ClC-7 is a slowly voltage-gated 2Cl − /1H + -exchanger and requires Ostm1 for transport activity
2011; Springer Nature; Volume: 30; Issue: 11 Linguagem: Inglês
10.1038/emboj.2011.137
ISSN1460-2075
AutoresLilia Leisle, Carmen Ludwig, Florian A Wagner, Thomas J. Jentsch, Tobias Stauber,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoArticle28 April 2011free access ClC-7 is a slowly voltage-gated 2Cl−/1H+-exchanger and requires Ostm1 for transport activity Lilia Leisle Lilia Leisle Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Carmen F Ludwig Carmen F Ludwig Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Florian A Wagner Florian A Wagner Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Thomas J Jentsch Corresponding Author Thomas J Jentsch Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Tobias Stauber Tobias Stauber Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Lilia Leisle Lilia Leisle Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Carmen F Ludwig Carmen F Ludwig Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Florian A Wagner Florian A Wagner Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Thomas J Jentsch Corresponding Author Thomas J Jentsch Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Tobias Stauber Tobias Stauber Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany Search for more papers by this author Author Information Lilia Leisle1,‡, Carmen F Ludwig1,‡, Florian A Wagner1, Thomas J Jentsch 1 and Tobias Stauber1 1Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany ‡These authors contributed equally to this work *Corresponding author. Physiology and Pathology of Ion Transport, Leibniz-Institut für Molekulare Pharmakologie (FMP), Max-Delbrück-Centrum für Molekulare Medizin (MDC), Robert-Roessle-Str. 10, 13125 Berlin, Germany. Tel.: +49 309 406 2961; Fax: +49 309 406 2960; E-mail: [email protected] The EMBO Journal (2011)30:2140-2152https://doi.org/10.1038/emboj.2011.137 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Mutations in the ClC-7/Ostm1 ion transporter lead to osteopetrosis and lysosomal storage disease. Its lysosomal localization hitherto precluded detailed functional characterization. Using a mutated ClC-7 that reaches the plasma membrane, we now show that both the aminoterminus and transmembrane span of the Ostm1 β-subunit are required for ClC-7 Cl−/H+-exchange, whereas the Ostm1 transmembrane domain suffices for its ClC-7-dependent trafficking to lysosomes. ClC-7/Ostm1 currents were strongly outwardly rectifying owing to slow gating of ion exchange, which itself displays an intrinsically almost linear voltage dependence. Reversal potentials of tail currents revealed a 2Cl−/1H+-exchange stoichiometry. Several disease-causing CLCN7 mutations accelerated gating. Such mutations cluster to the second cytosolic cystathionine-β-synthase domain and potential contact sites at the transmembrane segment. Our work suggests that gating underlies the rectification of all endosomal/lysosomal CLCs and extends the concept of voltage gating beyond channels to ion exchangers. Introduction CLC anion transport proteins (Jentsch, 2008), first identified by the cloning of the Cl−-channel ClC-0 from Torpedo (Jentsch et al, 1990), associate with dimers of identical or closely related subunits. Each CLC subunit contains an ion translocation pathway that is largely independent from the other subunit (Lorenz et al, 1996; Ludewig et al, 1996; Middleton et al, 1996; Weinreich and Jentsch, 2001; Dutzler et al, 2002; Robertson et al, 2010). Some CLC channels, however, display 'common gating' of both pores (Miller and White, 1984; Bauer et al, 1991; Accardi and Pusch, 2000). Eukaryotic CLC proteins have large cytosolic carboxyterminal domains comprising two CBS (cystathionine-β-synthase) domains that in some cases can bind nucleotides like ATP (Meyer et al, 2007) and have a poorly understood role in gating (Fong et al, 1998; Estévez et al, 2004; Bykova et al, 2006; Zhang et al, 2008; Zifarelli and Pusch, 2009b). Crystal structures of bacterial (Dutzler et al, 2002) and algal (Feng et al, 2010) CLC proteins, and of CBS domains from vertebrate CLCs (Meyer and Dutzler, 2006; Markovic and Dutzler, 2007; Meyer et al, 2007), have yielded important insights on how their structure relates to their biophysical properties. Intriguingly, the CLC gene family comprises both Cl− channels and electrogenic Cl−/H+-exchangers (Jentsch, 2008). The border between these different transport classes, however, is blurred, as gating of the ClC-0 Cl− channel may involve the transport of a proton (Lisal and Maduke, 2008) and because certain anions can uncouple anion flux from proton countertransport (Nguitragool and Miller, 2006; Zdebik et al, 2008; Bergsdorf et al, 2009; Zifarelli and Pusch, 2009a). Moreover, transport activity of mammalian ClC-3 to ClC-6 Cl−/H+-exchangers is strongly voltage dependent (Steinmeyer et al, 1995; Friedrich et al, 1999; Li et al, 2002; Neagoe et al, 2010). Their almost instantaneous deactivation at negative voltages precludes measurements of tail currents and it remains unresolved whether their voltage dependence results from a voltage sensitivity of the exchange process per se or from turning the transporter 'on' and 'off' ('gating') (Hebeisen et al, 2003; Zdebik et al, 2008; Picollo et al, 2010; Smith and Lippiat, 2010). Mammalian endosomal/lysosomal Cl−/H+-exchangers (ClC-3 to ClC-7) regulate vesicular H+ and Cl− concentration (Jentsch, 2007; Novarino et al, 2010; Weinert et al, 2010). Disruption of endosomal ClC-5 impairs renal endocytosis (Piwon et al, 2000) in Dent's disease (Lloyd et al, 1996), whereas mutations in lysosomal ClC-7 entail osteopetrosis and lysosomal storage disease (Kornak et al, 2001; Kasper et al, 2005). Similar phenotypes were observed when uncoupling point mutations converted these exchangers into pure anion conductors (Novarino et al, 2010; Weinert et al, 2010). ClC-7 needs Ostm1 as β-subunit for protein stability (Lange et al, 2006). Hence, disruption of Ostm1 results in osteopetrosis (Chalhoub et al, 2003) and lysosomal pathology (Lange et al, 2006; Pressey et al, 2010) just like a loss of ClC-7. It has remained unclear which parts of Ostm1 interact with ClC-7 and whether Ostm1 not only stabilizes ClC-7 but also modulates its ion transport activity. Apart from the acid-secreting membrane of osteoclasts (Kornak et al, 2001; Lange et al, 2006), ClC-7/Ostm1 is absent from the plasma membrane, severely limiting its biophysical characterization. Transport studies of native lysosomes (Graves et al, 2008; Weinert et al, 2010) suggest that ClC-7 mediates Cl−/H+-exchange. However, no currents could be measured, essential properties like voltage dependence, kinetics and substrate specificity have remained unknown, and no structure–function analysis could be performed. Here, we exploit the partial plasma membrane expression of recently described ClC-7 mutants which disrupted cytosolic sorting motifs (Stauber and Jentsch, 2010) to characterize the biophysical properties of ClC-7 and its functional interaction with Ostm1. The slow deactivation of ClC-7/Ostm1 resulted in tail currents that revealed functional features that could not be studied with other CLC anion/proton exchangers. Results Basic characterization of ClC-7/Ostm1 Cl−/H+-exchange Disrupting two dileucine lysosomal sorting motifs in the cytosolic N-terminus of rat ClC-7 (rClC-7) partially redirects the mutant protein (rClC-7LL23/24AA,LL36/37AA, in short rClC-7PM) to the plasma membrane (Stauber and Jentsch, 2010). Likewise human ClC-7 (hClC-7) carrying the mutations LL23/24AA and LL68/69AA (hClC-7PM) partially traffics to the plasma membrane, as ascertained in a chemiluminescence assay for an added extracytosolic HA tag (Figure 1A). This assay failed to detect hClC-7PM when co-expressed with Ostm1, possibly owing to a shielding of the epitope by the highly glycosylated N-terminus of Ostm1 (Lange et al, 2006). Indeed, both rClC-7PM/Ostm1 and hClC-7PM/Ostm1 gave robust plasma membrane currents (Figure 1B and C for rClC-7PM; Supplementary Figure S1A for hClC-7PM). Since currents of human and rat ClC-7PM were