Zentrum für Molekulare Neurobiologie Hamburg, Universität Hamburg, 20246 Hamburg, Germany
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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
-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.
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 M. 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.
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RESULTS |
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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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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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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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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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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.
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).
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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DISCUSSION |
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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--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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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