Determinants of slow gating in ClC-0, the voltage-gated chloride channel of Torpedo marmorata

Peying Fong, Annett Rehfeldtdagger , and Thomas J. Jentsch

Zentrum für Molekulare Neurobiologie Hamburg, Universität Hamburg, 20246 Hamburg, Germany

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Membrane hyperpolarization normally activates the slow gate of the Torpedo voltage-gated chloride channel (ClC-0). To elucidate the structural basis of this process, carboxy terminus truncation mutants and chimeras were constructed, expressed in Xenopus oocytes, and evaluated using a two-microelectrode voltage clamp. Introduction of stop codons at several positions between transmembrane domains 12 and 13 (D12 and D13) showed no expression, whereas a truncation just after D13 yielded wild-type currents. A chimera (022) entailing the substitution of the carboxy-terminal cytoplasmic tail after Lys-520 with the corresponding region of ClC-2 lacked slow gating, whereas a more conservative construct (chimera 002), in which D13 was replaced with its ClC-2 analog, retained its capacity to slow gate. These findings suggest that important structures reside within the interdomain stretch (IDS) between D12 and D13. Unlike ClC-2, in which transplantation of "ball" structures could restore gating to constitutively open mutants, transplantation of the ClC-0 IDS to the amino terminus of chimera 022 did not restore gating. Surprisingly, replacement of the IDS by the analogous regions of either ClC-1 or ClC-2 showed slow voltage-activated gating, although the gating was altered. Our findings lead us to conclude that both the functional expression and the slow voltage gating of ClC-0 rely on structures at the carboxy terminus of the channel.

voltage clamp; chimera; truncation

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE EXPRESSION CLONING OF the voltage-gated chloride channel of Torpedo marmorata (ClC-0) (9) has led to the molecular elucidation of an expanding family of chloride channels (11, 14, 31-34). These channels show different expression patterns and correspondingly play diverse physiological roles. Mutations in ClC-1, which is normally responsible for the substantial chloride conductance of skeletal muscle, result in a range of myotonias (12). ClC-2, a ubiquitous hypotonicity and hyperpolarization-activated member, may be important in cell volume regulation (7). Mutations in ClC-5 lead to the kidney stones associated with hereditary hypercalciuric nephrolithiasis (5, 14, 32). Recently, Bartter's syndrome was linked to disruptions in the ClC-Kb channel (30).

Biophysical descriptions of voltage gating by the chloride channel of Torpedo preceded its cloning by a decade and have contributed substantially to our knowledge of ion channel gating behavior (8, 22, 23, 35). To describe the unique bursting behavior and twin conductance states of chloride channels reconstituted into planar bilayers, Miller and White (23) proposed the "double-barreled" model of gating, which postulates the existence of two functional protochannel subunits. According to this model, each protochannel possesses an independent, depolarization-activated gate with rapid kinetics. At the same time, they share a common slowly gating mechanism that displays a reciprocal voltage dependence.

Until the cloning of ClC-0, structural analyses of these gating phenomena have not been feasible. Initial expression studies in Xenopus oocytes, assessed using two-microelectrode voltage-clamp measurements, showed macroscopic currents displaying both modes of gating behavior described previously for reconstituted channels. Bauer et al. (2) demonstrated that injection of ClC-0 mRNA alone was sufficient to produce single channels having conductance and kinetic properties identical to the reconstituted native channel, suggesting that additional subunits are not necessary. Voltage-clamp studies have indicated that ClC-1 normally possesses only a depolarization-driven fast gating mechanism. More recent studies have shown that, in oocytes expressing ClC-1, a component resembling the slow gate of ClC-0 is induced on exposure to low external pH (27). ClC-2 activates slowly on strong hyperpolarization and by external hypotonicity but has no fast gating. Gründer et al. (7) utilized mutagenesis strategies to demonstrate that this slow gating by ClC-2 is mediated by a position-independent, amino-terminal "ball" moiety. Recently, a potential receptor for this structure was identified within the transmembrane loop between domains 7 and 8 (D7 and D8) (10). In contrast, the amino terminus of ClC-0 does not play a critical role in its slow gating (6). More recent studies (16-18, 21, 25) have investigated the behavior of point mutants and concatameric constructs of the Torpedo channel. In addition to elucidating the multimeric structure of ClC-0 (17, 20, 21), studies of fast gating strongly suggest that this process is driven by the permeating anion, which supplies the gating charge (4, 26). Moreover, extensive kinetic analysis of these heterologously expressed channels has confirmed the independence of both permeation and fast gating by each constituent pore (18). The modest temperature dependence of the fast gate (Q10 ~2.2) is in striking contrast to that for the slow gate (Q10 ~40). From such measurements, Pusch et al. (25) conclude that slow gating involves complex rearrangements between the constituent monomers of ClC-0.