indistinguishable in both Xenopus oocytes and transfected mammalian cells, we refer to both as ClC-7PM in the following. Figure 1.Basic characterization of ClC-7PM/Ostm1 in Xenopus oocytes. (A) Chemiluminescence assay for surface detection of hClC-7 and hClC-7PM with an extracytosolic HA tag. hClC-7PM–exHA, but not hClC-7–exHA is detected at the surface of Xenopus oocytes. Co-expression with Ostm1 suppresses the luminescence signal although ClC-7PM/Ostm1 yields plasma membrane currents (in B, C). Mean luminescence intensity (error bars, s.e.m.) normalized to hClC-7PM—exHA from four independent experiments. (B, C) Two-electrode voltage-clamp analysis in Xenopus oocytes. Representative voltage-clamp traces (C) of rClC-7PM ('WT'), rClC-7PM(E245A) and rClC-7PM(E312A) co-expressed with Ostm1. Arrow indicates tail currents. Voltage was clamped from −80 to +80 mV in 2 s steps of 20 mV (inset). Mean±s.e.m. of currents reached after 2 s plotted (B) as function of voltage (rClC-7PM, n=20; rClC-7PM(E245A), n=13; rClC-7PM(E312A), n=11; uninjected, n=16 oocytes from at least three batches). Virtually identical results were obtained with hClC-7PM (Supplementary Figure S1A). (D, E) Intracellular pH changes of Xenopus oocytes co-expressing rClC-7PM ('WT' or glutamate mutants) with Ostm1 in response to a 10-s depolarization. Top traces, clamp currents; bottom traces, pH-dependent BCECF fluorescence measured with the Fluorocyte method (Zdebik et al, 2008). Increased fluorescence means alkalinization. Unless indicated otherwise, extracellular solution contained 96 mM Cl− at pH 7.4. For 0 Cl−, gluconate replaced Cl−. Left traces in (D) and (E) are from the same oocyte and centre and right recordings in (E) are from one oocyte as well. Similar results were obtained with at least five oocytes from three batches. Download figure Download PowerPoint Expression of ClC-7PM/Ostm1 in Xenopus oocytes (Figure 1B and C; Supplementary Figure S1A), tsA201 or HeLa cells (Supplementary Figure S1B) yielded strongly outwardly rectifying currents that activated slowly at voltages more positive than ∼+20 mV. In stark contrast to ClC-3 through ClC-6 (Friedrich et al, 1999; Li et al, 2002; Matsuda et al, 2008; Neagoe et al, 2010), full activation was not even observed after several seconds and slow deactivation resulted in tail currents at negative voltages (Figure 1C (arrow); Supplementary Figure S1A and B). Whole-cell patch-clamp experiments in HeLa cells showed that ClC-7PM/Ostm1 currents do not require intracellular ATP (Supplementary Figure S1C). We neither observed significant changes in current amplitudes like described for ClC-5 (Zifarelli and Pusch, 2009b) which is known to bind ATP by its CBS domains (Meyer et al, 2007), nor changes in voltage dependence. As typical for CLC antiporters (Friedrich et al, 1999; Li et al, 2002; Dutzler et al, 2003; Picollo and Pusch, 2005; Scheel et al, 2005; Zdebik et al, 2008; Bergsdorf et al, 2009; Neagoe et al, 2010), mutating the 'gating glutamate' (E245 in rat) of ClC-7PM to alanine resulted in almost ohmic, time-independent currents, and changing the 'proton glutamate' (E312 in rat) to alanine reduced currents to background levels (Figure 1B and C; Supplementary Figure S1A). ClC-7PM/Ostm1 mediated Cl−/H+-exchange as evident from depolarization-induced intracellular alkalinization of Xenopus oocytes expressing these proteins (Figure 1D). In these 'Fluorocyte' experiments, the pH-dependent fluorescence of BCECF previously injected into oocytes provides a semi-quantitative measure of cytosolic pH changes in response to depolarizing voltage steps. Depolarization not only activates ClC-7PM/Ostm1 but also provides a driving force for coupled H+-exit/Cl−-entry. Outward transport of protons required extracellular Cl− (Figure 1E), could occur against its electrochemical gradient (pHo=5.5, Figure 1E), and was abolished by either the E245A or the E312A mutation (Figure 1D). ClC-7PM/Ostm1 currents decreased upon replacing extracellular Cl− by I−, but unlike ClC-4 and ClC-5 (Friedrich et al, 1999), currents were not larger with