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To whom correspondence should be addressed: Leibniz-Institut für Molekulare Pharmakologie/Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str.10, D-13125 Berlin, Germany. Fax: 49-30-9406-2960;
Members of the CLC protein family of Cl− channels and transporters display the remarkable ability to function as either chloride channels or Cl−/H+ antiporters. Due to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these members of the late endosomal/lysosomal CLC branch in heterologous expression. Whereas recent data suggest that ClC-7 functions as an antiporter, transport characteristics of ClC-6 have remained entirely unknown. Here, we report that fusing the green fluorescent protein (GFP) to the N terminus of ClC-6 increased its cell surface expression, allowing us to functionally characterize ClC-6. Compatible with ClC-6 mediating Cl−/H+ exchange, Xenopus oocytes expressing GFP-tagged ClC-6 alkalinized upon depolarization. This alkalinization was dependent on the presence of extracellular anions and could occur against an electrochemical proton gradient. As observed in other CLC exchangers, ClC-6-mediated H+ transport was abolished by mutations in either the “gating” or “proton” glutamate. Overexpression of GFP-tagged ClC-6 in CHO cells elicited small, outwardly rectifying currents with a Cl− > I− conductance sequence. Mutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the “anion-coordinating” tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO3− conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl−/H+ antiporter.
), both channel- and exchanger-type CLC proteins share a glutamate in the permeation pathway that is involved in gating (in CLC channels) and in coupling chloride to proton countertransport (in CLC exchangers), respectively. Mutations in this gating glutamate profoundly affect CLC channel gating and uncouple anion from proton countertransport in CLC exchangers. All confirmed CLC antiporters display another glutamate (the proton glutamate) at their cytoplasmic surface that probably transfers protons to the central exchange site given by the gating glutamate (
). Because this proton glutamate is not found in confirmed CLC channels, its presence might indicate that the respective CLC is an exchanger.
Based on this hypothesis, ClC-3–7 should function as Cl−/H+ exchangers, but this remains to be shown for ClC-3, -6, and -7 by heterologous expression. Contrasting with ClC-4 and -5, which reach the plasma membrane to a degree that allows for detailed biophysical studies, currents mediated by ClC-3 were too low to determine whether it transports protons (
). On the other hand, ClC-3 is ∼80% identical in sequence to the established exchangers ClC-4 and ClC-5, with which it shares current properties that are similarly affected by mutations in the gating glutamate (
), in the plasma membrane. This may be a consequence of their efficient targeting to late endosomes and lysosomes, respectively. ClC-7 is the only member of the CLC family significantly expressed on lysosomes (
). Remarkably, this pathology is not associated with significant neuronal cell loss. A more severe form of lysosomal storage disease (and osteopetrosis) is observed with the loss of ClC-7 in mice and humans (
). For ClC-7, this hypothesis is strongly supported by mice carrying an uncoupling point mutation in ClC-7. These mice accumulate less Cl− in lysosomes and display severe lysosomal storage disease just like ClC-7 KO mice (
), it is of utmost importance to determine whether it likewise performs Cl−/H+ exchange. In this work, we achieve for the first time plasma membrane expression of wild-type and mutant ClC-6. We show unambiguously that ClC-6 mediates Cl−/H+ exchange and examine key mutants that change its coupling, conductance, and ion selectivity.
DNA-encoding human ClC-6 was cloned into the EcoRI site of pcDNA3 (Invitrogen) for expression in mammalian cells. For expression in Xenopus oocytes, ClC-6 was cloned into the pTLN vector (
). For expression of N-terminally GFP-tagged ClC-6 in mammalian cells, ClC-6-encoding DNA was inserted into pEGFP-C1 (Clontech) with the following linker sequence (including the initiator Met of ClC-6): SGLRSREFM. For the generation of the equivalent constructs for expression in Xenopus oocytes, EGFP was inserted first into pTLN (the resulting construct served as negative control), and subsequently, ClC-6 was inserted with the same linker sequence as in mammalian cell expression constructs. Point mutations were introduced by PCR with primers carrying the respective mutation. All constructs were confirmed by sequencing the complete open reading frame. The ClC-5 constructs have been described (
Electrophysiology and Qualitative pHi Determinations
Xenopus laevis oocytes were injected with cRNA (5 ng for ClC-5 constructs and 30–35 ng for GFP and ClC-6 constructs) transcribed with the mMessage Machine kit (Ambion) according to the manufacturer's instructions after linearization of the plasmid with MluI. Currents were measured using standard two-electrode voltage clamp at room temperature employing a TurboTec10C amplifier (npi electronic (Gmbh)) and pClamp10 software (Molecular Devices).
