Journal of Physiology (2002), 544.2, pp. 363-372
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.026096
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ABSTRACT |
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ClC-2, a chloride channel widely expressed in mammalian tissues, is activated by hyperpolarisation and extracellular acidification. Deletion of amino acids 16-61 in rat ClC-2 abolishes voltage and pH dependence in two-electrode voltage-clamp experiments in amphibian oocytes. These results have been interpreted in terms of a ball-and-chain type of mechanism in which the N-terminus would behave as a ball that is removed from an inactivating site upon hyperpolarisation. We now report whole-cell patch-clamp measurements in mammalian cells showing hyperpolarization-activation of rClC-216-61 differing only in presenting faster opening and closing kinetics than rClC-2. The lack of time and voltage dependence observed previously was reproduced, however, in nystatin-perforated patch experiments. The behaviour of wild-type rClC-2 did not differ between conventional and nystatin-perforated patches. Similar results were obtained with ClC-2 from guinea-pig. One possible explanation of the results is that some diffusible component is able to lock the channel in an open state but does so only to the mutated channel. Alternative explanations involving the osmotic state of the cell and cytoskeleton structure are also considered. Low extracellular pH activates the wild-type channel but not rClC-2
(Resubmitted 10 June 2002; accepted after revision 23 July 2002; first published online 16 August 2002)
Corresponding author F. V. Sepúlveda: Centro de Estudios Científicos, Avenida Arturo Prat 514, Casilla 1469, Valdivia, Chile. Email: fsepulveda{at}cecs.cl
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INTRODUCTION |
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The chloride channel ClC-2 belongs to the most widely distributed family of chloride channel proteins discovered so far. This ClC family consists of nine different mammalian members with sequence identity varying between 30 and 90 % (Jentsch et al. 1999). This diversity is increased further by alternative splicing, as demonstrated for ClC-2 and ClC-6 mRNA (Chu et al. 1996; Chu & Zeitlin, 1997; Eggermont et al. 1997; Cid et al. 2000). ClC-2 is expressed in many mammalian tissues and its physiological role is just beginning to be ascertained. In certain neurones, ClC-2 is thought to be implicated in the control of intracellular chloride concentration to regulate GABAA receptor action (Staley et al. 1996). ClC-2 has also been identified in the human colonic T84 epithelial cell line (Cid et al. 1995) where it was proposed to participate in fluid secretion not associated with the cAMP-dependent CFTR chloride channel (Fritsch & Edelman, 1996). Similar speculations have been raised for other epithelial cells (Carew & Thorn, 1996; Park et al. 1998; Schwiebert et al. 1998). More recently, a function in absortion has been surmised from a basolateral location in intestinal epithelium (Lipecka et al. 2002; Catalán et al. 2002). The problem of the possible physiological importance of ClC-2 has also been addressed studying ClC-2-deficient mice (Bösl et al. 2001; Nehrke et al. 2002). These animals show a severe degeneration of the retina and the testes that has been attributed to a defective transport by epithelia that would normally control the ionic environment of sensitive germ cells and photoreceptors (Bösl et al. 2001).
The physiological conditions which regulate the activity of ClC-2 and its gating mechanism have been addressed by several authors. ClC-2 shows low activity under resting conditions but opens slowly upon hyperpolarization (Thiemann et al. 1992). When expressed in amphibian oocytes, ClC-2 can be activated by hypotonic cell swelling but evidence against its role in regulatory volume adjustments has been reported (Bond et al. 1998; Nehrke et al. 2002). ClC-2 is also activated by extracellular acidification (Jordt & Jentsch, 1997; Schwiebert et al. 1998), and this property has been proposed to be important for its physiological function (Sherry et al. 2001). An interesting observation concerning the gating mechanism of ClC-2 came from mutagenesis work suggesting that the amino-terminus of this channel behaves as an inactivating region (Gründer et al. 1992). Deletion experiments demonstrated that ClC-2 channels lacking a cytoplasmic N-terminal domain (ClC-216-61) became constitutively active and independent of hyperpolarization, cell swelling and extracellular acidification. A potential receptor region in the linker between transmembrane domains 7 and 8, whose deletion also leads to loss of voltage dependence, has been identified (Jordt & Jentsch, 1997). It was speculated that these data could be explained by a mechanism akin to the 'ball-and-chain' hypothesis originally proposed to account for the gating of sodium channels (Armstrong & Bezanilla, 1977) and later demonstrated for potassium channels (Zagotta et al. 1990). In the case of ClC-2, the N-terminus would constitute a ball that at relatively positive voltages would interact with its receptor keeping the channel closed. Hyperpolarisation would remove the ball from its site of interaction in a voltage-dependent manner leading to slow activation. When the putative ball (amino acids 16-61) is removed by mutation, the channel shows voltage and time independence (Gründer et al. 1992). The ball-and-chain hypothesis, therefore, provides a 'common gating mechanism' that would account for the opening of ClC-2 by hyperpolarisation, cell swelling and extracellular acidification (Jordt & Jentsch, 1997).