NO3− (Figure 2A). Replacing a Cl−-coordinating serine by proline (rClC-7 (S202P)) increased the nitrate/chloride conductance ratio as with other CLC antiporters (Bergsdorf et al, 2009; Zifarelli and Pusch, 2009a; Neagoe et al, 2010) and with ClC-0 (Bergsdorf et al, 2009; Picollo et al, 2009). Akin to ClC-4, ClC-5 and ClC-6 (Friedrich et al, 1999; Neagoe et al, 2010; Picollo et al, 2010), currents were decreased by acidic extracellular pH (Figure 2B). In addition to a diminished driving force for Cl−/H+-exchange with increased extracellular [H+], faster activation kinetics at more alkaline pHo contributes to the pH dependence of ClC-7PM/Ostm1 outward currents (Figure 2C). Voltage-dependent current activation was also strongly dependent on temperature (Figure 2D). Mono-exponential fits yielded activation rate constants of 2.8±0.2 s−1 at 21°C and 16.6±1.9 s−1 at 37°C, giving an estimate of Q10≈3. Figure 2.Modulation of ClC-7/Ostm1 by anions, protons and temperature. (A) Relative anion conductance of oocyte-expressed rClC-7PM/Ostm1 in the presence of different extracellular anions (96 mM). Clamp protocol as in Figure 1C. Mean±s.e.m. of currents reached after 2 s at +80 mV was normalized to the current in Cl− for each oocyte (white bars) (Cl−, n=32 oocytes; Br−, n=7; NO3−, n=6; I−, n=5; gluconate (gluc−), n=5). Grey bar, NO3− conductance of rClC-7PM(S202P)/Ostm1 mutant measured and normalized as above (n=9). (B) Dependence of rClC-7PM/Ostm1 currents on pHo. I/V curves were obtained as in Figure 1B with currents normalized to those at pHo=7.4 and 80 mV; ⩾6 oocytes per data point. (C) Left, typical voltage-clamp traces (top right, protocol) obtained at different pHo values. Note different current scales that were chosen to normalize current amplitudes to the end of +80 mV pulse for better visualizing changes in activation kinetics. Right, τ was determined by single-exponential fit of the 80-mV traces for ⩾6 oocytes per pH value. Mean±s.e.m. as function of pHo. (D) Typical voltage-clamp traces of rClC-7PM/Ostm1 (protocol as in (C)) at different temperatures, representative for 11 oocytes in which temperature was changed between 21, 29°C (n=8) and/or 37°C (n=9). Download figure Download PowerPoint Slow ClC-7/Ostm1 gating allows characterization of an 'open exchanger' The slow deactivation of ClC-7PM/Ostm1 currents provides a unique opportunity to study mammalian Cl−/H+-exchange at negative membrane voltages. Using protocols developed for ion channels, we activated ('opened') ClC-7PM/Ostm1 by positive prepulses and measured tail currents from transfected HeLa cells at different test voltages (Figure 3A). We increased tail current amplitudes by including 121 mM Cl− in the patch pipette. Tail currents were extrapolated to the beginning of the test pulse to obtain the voltage dependence of 'open exchanger' currents (Figure 3A). Contrasting with the strong voltage dependence of pseudo-steady-state currents, 'open exchanger' currents displayed only very slight outward rectification. Figure 3.Tail current analysis of ClC-7PM/Ostm1. (A) After activating HeLa cell-expressed rClC-7PM/Ostm1 by pulses to +80 mV in whole-cell patch-clamp experiments, tail currents were measured at test voltages between −100 and +100 mV. Left, representative current traces (inset, clamp protocol). Right, I/V curve of 'open exchanger' obtained by extrapolation to the beginning of test pulses, shown together with 'pseudo-steady-state' currents measured after 2 s without preceding activation. Mean values±s.e.m. normalized to the current at +80 mV of 8 ('open exchanger') and 19 ('pseudo-steady-state') cells. Error bars are mostly smaller than symbols. (B) Determination of nCl−/H+-exchange stoichiometry from reversal potentials of tail currents. HeLa cell-expressed rClC-7PM/Ostm1 was clamped using a protocol as in (A), but tail currents were measured at only three voltages close to reversal potentials (−20 to +20 mV or 0 to +40 mV). The contribution of endogenous currents was estimated by short pulses from −80 to 0 mV before activating ClC-7PM/Ostm1 (see Materials