We report here our studies that investigate the role of carboxy-terminal structures in the slow voltage gating of ClC-0 by evaluating the functional consequences of introducing premature stop codons within this region. We also test the effects of chimeric substitution with the analogous 3' regions of other chloride channels. Several lines of evidence, including a recent rigorous study of the transmembrane topology of ClC-1 (7, 19, 28), indicate that the carboxy terminus localizes cytoplasmically. Our findings suggest that the region between D12 and D13 contains structures important to both functional expression and normal slow gating of chloride currents. The presence of a native D13, on the other hand, does not appear to be as critical to either expression or gating, although it may play an optimizing role in both. Moreover, data from some of the chimeric constructs reveal a novel process that, at very negative voltages, attenuates the currents carried by ClC-0.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Construction of mutant ClC-0 channels. The major portion (at least up to the unique Hind III site after D12 for carboxy terminus chimeras) of all mutant channels consisted of ClC-0 and from this point forth will be referred to as the backbone. Fragments for the construction of chimeric channels were prepared by restriction digestion of either ClC-1 or ClC-2. To introduce premature termination codons into ClC-0, primers incorporating the ochre mutation were used in the generation of fragments by PCR (Pfu DNA polymerase, Stratagene, La Jolla, CA). For all constructs, the restriction enzyme-digested fragments were ligated to the ClC-0 backbone using the T4 DNA ligase (GIBCO BRL). All constructs were cloned into either pBluescript (KS+, Stratagene) or pTLN, a pSP64T derivative that contains both the 5' and 3' untranslated regions of the Xenopus beta -globin gene (13, 15). Constructs in pBluescript incorporated the native 5' untranslated region of the Torpedo channel; this was not the case for inserts subcloned into pTLN. DNA prepared from positive colonies was analyzed by restriction digestion, and inserts were sequenced by the chain termination (dideoxynucleotide) method.

Preparation of mRNA and injection into Xenopus oocytes. Linearized template was treated with proteinase K (Boehringer Mannheim, Mannheim, Germany) and phenol/chloroform extracted before use in RNA synthesis and capping reactions (Stratagene, La Jolla, CA; Ambion, Austin, TX). Capped mRNA was resuspended in diethyl pyrocarbonate-treated water and divided into aliquots before storage at -80°C.

After surgical removal of ovarian lobes from Xenopus laevis, stage V and VI oocytes were manually defolliculated and kept in a modified Barth's solution [in mM: 90 NaCl, 1 KCl, 1 CaCl2, 0.33 Ca(NO3)2, 0.82 MgSO4, and 10 NaHEPES; pH 7.6] at 4°C. Oocytes were injected with mRNA using a positive displacement pipettor (Drummond Scientific, Broomall, PA). After injection, oocytes were stored in modified Barth's solution, with daily changes of the medium, in a 16°C incubator. Wild-type ClC-0 mRNA expressed robustly in oocytes as early as 1 day postinjection, whereas mutants displayed a broad range of expression levels. Inserts in the pTLN vector displayed levels of expression exceeding those in pBluescript, with no detectable changes in channel properties. Uninjected and water-injected oocytes served as negative controls.