It is of great potential interest to understand the possible physiological role of ClC-2 and particularly its gating mechanism to design possible pharmacological ways to affect its activity. The possibility of altering the gating of ClC-2 is relevant given its potential as an alternative to CFTR in the impaired secretory state of cystic fibrosis patients (Schwiebert et al. 1998; Mohammad-Panah et al. 2001). Human and guinea-pig intestinal epithelial tissue express an N-terminus-modified (77-86) splice variant of ClC-2, which has altered function (Cid et al. 2000). Its activity has not been directly compared with that of the ClC-216-61 mutant.
The ball-and-chain hypothesis for ClC-2 gating has not been evaluated in a mammalian expression system. This might be of interest as using inside-out patches from rClC-216-61-expressing oocytes has produced surprising results. In fact, the abolition of voltage and time dependence of the N-terminus-deleted mutant was not observed under this recording configuration (Pusch et al. 1999). An explanation for this discrepancy with the intact oocyte experiments could be that using inside-out patches some intracellular component essential for confering lack of time dependence is lost. A way to assess this possibility would be to compare conventional whole-cell patch-clamp recordings with similar experiments using nystatin-perforated patches (Horn & Marty, 1991), which afford access to the cell with dialysis of monovalent cations only.
In the present communication we evaluate the behaviour of the N-terminally deleted ClC-2 expressed in HEK 293 cells by conventional or nystatin-perforated patch-clamp recordings. We also study an intestinal splice variant presenting a deletion near the putative ball region, which could therefore present altered gating relevant to its physiological function. It is demonstrated that, though the N-terminus is relevant to gating, its deletion does not lead to loss of voltage dependence. Extracellular pH, on the other hand, appears to affect ClC-2 by a mechanism that is independent of the presence of the 16-61 amino acid region. The results are incompatible with the ball-and-chain model as previously proposed for ClC-2.
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METHODS |
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The cDNAs of rClC-2 and rClC216-61 were kindly provided by Dr Thomas J. Jenstch in vector pTLN. The subcloning into the mammalian expresion vector pCR 3.1 (Invitrogen) was made by digestion with restriction enzyme EcoRI and then a partial digestion with restriction enzyme HindIII. The cDNAs for gpClC-2 and gpClC-277-86 are those described previously (Cid et al. 2000). The 16-61 deletion in gpClC2 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 to identify effectively transfected cells as described previously (Cid 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 (35 mM chloride) contained (mM): 100 sodium gluconate, 33 CsCl, 1 MgCl2, 2 EGTA, and 10 Hepes pH 7.4 adjusted with Tris. Low Cl- solutions were made by equimolar replacement of 130 mM NaCl with the corresponding foreign anion Na salt. Changes in liquid junction potential were calculated (Barry, 1994) and corrected for when necessary.
Standard whole-cell patch-clamp recordings were performed as described elsewhere (Díaz & Sepúlveda, 1995) using an Axopatch 200B (Axon Instruments, Foster City, USA) amplifier. The bath was earthed via an agar-3 M KCl bridge. Patch-clamp pipettes had resistances of 2-4 M
. The perforated-patch configuration was obtained by supplementing the pipette internal solution with nystatin (Horn & Marty, 1991). Nystatin stock solution was freshly made up on each day of the experiments in dimethyl sulphoxide (DMSO) at 60 mg ml-1. Aliquots of stock solution were added to the pipette solution to obtain a final concentration of 800 or 1000 µg ml-1. To allow the formation of the giga-seal, the pipette tip was filled with nystatin-free solution, whereas the pipette bulk was backfilled with the nystatin-containing solution. Brief steps to -120 mV were used to monitor the gradual increase of the current. Usually after ~20 min a stable current was achieved, with an access resistance (Ra) of 15 ± 8 M (mean ± S.D.). This could be measured accurately only in non-16-61 channels that have no current at around 0 mV. We have not applied a correction for access resistance and have tried to use experiments with a maximum current of 1 nA in nystatin experiments. This will introduce an error in membrane potential (Vm), on average, of ~8 % at the highest potential used (-180 mV). As accurate quantitative estimation of voltage dependence is not a central aspect of the interpretation, it has been neglected. The voltage pulse generator and analysis programs were from Axon Instruments. Unless otherwise stated, when giving trains of pulses, an interval of 60 or 90 s between pulses was left at the holding potential to allow for complete current deactivation. 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 fitted to double exponential plus a constant term equations as described previously (Cid et al. 2000). The tail current as a function of voltage was adjusted to a Boltzmann distribution of the form: G = G0 + Gmax/(1 + exp((V - V0.5)/k) where G, G0 and Gmax are conductance as a function of voltage, residual conductance independent of voltage, and maximal conductance at full activation (extrapolated), respectively. V0.5 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:
Px/PCl = {1[Cl]oexp(-FErev /RT) - 2[Cl]o}/[X]o,
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. Erev is the new reversal potential minus the original, and R, T and F have their usual meanings.