and methods). [Cl−]o was shifted from 139 to 39 and/or 19 mM Cl− (top, representative traces from one cell) and pHo from 7.4 to 6.4 and/or 8.4. Bottom, reversal potentials corrected for background currents and liquid-junction potentials. Crosses, individual measurements. Filled circles and error bars, mean±s.d. Lines, predictions for an nCl−/H+-exchanger with n=1, 2 and 3, and for a Cl−-channel (1:0) under our experimental conditions. Dashed lines in (A) and (B), I=0. (C) Tail current analysis of hClC-7PM(R762Q)/Ostm1 expressed in HeLa cells to determine Popen(V). Clamp protocol at bottom. (D) Apparent open probability Popen as function of prepulse voltage, determined from tail currents as shown in (C). The line shows the fit by the Boltzman function Popen=1/(1+exp(zn × e0(V½−V)/kT)), which yielded zn=1.32 and V½=82 mV. Values are mean of five experiments. Error bars represent s.e.m. Download figure Download PowerPoint Whereas the strong rectification and near-instantaneous deactivation of ClC-4 and ClC-5 precludes measurements of reversal potentials (Steinmeyer et al, 1995; Friedrich et al, 1999), ClC-7PM/Ostm1 tail currents allowed us to determine Cl−/H+-coupling ratios from Cl− and H+-dependent shifts in reversal potentials (Figure 3B). Our results were best fitted by a 2Cl−:1H+ stoichiometry. The apparent deviation from this stoichiometry at nominal pHo of 8.4 might be explained by depolarization-induced outward transport of protons through the exchanger (Zifarelli and Pusch, 2009a). This process is expected to cause a larger deviation of actual from nominal pH at the lower H+ concentrations of more alkaline pH. Whereas the tail current analysis of instantaneous 'open exchanger' currents requires the same open probability Popen at the beginning of test pulses (as indicated above by identical macroscopic currents), Popen must have reached steady state at the respective voltage when Popen is determined as a function of voltage by tail currents. The slow voltage-dependent activation of ClC-7PM/Ostm1, however, precluded reliable measurements of steady-state currents that are needed for this analysis. We, therefore, resorted to a ClC-7 point mutant (R762Q; described below) that drastically accelerates activation. ClC-7PM(R762Q)/Ostm1 currents reached steady state already ∼400 ms after the beginning of voltage steps (Figure 3C). At t=500 ms, tail currents were measured at a constant test voltage (+80 mV) as function of the voltage of the preceding pulse (between −40 and +140 mV). After correcting for endogenous HeLa cell currents, apparent Popen(V) was obtained by extrapolating tail currents to the time of the voltage step (see Materials and methods). Boltzmann fits (Figure 3D) revealed a voltage of half-maximal activation V½≈82 mV and an apparent gating charge of zn≈1.32. Although we performed this tail analysis study with a mutant, we expect the values of 'WT' ClC-7/Ostm1 to be similar. Structural basis and functional consequences of Ostm1–ClC-7 interactions So far, all experiments on ClC-7PM were performed in co-expression with Ostm1. When we expressed ClC-7PM with or without Ostm1 in HeLa cells (Supplementary Figure S1D) or Xenopus oocytes (not shown), ClC-7PM yielded currents only together with Ostm1. Since ClC-7PM clearly reaches the plasma membrane also without Ostm1 (Figure 1A; Stauber and Jentsch, 2010), these results indicate that Ostm1 is needed to activate ClC-7 ion transport. We next asked which parts of Ostm1 interact with ClC-7. We constructed chimeras with CD4, a protein that shares the type I transmembrane topology of Ostm1 (Lange et al, 2006) but traffics to the plasma membrane by default. The extracellular, transmembrane and intracellular domains of Ostm1 were replaced by those of CD4 either individually or in combination. Without ClC-7, Ostm1 stays in the endoplasmic reticulum (ER), whereas a portion of Ostm1 reaches lysosomes upon co-expression with ClC-7 (Lange et al, 2006). We first ascertained that Ostm1 and Ostm1/CD4 chimeras carrying C-terminal green fluorescent protein (GFP) tags were confined