Oocytes were superfused with modified ND96 saline (96 mm NaCl, 2 mm potassium gluconate, 1.8 mm calcium gluconate, 1 mm magnesium gluconate). pH was buffered with 5 mm HEPES, MES, or Tris as appropriate. Ion substitutions were done by replacing NaCl with equimolar amounts of NaNO3 or NaI. Proton transport activity was measured with a fluorescence-based device called Fluorocyte (
) and allowed sensitive, qualitative recording of intracellular pH (pHi) changes based on the pH-sensitive excitation of BCECF (injected 10–30 min prior to the experiment). These measurements do not reflect pHi averaged over the whole volume of the oocytes, but report pHi changes close to the plasma membrane. Unless stated otherwise, the stimulation protocol consisted of depolarizing pulses to +90 mV for 400 ms interrupted by 100-ms pulses to −60 mV to avoid the activation of endogenous currents by prolonged depolarization (
). Current activation by standard two-electrode voltage clamp and pHi changes were recorded simultaneously with the pClamp10 software. A relative measure of the alkalinization rate was determined semiquantitatively as the difference of the slopes of the normalized fluorescence values after and before the depolarization protocol.
Patch clamp measurements on CHO cells were performed by standard methods (
). Patch pipettes of 3–5 megohm resistance were filled with 110 mm CsCl, 8 mm NaCl, 0.5 mm CaCl2, 1 mm EGTA, 2 mm Mg-ATP, 20 mm HEPES, pH 7.2. The calculated free Ca2+ concentration was 190 nm. The bath solution contained 118 mm NaCl, 5.6 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 15 mm glucose, 5 mm HEPES, pH 7.5, with NaOH. Osmolarity was adjusted to 270 mosmol/kg for the pipette solution and 290 mosmol/kg for the extracellular solution by adding sucrose. Data were acquired with an EPC-10 double amplifier and Pulse software (HEKA). Whole-cell currents were measured by sequential 20-mV voltage steps from −100 to +100 mV. The Nernst equation (VNernst = −RT/F ln ([Cl]o/[Cl]i)) was used for reversal potential calculation. Reversal potentials measured with gluconate-containing solutions were corrected for liquid-junction potentials.
A membrane-impermeable NH2-reactive biotin ester (sulfo-N-hydroxysuccinimide-LC-biotin; Pierce) was used to label plasma membrane proteins of Xenopus oocytes expressing ClC-6 constructs. Oocytes were incubated for 20 min at 4 °C in ND96 with 1 mg/ml biotin ester. After washing in ice-cold ND96, cells were homogenized in lysis buffer containing 150 mm NaCl, 20 mm Tris, pH 7.6, 1% Triton X-100, and protease inhibitor mixture (Complete+Pefa, Roche Applied Science). After centrifugation at 4000 × g for 1 min, the supernatant was precipitated with streptavidin beads (Pierce). Bound protein was eluted and separated by SDS-PAGE. For immunoblotting, we used rabbit antibodies directed against the C terminus of ClC-6 (6C3; supplemental Fig. 1) and actin (Sigma), respectively. Surface biotinylation of transfected CHO cells was performed similarly.
Plasmid DNA encoding the respective constructs was transfected using FuGENE6 (Roche Applied Science) according to the manufacturer's instructions, and cells were grown in a humidified 5% CO2 incubator at 37 °C for a further 36 h before fixation with 4% paraformaldehyde in phosphate-buffered saline for 15 min. For immunostaining, cells were incubated with 30 mm glycine in phosphate-buffered saline for 5 min and permeabilized with 0.05% saponin in phosphate-buffered saline for 10 min. Both primary and Alexa Fluor-coupled secondary (Molecular Probes) antibodies were applied in phosphate-buffered saline/0.05% saponin supplemented with 3% bovine serum albumin. Untagged ClC-5 and ClC-6 were immunostained with rabbit antibodies directed against the N terminus of ClC-5 (5A2) (
). When we expressed ClC-6 heterologously in mammalian cells, it localized predominantly to punctate intracellular structures and possibly to a very small extent to the plasma membrane (Fig. 1A). Agreeing with a previous study (
), heterologously expressed ClC-6 colocalized with markers of early and recycling endosomes (EEA1 and transferrin receptor, respectively) (data not shown). By contrast, native ClC-6 localizes to late endosomes of neuronal cells (
). We observed that the fusion of GFP to the N terminus of ClC-6 increased its residence in the plasma membrane in several cell lines, including CHO cells (Fig. 1A), HeLa, HEK293, and COS7 cells (data not shown). GFP did not affect the localization of ClC-6 when fused to the C terminus of the protein (data not shown). A similar effect of an N-terminal GFP tag had been found previously for aquaporin 6. This water channel normally resides in intracellular compartments and is mislocalized to the plasma membrane with an N-terminal (but not with a C-terminal) GFP tag (
). The enhanced plasma membrane localization observed with the N-terminally tagged ClC-6, henceforth referred to as GFP-ClC-6, suggested that we might be able to study its biophysical properties at the plasma membrane.