Significance of differences between means was determined using Student's unpaired t test.
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RESULTS |
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Effect of 16-61 deletion on the kinetics of activation of rClC-2
Functional assays of rClC-2 were conducted by whole-cell patch-clamp examination of acutely transfected HEK 293 cells. Figure 1A shows currents elicited by voltages between -180 and 0 mV, for rClC-2. As described before, currents were small at positive or moderately negative potentials, but activated slowly with strong hyperpolarisation. Equivalent data for rClC-216-61-transfected cells are shown Fig. 1B. There was no apparent difference between the variants in the degree of rectification as the current-voltage relations for measurements at the end of the main voltage pulses showed strong inward rectification for both channel types. The rate at which currents were activated by hyperpolarisation and recovered after returning to a depolarised voltage, however, appeared faster for the deleted form. To quantify the changes in activation and deactivation kinetics between the two channel types, their time dependence was analysed by fitting a two-exponential plus a time-independent model to the relaxations (Cid et al. 2000). Figure 1C and D shows the time constants obtained. They were both voltage dependent, becoming faster with hyperpolarisation, and were markedly faster for rClC-216-61 than for the wild-type. Fractional amplitudes are shown in Fig. 1E and F. The amplitude of the slow component of gating (A1) was larger for rClC-2 than for rClC-216-61, and the reverse was true for the fast component (A2). The fractional amplitude of the instantaneous component was largely voltage independent and not different between the two channel types. In Fig. 2 a summary for the analysis of the deactivation process of rClC-2 and rClC-216-61 is shown. Deactivation at 40 mV was a slow process that could be fitted by a double exponential time course. Both time constants were markedly faster for rClC-216-61 with similar relative contributions, as seen in the plot of fractional amplitudes.
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Figure 1. Gating of rClC-2 and rClC-2 A and B, representative current traces, elicited from a holding potential (Vh) of 0 mV in response to test pulses, delivered every 60 s, ranging from 0 to -180 mV in 20 mV steps. These were followed by a pulse to 40 mV. The duration of the main pulses was increased at less positive voltages in order to approximate full activation of the conductance. For the purposes of illustration, the beginning of the tail currents at 40 mV were set at the same time. A, rClC-2. B, rClC-2 | ||
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Figure 2. Time course of deactivation of rClC-2 or rClC-2 Time constants and fractional amplitudes for the fit of the deactivation at 40 mV to a double exponential model are shown for rClC-2 (open columns) and rClC-2 | ||
Effect of 16-61 deletion on the kinetics of activation of rClC-2 recorded with nystatin-perforated patches
The time and voltage dependence of rClC-216-61 observed here is in marked contrast to results obtained by voltage clamp with intracellular microelectrodes reported for the channel expressed in oocytes (Gründer et al. 1992), which under those conditions showed voltage and time independence. When using inside-out patches from rClC-216-61-expressing oocytes, however, clear time dependence could be seen (Pusch et al. 1999). An explanation for these discrepancies could be that in the whole-cell recording mode of the patch-clamp technique used here, as in the inside-out patch experiment, some intracellular component essential for confering lack of time dependence is lost. As a way of assessing this possibility, recordings were obtained by patch-clamping using nystatin-perforated patches (Horn & Marty, 1991). The currents associated with rClC-2 expression using this recording method did not differ in any way from those recorded by the conventional approach. Figure 3 shows a nystatin-perforated patch recording of rClC-2 exhibiting slow activation by hyperpolarisation (A) and an inwardly rectifying current-voltage relation (B). No statistically significant differences with rClC-2 recorded conventionally were observed after quantifying the activation and deactivation processes as described above (Fig. 3C and D). Results obtained with rClC-216-61 were strikingly different: no time or voltage dependence was observed over a wide voltage range (Fig. 4A) yielding a linear steady-state current-voltage relation (Fig. 4B). ClC-2 is known to be inhibited by Cd2+ (Clark et al. 1998) and has a characteristic permeability sequence (Thiemann et al. 1992; Furukawa et al. 1998). These properties were present in nystatin-perforated patch-clamp recordings of rClC-216-61 currents. The current thus recorded was Cd2+ sensitive as shown in Fig. 4B.