Voltage-clamp current measurements. We performed measurements with ND-96 (in mM: 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 NaHEPES; pH 7.5) as the perfusing bath solution. Resistances of voltage and current microelectrodes ranged between 1 and 2 MOmega . Data were acquired using a TurboTec 01C voltage-clamp amplifier (NPI, Tamm, Germany) interfaced to a 486 computer (Escom) by a Tecmar analog-to-digital/digital-to-analog board (Scientific Solutions, Solon, OH). Acquisition and analysis were facilitated by using commercially available programs (pCLAMP 5.5, Axon Instruments, Foster City, CA; Sigma Plot, Jandel Scientific, Corte Madera, CA), as well as several fit routines written by Dr. Michael Pusch. Normalized current responses were calculated using the relationship Inorm = (Imeas - Imin)/(Imax - Imin), where Inorm refers to the normalized current, Imeas is the current value of interest, Imin is the minimum current, and Imax represents the maximum current.

Statistical analysis. Xenopus oocytes are notorious for the variability of their endogenous currents between batches. Thus, although a larger body of data exists that supports the conclusions of this study, most analyses presented were limited to only those comparisons of test and control oocytes that can be paired on the basis of the oocyte batch. The exceptions are explicitly noted. To provide a more accurate impression of the data scatter, all summary values are presented as means ± SD.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ClC-0 activates slowly on hyperpolarization (9). This gating can be assessed by measuring pseudo-steady-state currents in response to a constant test pulse after application of long, progressively hyperpolarizing prepulses (Fig. 1A). The time point at which these currents are measured (7.2 s) allows for a full relaxation of the fast gate without changing the open probability of the slow gate and therefore permits the assessment of the slow gate in isolation. Figure 1B illustrates the response of ClC-0 to this protocol in a typical experiment. Because expression levels show variability even within batches, test responses routinely are normalized to facilitate ready comparisons between individual oocytes. Figure 1C shows the normalized steady-state currents derived from the experiment shown in Fig. 1B plotted as a function of the prepulse voltage, compared with the mean values from four experiments. Such steady-state current-voltage (I-V) plots can be fitted by a Boltzmann function and, for the wild-type Torpedo channel, is characterized by a half-maximal voltage of gating (V1/2) of approximately -100 mV and a nominal gating charge of ~2 (Fig. 1C).


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Fig. 1.   A: pulse protocol used to evaluate slow gating of expressed ClC-0 channel currents. Prepulse potentials (6 s) started at a level of +60 mV and were stepped in -20-mV increments after each subsequent test pulse (2-s duration) to +40 mV. The most negative prepulse potential applied was -160 mV. Oocytes were clamped at their respective resting membrane potentials for 1 s before and 1 s after these steps. For expressing cells, this usually ranged between -20 and -30 mV. B: typical ClC-0 currents measured 1 day postinjection. Increased currents in response to constant test pulse reflect activation of slow gate. Note that ClC-0 currents express abundantly enough that contributions by native hyperpolarization-activated chloride channels do not obscure measurements appreciably. Presence of such endogenous currents in this oocyte can be seen in slowly developing inward currents in response to hyperpolarizing prepulses. C: currents measured during test pulse period are normalized (norm) as described in MATERIALS AND METHODS and plotted as a function of prepulse potential. bullet , Normalized data from trace shown in B. square , Mean normalized values from 4 oocytes (including the one represented in B) taken from 4 batches, 1 day postinjection. Error bars indicate SD of mean normalized currents. A Boltzmann fit of these averaged points (no symbols, solid line) is shown for comparison. The form of equation used was open probability (Po) = 1/{1 + exp[zneo(V1/2-V)/kT]}, where zn represents nominal gating charge, eo is the elementary charge, V1/2 is half-maximal voltage of gating, V is voltage, k is Boltzmann constant, and T is temperature. Po is directly related to macroscopic current (I) by relationship I = nPoi, where n is number of channels in oocyte and i is single-channel current. Values of zn and V1/2 obtained are, respectively, 1.93 elementary charges and -98.66 mV. Vhold, holding potential.