We expressed this construct in oocytes from albino Xenopus laevis frogs and recorded simultaneously currents and changes in intracellular pH (pHi) in response to changes in plasma membrane voltage. Currents were measured using the two-electrode voltage clamp technique. pHi was measured qualitatively in our “Fluorocyte” device (
) that measures the pH-sensitive fluorescence of BCECF, with which oocytes had been injected before the experiment. While these nonratiometric measurements suffer invariably from a drift in fluorescence (
), it allows us to detect fluorescence changes induced by experimental maneuvers (e.g. depolarization) with extraordinary sensitivity. To minimize the activation of endogenous oocyte transport processes, oocytes were not depolarized continuously, but by a train of depolarizing pulses (
). Whereas no currents above endogenous background levels were observed in oocytes expressing untagged ClC-6 or GFP-ClC-6, trains of depolarizing pulses (to +90 mV) induced cytosolic alkalinization (i.e. proton extrusion). Fig. 1B shows typical traces for GFP-ClC-6, ClC-6, and ClC-5. For ClC-5, both proton transport activity (Fig. 1B) and currents (data not shown) were detectable already after 2–3 days, whereas transport activity of GFP-ClC-6 became measurable only 5 days after cRNA injection. We also detected very weak depolarization-induced alkalinization in a few oocytes expressing wild-type ClC-6 (Fig. 1B). To ascertain that the observed pHi changes are mediated by GFP-ClC-6 rather than result from nonspecific activation of endogenous oocyte transporters (
), we inserted prototypical mutations into ClC-6 that are known to abolish proton transport activity in established CLC antiporters. When either the gating or the proton glutamate of GFP-ClC-6 were mutated to alanine, depolarizing pulses no longer induced cytoplasmic alkalinization (Fig. 2A). This loss of H+ transport could not be attributed to reduced protein expression or diminished presence at the surface of oocytes (Fig. 2B). Likewise, the mutant proteins reached the plasma membrane in transfected mammalian cells (Fig. 2C). Hence, GFP-ClC-6 itself mediates proton transport.
As expected for a Cl−/H+ exchanger, replacing Cl− with impermeable gluconate abolished depolarization-induced proton extrusion by both GFP-tagged (Fig. 3A) and untagged ClC-6 (supplemental Fig. 2). By contrast, proton extrusion was observed in the presence of external NO3− and I−, suggesting that GFP-ClC-6 can exchange these anions for protons (Fig. 3B). Unfortunately, the inability to record currents above background levels precluded the estimation of anion-to-proton coupling ratios. Depolarization-induced alkalinization occurred also against electrochemical proton gradients (Fig. 3C) in a direct demonstration of chloride-driven secondary active transport.