to the ER and/or plasma membrane of transfected HeLa cells (Figure 4A). We then co-transfected GFP-tagged Ostm1/CD4 chimeras with ClC-7 and assayed the co-localization of GFP fluorescence with the lysosomal marker LAMP-1 as read-out for Ostm1–ClC-7 interaction (Figure 4B and C; Supplementary Figure S2). The transmembrane domain (TMD) of Ostm1 was necessary and sufficient for Ostm1 constructs being carried to lysosomes by ClC-7 (Figure 4C). Figure 4.Domains of Ostm1 that interact with ClC-7. (A) When transfected into HeLa cells, Ostm1–GFP localizes to the ER, CD4–GFP mostly to the plasma membrane and a GFP-tagged CD4 chimera containing the TMD of Ostm1 (COC–GFP) to the ER and plasma membrane. (B) When co-transfected with HA-tagged rClC-7, Ostm1–GFP and COC–GFP, but not CD4–GFP, co-localized with rClC-7–HA (immunolabelled for the HA epitope) to late endosomes/lysosomes (marked by immunolabelling for LAMP-1). (C) Statistical analysis of lysosomal targeting of Ostm1, CD4 or chimeras thereof expressed either without (−) or with (+) rClC-7–HA assayed as in (A) and (B). In the 3-letter abbreviations, C means CD4, O means Ostm1, in the sequence extracytosolic N-terminal part, TMD, and cytoplasmic C-terminus. Means of 3–4 independent experiments with >100 cells each evaluated. Error bars represent s.e.m. Constructs containing the TMD of Ostm1 localized to lysosomes upon co-expression with rClC-7. (D) Typical current traces of Xenopus oocytes co-expressing rClC-7PM with Ostm1, CD4 or CD4/Ostm1 chimeras. Expression of significant currents required the presence of both the N-terminus and TMD of Ostm1. Similar results were obtained with at least 10 oocytes of at least 3 batches. Download figure Download PowerPoint While these experiments suggested that the TMD of Ostm1 binds ClC-7, other parts of Ostm1 may modulate ClC-7 transport activity. We, therefore, assayed currents of Xenopus oocytes co-expressing ClC-7PM and Ostm1/CD4 chimeras (Figure 4D). As expected from our localization assay, currents were not detectable when the TMD of Ostm1 was replaced by that of CD4. Even larger currents were observed when ClC-7PM was co-expressed with a chimera (OOC), in which the Ostm1 C-terminus was replaced by that of CD4. By contrast, currents were indistinguishable from background with chimeras lacking the Ostm1 N-terminus (Figure 4D), even though ClC-7PM was still able to reach the plasma membrane (Supplementary Figure S3). Hence, both the N-terminus and TMD of Ostm1 are required for ClC-7 transport activity. Functional effects of human CLCN7 mutations underlying osteopetrosis The plasma membrane expression of ClC-7PM/Ostm1 allowed us for the first time to study functional consequences of disease-causing CLCN7 mutations (Cleiren et al, 2001; Kornak et al, 2001; Frattini et al, 2003; Waguespack et al, 2003; Letizia et al, 2004; Pangrazio et al, 2010; Phadke et al, 2010), which we selected from different categories based on the mode of inheritance (recessive versus dominant) and on their location within the protein (transmembrane region or cytoplasmic CBS domain) (Figure 5A; Supplementary Figure S4). Figure 5.Characterization of osteopetrosis-causing mutations in human ClC-7. (A) Position of analysed dominant and recessive osteopetrosis-causing mutations (solid and open stars, respectively) in a CLC topology model (Dutzler et al, 2002). Mutations yielding no currents shown in purple (when retained with Ostm1–GFP in the ER of HeLa cells) and red (when exported from the ER), those with apparently normal currents in green, and those with accelerated activation in blue (see (C, D) and Supplementary Figure S5). (B) Close-up of X-ray structure of CmClC (Feng et al, 2010) displaying the location of ClC-7 residues that accelerate gating when mutated. Except for L213 (corresponding to L174 in CmClC), the depicted ClC-7 residues are not identical to those of CmClC at these positions (R286, P619, R762 and R767 of hClC-7 correspond to L241, R532, V680 and S685, respectively, in CmClC) (Feng et al, 2010). The transmembrane part of one subunit is shown in