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Figure 3. Gating properties of rClC-2 in nystatin-perforated whole-cell patch-clamp recordings A, currents elicited from 0 mV in response to the same protocol used in Fig. 1. B, current-voltage relation for the currents measured at the end of the voltage steps. C, time constant for the slow ( | ||
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Figure 4. Behaviour of rClC-2 A, voltage protocol with the respective currents obtained in a cell transfected with rClC-2 | ||
The reversal potentials for the currents mediated by rClC-2 and rClC-216-61 in conventional whole-cell recording, and by rClC-216-61 in nystatin-perforated patch recordings, were -41 ± 4, -37 ± 2 and -36 ± 4 mV, respectively (mean ± S.D.). With 35 mM intracellular Cl-, the predicted reversal potential would be -37 mV for a membrane perfectly selective for Cl-. This suggests a negligible permeability for gluconate. Relative permeability to other anions, PX/PCl, where X stands for the foreign anion, was studied by partial replacement of extracellular Cl- as shown in Fig. 4C and D. The permeability sequence was Cl- > Br- > NO3- > I-. This did not differ from either rClC-2 or rClC-216-61 recorded in the conventional way (Fig. 4D). Slope conductances at the reversal potential were also measured under these partial replacement conditions and are reported as *GX/GCl in Fig. 4D (lower panel). These do not represent strictly the relative conductances for the foreign anions as in all measurements 16 mM Cl- remained in the medium.
There was no significant difference in the voltage dependence of the apparent conductances measured from the tail currents at 40 mV, between rClC-2 or rClC-216-61 studied in conventional whole-cell recording, or nystatin-perforated patch recording for rClC-2 (Fig. 5).
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Figure 5. Voltage dependence for the activation of the conductance of rClC-2 and rClC-2 The apparent conductance was calculated from experiments like that illustrated in Fig. 1A by taking the current at the beginning of a pulse to 40 mV given after various conditioning prepulses to the voltages in the abscissa. Results are means ± S.E.M. of four, five and seven separate experiments, respectively, for rClC-2 measured with nystatin-perforated patches and rClC-2 and rClC-2 | ||
The different behaviour of rClC-216-61 in nystatin-perforated patch compared with conventional whole-cell recordings might be due to a change in cytoplasmic composition between the two configurations. If this were the case, the mutant ought to have voltage-independent behaviour in the intact cell and a shift to a voltage-dependent mode upon dialysis. Depending on the speed of this change, evidence for it might be seen early in a conventional whole-cell recording experiment. Figure 6 shows experiments attempting to observe this shift. In Fig. 6A, a continuous recording in a cell transfected with rClC-216-61 is shown. At the time indicated by the arrow, access to the cell was gained at a holding potential of -20 mV and the asterisks indicate times at which the cell was subjected to voltage stimulation. Immediately upon access there was inward current at the holding potential which was markedly decreased with time. The currents elicited during the first three sweeps, started as soon as possible after access, are shown superimposed in Fig. 6B. The first sweep had an important component of instant current elicited by the main pulse to -120 mV and a sustained current at the post-pulse to 40 mV. The reversal potential for the current measured during the ramp given from -120 mV varied from -12 to -28 and then to -38 mV for sweeps 1, 2 and 3, respectively. This result can be interpreted to mean, first, that the channel was open at rest, and second, that the intracellular Cl- concentration was high, probably as a consequence. The first contention is supported by the fact that there was a sizeable current at the holding potential and a large instant current upon hyperpolarisation. The second, by the fact that the reversal potential was initially rather depolarised and became more negative with time in the whole-cell mode, presumably as equilibration with the 35 mM Cl- of the pipette took place. Changes such as these were not seen in cells transfected with non-mutated rClC-2 as shown in Fig. 6C and D. The current at the holding potential was initially small and remained constant and the activation and deactivation processes characteristic of rClC-2 channels were similarly unaltered. A summary of these results is shown in Fig. 6E by reporting the average values of currents at the holding potential for early times after access for rClC-2 and rClC-216-61, showing low, constant current for the former and large initial current vanishing with time for the latter.