In the Xenopus expression system, determinations of pHi by the Fluorocyte device were more sensitive in detecting Cl−/H+ exchange (i.e. it suffered less from background problems) than measurements of electrical currents. To determine electrical properties of ClC-6 we therefore turned to other expression systems and tested several mammalian cell lines for better signal-to-noise ratios. Surface expression of GFP-ClC-6 could be detected in several cell lines, including CHO (Fig. 1A). We finally chose CHO cells for expression as our previous study had shown that this cell line is virtually devoid of the Cl−/H+ exchanger ClC-5 (
). Transient expression of GFP-ClC-6 in CHO cells resulted in currents that were significantly above background, although their magnitudes were low (Fig. 4A). These currents showed less outward rectification than ClC-5. As expected for a chloride conductance, replacing extracellular chloride with gluconate shifted the reversal potential to positive values (data not shown). Replacing chloride with other anions revealed a Cl− ∼ NO3− > I− conductance sequence (supplemental Fig. 3). The low magnitudes of the currents and their rectification precluded a reasonably accurate determination of reversal potentials and permeability ratios. No currents could be elicited by the proton glutamate mutant E267A (Fig. 4A) and the gating glutamate mutant (E200A) yielded currents with a roughly linear current-voltage relationship (Fig. 4B), agreeing with the behavior of the equivalent ClC-5 mutant (
). These mutations are thought to remove an external gate embodied by the gating glutamate and an internal gate represented by a Cl−-coordinating tyrosine. When we inserted equivalent mutations into GFP-ClC-6, the resulting mutant, GFP-ClC-6(E200A, Y576S), yielded currents that were about three times larger than those of the single gating glutamate (Fig. 4B), further supporting the notion that the observed currents are mediated by ClC-6. These currents showed both slight inward and outward rectification like those observed previously with gating glutamate mutations in ClC-4 and -5 (
). Extracellular application of the anion transport inhibitor DIDS (1 mm) nearly abolished the outwardly rectifying currents elicited by GFP-ClC-6 (Fig. 4C). As expected for a Cl−/H+ exchanger and similar to results obtained previously for ClC-4 and ClC-5 (
), extracellular alkalinization increased (Fig. 4D), whereas extracellular acidification decreased (Fig. 4E) GFP-ClC-6 currents. As expected with an uncoupling “gating glutamate” mutation, currents of the GFP-ClC-6(E200A, Y576S) were insensitive to changes in pHo (Fig. 4F). We neither detected proton transport with the Fluorocyte device when this mutant was expressed in Xenopus oocytes (data not shown). Unfortunately, the conductance of the outwardly rectifying GFP-ClC-6 was close to background conductance at the voltages where reversal potentials may be determined. We were therefore unable to reliably determine a shift in reversal potential with changes in extracellular pH, which is expected for Cl−/H+ exchangers (
) proved to be too insensitive to yield reliable results for GFP-ClC-6.
However, the increased current magnitudes of the GFP-ClC-6(E200A,Y576S) mutant allowed us to measure similar currents also in Xenopus oocytes (Fig. 5A). Ion substitution experiments revealed an NO3− > Cl− > I− conductance sequence (Fig. 5A). In the presence of nitrate, the mutant protein showed outward rectification at positive potentials (Fig. 5, A and B). A similar change in rectification with nitrate had been observed previously in a gating glutamate mutant of ClC-5 (
). As expected for a pure anion conductance, substituting extracellular chloride for impermeable gluconate shifted the reversal potential to positive values according to the Nernst potential (Fig. 5C). To further substantiate that these currents were mediated by GFP-ClC-6(E200A,Y576S), we changed the Cl−-coordinating serine 157 to proline. Equivalent mutations in ClC-5 (
). The triple mutant (S157P,E200A,Y576S) was compared with the starting construct GFP-ClC-6(E200A,Y576S) after transfection in CHO cells (Fig. 5, D and E). Patch clamp measurements in the presence of different extracellular anions indeed showed that the S157P mutation increased the nitrate/chloride conductance ratio.
ClC-6 Is a Cl−/H+ Antiporter
We have recorded for the first time the transport activity of ClC-6. Using a fusion protein with enhanced surface expression and point mutations that changed transport properties in a manner that is typical for other CLC exchangers, we obtained compelling evidence that ClC-6 acts as an electrogenic anion/proton exchanger. Mutations of the gating and proton glutamates resulted in changes in charge and H+ transport that were similar to those found in other eukaryotic CLC exchangers (
). Moreover, the S157P mutant increased nitrate conductance of an uncoupled ClC-6 mutant. Taken together, the effects of these mutations very strongly bolster the conclusion that we have measured the transport activity of ClC-6 rather than transporters endogenous to the expression system.
To reach this conclusion, we had to resort to two different expression systems. Xenopus oocytes proved to be superior for the detection of small changes in pHi resulting from proton transport, whereas exchanger currents could only be detected in transfected CHO cells. The detection of Cl− currents in Xenopus oocytes is hampered by their large Ca2+-activated Cl− conductance. Because the Fluorocyte measures pHi close to the plasma membrane, the large volume of the oocytes does not preclude sensitive measurements of plasma membrane proton transport. Although the volume of CHO cells is much smaller than that of oocytes, even the perforated patch clamp technique we have used previously (
) does not totally prevent an equilibration of pHi with the pH of the patch pipette.
In most cases, we examined a GFP-ClC-6 fusion protein that yielded larger transport rates because of increased surface expression. We are confident that the measured transport qualitatively reflects that of native ClC-6 because a) we did not observe changes in ClC-5 transport properties when it was tagged with GFP at its amino terminus,
and b) we could observe very small, but similar depolarization-induced alkalinization in a few oocytes overexpressing wild-type ClC-6.