Stop codon mutations implicate an involvement of carboxy-terminal structures in expression. Table 1 summarizes the data obtained from oocytes injected with cRNAs encoding ClC-0 into which a series of premature termination codons had been introduced. TAA 3.5, a mutation of Lys-567 to the ochre stop codon, expressed currents indistinguishable from levels seen in water-injected controls. Similarly, no currents were detectable in oocytes injected with cRNAs encoding constructs with stop codons further 3', at positions Ser-600 and Ser-674 (TAA 3.6 and TAA 3.7). In contrast, injection of a construct entailing the placement of a stop codon after D13 (Tyr-772) produced wild-type currents in all cases examined. These experiments therefore suggest that the carboxy terminus is important for expression of normal channels.

                              
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Table 1.   ClC-0 stop codon mutants

Lack of slow gating in a carboxy terminus chimera of ClC-0 and ClC-2. To further investigate the role of the carboxy terminus in slow gating, currents resulting from the expression of a chimeric channel in which the 3' cytoplasmic tail starting from the unique Hind III site after D12 (at position 520) was replaced with the analogous portion of ClC-2 (chimera 022; see Fig. 2) were measured. As can be seen from the unchanged test current responses to the full range of prepulses, this chimeric channel totally lacked slow gating (Fig. 3a), suggesting that the native ClC-0 carboxy terminus may contain structures relevant to this process. Note the robust expression of this construct. Taken together with the inability of TAA 3.5, TAA 3.6, and TAA 3.7 to express, this observation suggests that conserved carboxy-terminal structures distal to Lys-567 are essential to functional expression of currents. However, such replacements evidently are not sufficient for normal slow gating.


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Fig. 2.   A: working model for transmembrane topology of ClC-0 (based on model described in Ref. 28). Note that both amino and carboxy termini are located at cytosolic aspect. Sites relevant to construction of chimeras described are indicated by dashed lines. Domains are numbered 1-13. Note that region referred to as interdomain stretch (IDS) is defined by Hind III and Bgl II restriction sites, whereas area designated D13+ actually includes short (38 amino acids) stretch after Bgl II site. A glycosylated site is indicated by asterisk. B: carboxy terminus chimeras considered in this study, as well as 2 that were constructed but did not warrant further scrutiny (chimeras 001 and 011). WT, wild type; 0, 1, and 2 denote regions from ClC-0, ClC-1, and ClC-2, respectively. Oocytes injected with chimera 001 did not express currents that could be distinguished reliably from those generated by endogenous, hyperpolarization-activated currents. Preliminary experiments have shown that chimera 011 expressed currents resembling those seen previously in ClC-0 point mutations (16) with respect to their strong inward rectification, as well as lack of slow gating (data not shown). This mutation was not studied further.


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Fig. 3.   A and B: raw current responses showing that replacement of region following Hind III site (chimera 022; A) results in loss of slow gating and that slow gating is conserved in chimera 002 (B). Evaluation of steady-state current-voltage (I-V) plot parameters indicate slow gating of chimera 002 resembling that of wild-type ClC-0 (see Table 2 for results of paired analyses). Note that only responses to prepulses to +60, +20, -20, -60, -100, and -140 mV are shown. C: for purposes of comparison, wild-type current responses are plotted similarly. All 3 oocytes originated from same batch. A and B were taken on same day (day 1) postinjection, whereas C was measured on day 2. D: experimental record showing that transplantation of a large portion of ClC-0 IDS to amino terminus of chimera 022 does not restore slow gating. A 3-stage recombinant PCR reaction produced a fragment that subsequently was subcloned into amino terminus of chimera 022. This yielded a construct (B6) that incorporated IDS region between Ser-530 and Ile-700 just after initiator Met.

To study the role of the carboxy terminus further, we next divided it into two regions and exchanged these individually to construct chimeric cDNAs (Fig. 2). Interestingly, a more conservative chimera (002), in which only the D13+ of ClC-0 was replaced with that of ClC-2, retains its ability to gate slowly (Fig. 3B), although the currents that develop during the conditioning pulse period often appear to take on a more pronounced inward component. These data suggest that loss of slow gating by chimera 022 is not due to replacement of D13+, and hint that ClC-0-specific structures important for gating may reside within the interdomain stretch (IDS).