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Figure 6. Evolution of currents at early times after breaking into whole-cell recording mode in rClC-2- or rClC-2 A and C show current measured continuously at the holding potential (-20 mV) before and after gaining access to the cell. Break-in is indicated by the arrows and asterisks indicate times at which voltage stimulation was applied. In B and D the currents elicited by voltage stimulation during the first three pulses is shown for both rClC-2 and the deleted mutant. The protocol took the potential from the holding potential to -120 mV, with an interruption for a ramp taking the potential to 40 mV. After return to -120 mV, a square post-pulse to 40 mV was given. E, summary of measurements of current at the holding potential taken just before each of the first three sweeps. Values are means ± S.E.M. of eight and seven experiments for rClC-2 and rClC-2 | ||
Effect of 16-61 deletion on the dependence of rClC-2 activity on extracellular pH
Differences in the pHo response of rClC-2 and rClC-216-61 expressed in amphibian oocytes have also been reported (Jordt & Jentsch, 1997). Results in Fig. 7A confirm the pH dependence of rClC-2, which is activated at acid and inhibited at alkaline extracellular pH. Unlike previous reports, in the mammalian expression system used here rClC-216-61 recorded conventionally (Fig. 7C) or with nystatin-perforated patches (Fig. 7D), also responded to extracellular pH similarly to the non-mutated rClC-2. These results are summarised in Fig. 7E. The increase in current elicited by acidification appears to be on ClC2-mediated currents as it did not take place in mock-transfected cells. This is shown in Fig. 7B, with similar results being obtained in four separate experiments.
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Figure 7. Effect of changes in extracellular pH upon rClC-2 and rClC-2 A, measurements in rClC-2-expressing cells recorded conventionally. C and D, measurements in rClC-2 | ||
Effect of 16-61 deletion on gpClC-2, the guinea-pig ClC-2 orthologue
As was the case for rClC-216-61, the equivalent deletion in (guinea-pig) gpClC-2 did not yield dramatic changes in voltage dependence of the channel. Expression of gpClC-216-61 gave rise to slowly developing, strongly inwardly rectifying currents in conventional whole-cell recordings (data not shown). Save for faster kinetics, similar to what is described above for the rat orthologue, there was no marked difference in behaviour with non-mutated gpClC-2 (Cid et al. 2000). The apparent voltage dependence of gpClC-216-61 conductance, gauged from the tail currents at 40 mV, was not different from previously published data for gpClC-2 (Cid et al. 2000).
As with the rat channel, whole-cell recordings in nystatin-perforated patches, whilst not markedly affecting gpClC-2, led to a loss of voltage dependence for gpClC-216-61. The lack of voltage dependence of gpClC-216-61 could be interesting in a physiological context if a naturally existing equivalent could be found. The presence of an N-terminal truncated form of ClC-2 was reported in rabbit heart suggesting strongly a variant with open channel phenotype. This, however, was later demonstrated to be a consequence of a cloning artifact (Furukawa et al. 1995). A genuine ClC-2 splice variant has been found in human and guinea-pig intestinal tissue which presents a deletion of amino acids 77-86. The variant gpClC-277-86, measured in conventional whole-cell recordings, was not affected in its voltage dependence and only differed in deactivation kinetics (Cid et al. 2000). It could, nevertheless, behave differently in non-dialysed cells and this was therefore tested. Unlike what was seen with rClC-216-61, recording with nystatin-perforated patches did not cause a significant change in voltage dependence of gpClC-277-86 (Fig. 8).