In many respects, the transport properties of ClC-6 resembled those of other eukaryotic CLC exchangers. GFP-ClC-6 transport depended on the presence of anions. It displayed tight anion-proton coupling as demonstrated by its ability to transport protons against an electrochemical gradient. Proton transport could also be observed when chloride was replaced with iodide or nitrate ions that partially uncouple other CLC exchangers (
). Unfortunately, the low surface expression levels even of GFP-ClC-6 precluded the determination of coupling ratios. Therefore, we cannot make any statement as to a partial uncoupling of anion from proton fluxes with those ions. Like other CLC exchangers and channels, ClC-6 displayed a higher conductance with Cl− than with I−. In contrast to mammalian ClC-4 and -5 and to plant AtClC-a, nitrate conductance did not seem larger than chloride conductance. However, this observation must be viewed with caution because of the low expression levels. Rather unexpectedly, we found that 1 mm DIDS strongly inhibited GFP-ClC-6 currents. This contrasts with the insensitivity of ClC-5 to that anion transport inhibitor (
In addition to demonstrating that the observed transport activity is mediated by ClC-6, the present mutations in the ClC-6 backbone underpin the importance of certain key residues for Cl−/H+ exchange activity and selectivity. Just as in other CLC antiporters, neutralizing the gating glutamate converted ClC-6 into a pure anion conductance that was uncoupled from protons. Likewise, this mutation changed its voltage dependence to a slight inward and outward rectification like equivalent mutations in ClC-4 and -5 (
). This loss of function could not be attributed to a decreased plasma membrane expression. We additionally mutated both the gating glutamate and a tyrosine that is involved in coordinating a chloride ion in the central binding site of EcClC-1 (
). These amino acids were hypothesized to form external and internal gates that open alternatively during exchange cycles. In support of this hypothesis, EcClC-1 showed largely increased transport rates in the (E148A,Y445S) double mutant (
). We tested this idea for the first time for a eukaryotic CLC exchanger. In ClC-6, the combination of these mutations also increased currents, albeit only by a factor of three compared with the single gating glutamate mutant. Based on the moderate increase in currents with the double mutant, we hesitate to call it “channel-like,” as has been proposed for the equivalent EcClC-1 mutant (
). Finally, we showed that in the background of this double mutant an exchange of the Cl−-coordinating serine for proline increased its NO3− conductance. This result extends previous observations with ClC-5 (
) that a proline exchange at this position enhances nitrate selectivity. It also agrees with the earlier observation that even the conservative mutation of Ser123-to-Thr changed the halide selectivity of ClC-0 (
). We conclude that this residue is a critical determinant of ion selectivity in both CLC channels and exchangers.
Currents recorded from GFP-ClC-6-transfected CHO cells were outwardly rectifying. Although the low signal-to-background ratio of these currents warrants a cautious interpretation, their rectification seemed less pronounced than those of ClC-3 to ClC-5 (
). The rectification of intracellular CLC transporters is of critical importance for understanding their role in intracellular organelles, which are mostly believed to be inside-positive.
Cell Biological Implications
Intracellular CLCs are thought to form functional modules with the vesicular H+-ATPase. These vesicular CLCs may provide counterion conductances for electrogenic proton pumping, thereby facilitating acidification of the respective compartment. Indeed, ClC-3, -4, and -5 have been shown to be required for proper acidification of endosomes (
), it has been speculated that intracellular CLCs in general may not only provide countercurrents for proton pumping but may increase the luminal chloride concentration in exchange for protons by secondary active transport. Supporting this hypothesis, organellar nitrate accrual has recently been shown for the plant CLC AtClC-a (
In the present work, we have provided compelling evidence that ClC-6 functions as a Cl−/H+ exchanger and have used mutagenesis to show that its transport properties rely on certain key residues in a fashion similar to other CLC antiporters. Together with previous work (
), the present results suggest that all mammalian vesicular CLCs function as anion-proton antiporters and may have a dual role in vesicular acidification and chloride accumulation. Similar to lysosomal Cl− accumulation by ClC-7 (
) might be due to a reduced Cl− concentration in late endosomes.
We thank P. Seidler, S. Wernick, and S. Zillmann for technical assistance and Anselm A. Zdebik for suggesting that we use the Fluorocyte to investigate ClC-6, for building the device, and for help in its implementation.