Transplantation of the ball region of the ClC-2 gate to the carboxy terminus of a constitutively open mutant can restore slow gating (7). By analogy, if the IDS indeed contributes to slow voltage-dependent gating, can this attribute be restored by reintroduction of this region to the amino terminus of a nongating mutant such as chimera 022? A chimera entailing transplantation of a large portion of the IDS (between positions Ser-530 and Ile-700) to the amino terminus of chimera 022 was constructed, and the current responses to the standard pulse protocol were measured (Fig. 3D). As can be seen by the lack of change in currents measured at the +40 mV test pulse, this does not restore the slow gate. These observations therefore argue against the notion that the IDS acts as a discrete, position-independent gating moiety.

Effects of replacing the IDS. To test further the involvement of the IDS in slow gating, we constructed a chimeric channel in which the IDS, defined at the 5' end by the Hind III site and at the 3' end by a Bgl II site, was replaced by the corresponding region of ClC-2 (chimera 020; see Fig. 2). Surprisingly, slow gating is present, although altered, in this construct (Fig.4, A and B; Table 2). Taken together with the persistence of slow gating in chimera 002, these findings strongly suggest that abolishment of slow gating in chimera 022 is not the consequence of losing discrete, essential, ClC-0 carboxy terminus-specific structures. Instead, the findings necessitate consideration of the possibility that higher order, possibly cooperative, interactions mediate this gating process.


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Fig. 4.   A: current responses of chimera 020, a chimera entailing substitution of ClC-0 IDS with that of ClC-2. For clarity, responses to only 6 prepulses (in 40-mV, rather than 20-mV, increments) are shown. Amplitudes achieved after prepulses to -60, -100, and -140 mV are indicated. These data, together with plot of normalized current responses vs. prepulse potential (B), clearly illustrate change in slow gating profile from sigmoidal to bell-shaped. Chimeric constructs 010 (C) and 012 (D) show similar steady-state I-V responses. In B-D, batch-paired wild-type responses (black-square) are shown for comparison with those of test chimeras (bullet ).

                              
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Table 2.   Paired comparisons of mean normalized currents

Replacement of the D12-D13 stretch reveals a novel current attenuation process. A chimera (010) in which IDS was replaced with the corresponding region of ClC-1, as well as chimera 012, showed alterations in gating similar to those observed for chimera 020 (Fig. 4, C and D; Table 2). Although slow gating was present, all of these chimeric channels displayed strong attenuation of current in response to hyperpolarizing conditioning pulses in excess of approximately -100 mV. We operationally refer to this process as hyperpolarization-induced current decrease (HICD). Although wild-type channels sometimes showed a very slight HICD (see Fig. 1C, control traces in Fig. 4, B-D, and Table 2), this never occurred in response to prepulse potentials more positive than -140 mV.

To obtain information about the time dependence of HICD, we maximally activated the slow gate by a 4-s hyperpolarization to -100 mV and then measured the current (at a test pulse of +40 mV) in response to increasing the duration of a constant level prepulse (to -140 mV, a potential at which HICD is notable). Our preliminary analysis of the resultant current decline suggests a time constant on the order of seconds (data not shown).

IDS chimeras display an apparent shift of the V1/2 to more positive potentials. However, our estimation of V1/2 for the slow gate (see MATERIALS AND METHODS) depends on the position of the current maximum along the voltage axis. If HICD represents a separate process that overlaps with the voltage-dependent opening of the slow gate and its V1/2 is shifted in the positive direction, then the overall current maximum is also shifted to more depolarized values. Estimations of the slow gate V1/2 based on such measurements therefore may not represent true shifts in voltage dependence of the slow gate per se.

HICD also presents independently of an apparent shift in V1/2 and may be modulated by D13. That replacement of D13+ may influence gating is best seen by comparing the gating behavior of chimera 020 and 022 (Figs. 3A and 4A). The test period current responses of chimera 002, a chimera with only D13 exchanged, are not readily distinguishable from those of wild-type ClC-0 in most cases (compare Fig. 3, B and C). Like the wild-type ClC-0, in a few oocyte batches chimera 002 also displayed HICD following prepulses to -160 mV, but this drop was independent of any apparent shifts in V1/2. However, Table 2 shows that the variability of the chimera 002 current decrease, as indicated by the SD (±0.394), exceeds that of the paired wild-type oocytes (±0.077).