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Figure 8. Gating of (guinea-pig) gpClC-2 A, representative current traces, elicited from a Vh of 0 mV in response to test pulses, delivered every 60 s, ranging from -180 to -20 mV in 20 mV steps. These were followed by a pulse to 40 mV. 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 beginning of the tail currents at 40 mV were set at the same time. B, current taken at the end of pulses is plotted against voltage. C, current at the beginning of pulse to 40 mV as a function of the preceding pulse. Similar results were obtained in four separate experiments. The fit was obtained with a V0.5 of -107 mV and a slope factor of 25 mV. | ||
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DISCUSSION |
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The experiments reported above, showing significant voltage dependence for 16-61-ClC-2 currents measured in conventional whole-cell mode, are not compatible with a ball-and-chain model in which residues 16-61 would be an essential part of the ball domain. This discrepancy with previously observed results obtained in amphibian oocytes using a two intracellular microelectrode approach is explained by our present experiments showing that when recording with nystatin-perforated patches, the oocyte results are reproduced. This observation provides a rationale to account for the puzzling result obtained in isolated membrane patches from oocytes showing voltage-dependent gating of rClC-216-61 (Pusch et al. 1999). Present and previous results could be explained by postulating a diffusible intracellular component that interacts with the deleted form of the channel to block voltage-dependent gating, locking it in the open state. This putative component fails to affect the wild-type channel, therefore one must assume that the putative site of interaction becomes exposed only after mutation. The intracellular factor, if it exists, does not affect the wild-type channel or a partially N-terminal deleted, naturally occurring ClC-277-86 splice variant. This would suggest that the effect might be artifactual rather than physiological. Another possible explanation for the results might involve a difference in the osmotic forces in the cytoplasm of conventionally recorded nystatin-permeabilised patch cells. This explanation finds some support in the reported osmosensitivity of the channel expressed in amphibian oocytes (Gründer et al. 1992). Finally, it is possible that the dual behaviour might be related to a different channel-cytoskeleton interaction in the mutant. Again, it has been suggested that an interaction of the cytoskeleton with the N-terminus might take place and it has been proposed to account for the different kinetic behaviour of the N-terminus-deleted channel (Ahmed et al. 2000).
The deletion is not without effect on the kinetics of the channel, leading to an acceleration of both opening and closing. The rate constants for the opening of 16-61-ClC-2 were less voltage dependent than those of the wild-type channel. The fast (considered instantaneous here) and very fast components made up 67 % of the total current for 16-61-ClC-2 compared with only 43 % for the non-mutated channel. Not much is known about the gating processes of ClC-2. The work of Miller originally showed that ClC-0 had a double-barrelled structure with separate pores that have individual gates, and a common gate that affects both pores simultaneously (Miller, 1982; Miller & White, 1984; Middleton et al. 1996). A similar structure has been proposed for ClC-1 (Saviane et al. 1999). There is also recent evidence for a double-barrelled structure of ClC-2 obtained from tandem constructs ClC-2/ClC-2 and ClC-2/ClC-0 (Weinreich & Jentsch, 2001), but whether the gating process for ClC-2 is similar to that in ClC-0 and -1 is not yet known. The dimeric structure has had recent confirmation from structural data on other ClC channels (Dutzler et al. 2002). The changes in kinetics of the channel do suggest a participation of the N-terminus in the gating process and changes in kinetics have also been shown for the 77-86 alternatively spliced channel (Cid et al. 2000). The lack of a realistic model for ClC-2 gating prevents further speculation of its specific function.
The similar effect of extracellular pH upon ClC-2 and ClC-216-61 recorded here conventionally or with nystatin-perforated patches was surprising in view of previous results on the oocyte system. The effect of extracellular pH on ClC-2 has been described in oocyte expression experiments as a marked change in voltage dependence with acid pH facilitating channel opening (Jordt & Jentsch, 1997). The N-terminal-deleted ClC-2 was described as pH insensitive and an effect of pH on the open pore was discarded on this basis. Instead the effect of pH was proposed to be on the same gating mechanism that was affected by hyperpolarization, i.e. the ball-and-chain mechanism. Our results show that both channel forms are affected similarly by extracellular pH. Even when the recordings of rClC-216-61 were performed with nystatin-perforated patches, that is, in the absence of voltage-dependent gating, a similar effect of extracellular pH was observed. Thus the effect of pH is not mediated by interaction with the gating process governed by voltage, and whatever gating process the N-terminus is involved in, the effects of protons are exerted independently.
In summary, the data presented here are incompatible with a simple ball-and-chain gating mechanism, although they point to an important role for the N-terminus of ClC-2 in voltage-dependent gating. Gating by pH is probably a separate process that has to be related to the interaction of protons with a region of the protein separate from the deleted region.
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REFERENCES |
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Acknowledgements
We are grateful to T. J. Jentsch for a gift of rClC-2 constructs and for comments on an early version of the manuscript. This work was supported by grant Fondecyt-Chile 1000622. Institutional support to the Centro de Estudios Científicos (CECS) from Empresas CMPC is gratefully acknowledged. F.V.S. was an International Scholar of the Howard Hughes Medical Institute and holds a J. S. Guggenhein Memorial Foundation fellowship. CECS is a Millennium Science Institute.
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1) and fast time constant (