Interestingly, chimera 012, an IDS chimera also incorporating a substitution of ClC-0 D13+ with that of ClC-2, shows HICD to a greater degree than chimera 010 (Fig. 4D; Table 2). In keeping with the comparison between chimera 002 and wild-type ClC-0, the apparent V1/2 of chimera 012 does not differ from that of chimera 010. Taken together, these observations hint that the D13+ region may modulate HICD.

The fast gate remains unchanged in chimeric channels. Despite a noticeable reduction in the expression level, the fast gate of these chimeras and all other chimeras studied was not changed. To obtain the instantaneous I-V, we used the protocol described by Pusch et al. (26). Briefly, the oocytes were clamped to a hyperpolarizing potential (typically -140 mV) for ~10 s to maximally activate any slow gating process. Deactivating currents in response to test pulses (20 ms) ranging from -160 mV to +60 mV then were measured after a constant +60-mV conditioning pulse (20 ms). The currents were extrapolated to time 0 of the test pulse period by an exponential fit routine. The resultant open channel I-V relationships remained linear (Fig. 5), suggesting that permeation was unaffected as well. In addition, we observed no remarkable alterations in ionic selectivity (data not shown). These observations are noteworthy because Lys-519, a residue found to be important in fast voltage activation, ion selectivity, and permeation (26), immediately precedes the Hind III site used in the construction of chimera 022 and most other carboxy terminus chimeras considered in this study.


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Fig. 5.   Instantaneous currents of carboxy terminus chimeras 022 (black-square), 002 (black-triangle), 020 (down-triangle), and 012 (star ) remain linear. Inset: measurements were obtained from single oocytes of same batch, using a procedure similar to that described by Pusch et al. (26), outlined in RESULTS. After an initial 10-s conditioning period during which oocyte membrane was clamped at -140 mV (A; dotted lines), oocytes were stepped in 12 1-s periods consisting of a 200-ms prepulse (B) to +60 mV followed by a 200-ms test pulse (C; at a voltage between -160 mV and +60 mV in 20-mV increments) and flanked by recovery periods (100 ms clamped at -100 mV and 500 ms at a Vhold equal to initially measured resting level). I-V plot shows instantaneous currents obtained by extrapolation to time 0 of test period (arrow in inset). ClC-0 wild-type currents (bullet ), also from a batch-paired oocyte, are shown for comparison. Note that differences in slope conductance reflect varying levels of expression.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The voltage-gated chloride channel of Torpedo serves to maintain the high conductivity of the noninnervated membrane of the electrocyte during electric discharges by the electroplax. Its unusual gating was first described and modeled by Miller and co-workers (8, 22, 23, 35), who observed novel behavior on a single-channel level in a reconstituted bilayer system. The observation of bursts of activity, always exhibiting rapid transitions to two equal conductance levels and separated by long quiescent periods, led to the development of the double-barreled model of channel gating (23). Although the two identical protochannels independently gate via a fast, depolarization-dependent mechanism, ion conduction can proceed only after the opening of a slow, hyperpolarization-activated gate common to both functional subunits.

The cloning and initial characterization of ClC-0 (9) have permitted the homology cloning of several physiologically significant members of a new ion channel family (11, 31-34). An understanding of the multimeric composition of this double-barreled channel has been facilitated as well (17, 20, 21). Moreover, primary structural information has made possible the further elucidation of the unique gating mechanisms of ClC-0 by enabling the commencement of structure-function studies. Here, we probe the structural basis underlying the process of slow gating.

The lack of expression by TAA 3.5, TAA 3.6, and TAA 3.7, constructs entailing the introduction of a stop codon after D12, implicates the necessity of carboxy-terminal structures to functional expression. A chimeric construct that substitutes the entire carboxy-terminal cytoplasmic tail with the corresponding region of ClC-2 expressed robustly but lacked slow gating. Taken together, these observations suggest that the structural constraints for functional expression are more relaxed than those for gating.

Interestingly, Maduke and Miller (19) recently reported that the introduction of a soluble ClC-0 carboxy terminus peptide into oocytes injected previously with apparently nonexpressing truncation constructs rescued expression of chloride currents. These observations underscore the importance of this region to channel function and furthermore suggest that carboxy terminus truncation products constitute an inactive pool that can assemble with the injected peptide to form functional channels. On introduction of carboxy terminus peptide, these truncated precursors can form active channel complexes. These findings are in agreement with those previously reported for ClC-1 (29), which showed that the truncated ClC-1 channel could be rescued by coinjecting cRNA encoding for the missing part of the carboxy terminus.

Considerable attention has been paid recently to the identification of two CBS domains (named for cystathionine-beta -synthase, one protein in which such motifs also have been found) in the carboxy terminus of ClC channels, including ClC-0 (1, 24). The presence of these motifs raises interest because the crystal structure of another protein containing these domains, inosine monophosphate dehydrogenase, now is known. Coincidentally, the region we have defined in our studies as the IDS includes one CBS domain, whereas D13+ encompasses the second CBS domain. However, the function of CBS domains remains unclear.

If one were to subscribe to the notion that slow gating is determined by discrete regions within the carboxy terminus, then the contrasting behavior of chimeras 022 and 002 would lead to the hypothesis that the presence of the ClC-2 IDS would eliminate this process. However, the response of chimera 020 confounds this prediction, since it displays not an elimination of slow gating but an alteration. We therefore favor the interpretation that the IDS and D13+ cooperate to produce normal slow gating. Because this gate operates simultaneously on both protochannels in the double-barreled channel (23) and requires a high energy of activation (25), the involvement of a concerted conformational change in the multimer is likely. In chimera 022, the addition of large cytoplasmic regions of ClC-2 could make gating transitions energetically unfavorable, perhaps by influencing monomer interactions.

What underlies HICD? Wild-type ClC-0 sometimes shows a very slight drop in current responses after prepulses to -160 mV (see Fig. 1C, control traces in Fig. 4, B-D, and Table 2). This suggests that HICD represents another voltage-dependent gating process present in wild-type ClC-0 that is more conspicuous in the carboxy terminus mutants, perhaps due to a shift of its gating to more positive voltages. A possible hypothesis is that HICD reflects a disassembly of channel subunits, the association of which is determined by the carboxy terminus. Thus substitutions within this region could lead to structures that are more labile when presented with strongly hyperpolarized membrane potentials, resulting ultimately in the current attenuation characteristic of HICD. It is interesting to note that precedent exists for dissociation mechanisms in channel gating. For example, the slow closure of gap junction channels is thought to proceed by a CO2-sensitive uncoupling of the connexon hemichannels (3). However, in the absence of more direct measurements, we only can speculate on a role for such a mechanism in mediating HICD, which alternatively may just represent a conformational change of the channel protein. We also cannot rule out possible effects of reversible dielectric breakdown of the membrane on slow gating, although such processes usually mediate increases in conductance.

In summary, we draw the following main conclusions from this study. First, truncation studies indicate that the presence of the carboxy terminus is necessary for functional expression. Second, the substitution of native carboxy-terminal regions by the corresponding parts of related channels may interfere with the transitions necessary for normal slow gating. Finally, some chimeras display HICD, a diminishment of macroscopic currents on strong hyperpolarization that may reflect the dissociation of dimers. HICD may also be intrinsic to wild-type ClC-0 but cannot be observed within the voltage range in which the slow gate is normally studied.

    ACKNOWLEDGEMENTS

We acknowledge the helpful comments of Drs. W. Günther, C. Lorenz, and B. Schwappach, who read earlier drafts of this paper, and thank Drs. M. Pusch and K. Steinmeyer for many helpful and stimulating discussions.

    FOOTNOTES

dagger Deceased April 1994. 

P. Fong was supported by an International Human Frontiers in Science Long-Term Fellowship during the course of this work. Research in Prof. Jentsch's laboratory is supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemische Industrie.

Portions of this work were presented previously in abstract form (6).

Present address of P. Fong and address for reprint requests: Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510.

Received 16 September 1997; accepted in final form 2 December 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 274(4):C966-C973
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