grey, CBS2 and the linker to CBS1 of that subunit in red and yellow, respectively. Green helices at left are from the second subunit of the homodimer. (C) Representative current traces of hClC-7PM ('WT' or selected osteopetrosis-causing mutants) upon expression with Ostm1 in Xenopus oocytes. Mutants yielded either no or very low currents (R526W and L490F), apparently normal currents (S744F), or displayed accelerated activation (R762Q). (D) Subcellular localization of hClC-7 and selected mutants co-expressed with Ostm1–GFP in HeLa cells. Cells were immunolabelled for hClC-7 and LAMP-2. In most cases, hClC-7 co-localized with Ostm1–GFP to LAMP-2-positive structures in addition to localizing to ER-like structures. However, hClC-7(R526W) remains with Ostm1–GFP in the ER (highlighted by nuclear envelope staining). In cells with a clear excess of Ostm1–GFP (asterisk), it predominantly localizes to the ER. Download figure Download PowerPoint Within all CLCN7 mutation categories mentioned above, we found mutations that abolished or strongly reduced currents (Figure 5C; Supplementary Figure S5A). Surprisingly, other mutations either left ClC-7PM currents virtually unchanged (e.g., S744F; Frattini et al, 2003) or accelerated their activation kinetics between moderately (e.g., the frameshift mutation G796fs; Cleiren et al, 2001) and dramatically (e.g., R762Q; Kornak et al, 2001) (Figure 5C; Supplementary Figure S5A). Mutants that yielded plasma membrane currents also transported H+ as revealed by Fluorocyte (Zdebik et al, 2008) experiments (not shown). To test whether changed subcellular targeting of ClC-7/Ostm1 might explain the disease-causing effect of those mutants, we inserted them into hClC-7 instead of hClC-7PM and co-expressed them with GFP-tagged Ostm1 in HeLa cells. However, in addition to a pronounced ER-like labelling, all mutants that gave currents (in hClC-7PM) reached their normal destination (lysosomes) where they co-localized with Ostm1 (Figure 5D; Supplementary Figure S5B). Only three of the mutants with reduced currents were retained in the ER, whereas the other six partially localized with Ostm1 to late endosomes/lysosomes (Figure 5D; Supplementary Figure S5B). In the absence of lysosomal targeting sequences, ClC-7 reaches the plasma membrane by default once it has left the ER (Stauber and Jentsch, 2010). Hence, normal lysosomal targeting (in hClC-7) of the V297M, F318L, L490F, L651P, R767P and R767W mutants, all of which reduce or abolish plasma membrane currents in hClC-7PM, suggests that these mutations may interfere directly with the ion transport of ClC-7 or with the mechanism by which Ostm1 activates ClC-7. One should note, however, that all these mutants were able to carry Ostm1 to lysosomes. A reduction in the expression level due to limited stability may also contribute to reduced currents. Western blot analysis showed that this was not the case for the V297M and F318L mutants, whereas protein levels were markedly reduced with the L490F mutant (Supplementary Figure S6). Discussion Despite the medical importance of ClC-7/Ostm1 and its crucial role in lysosomal function (Kornak et al, 2001; Kasper et al, 2005; Lange et al, 2006; Wartosch et al, 2009; Weinert et al, 2010), the only available information concerning its biophysical properties has remained its ability to perform Cl−/H+-exchange (Graves et al, 2008; Weinert et al, 2010). Acid-activated currents previously ascribed to ClC-7 (Diewald et al, 2002) most likely represent currents endogenous to the expression systems (Jentsch, 2008). Exploiting the partial plasma membrane localization of ClC-7 mutants which we have recently described (Stauber and Jentsch, 2010), we could now study for the first time important details of ion transport properties, investigate effects of human disease-causing mutations, and show that ClC-7 needs specific domains of the Ostm1 β-subunit not only for protein stability (Lange et al, 2006), but also for ion transport activity. Voltage gating of intrinsically linear voltage-dependent 2Cl−/H+-exchange Several properties of ClC-7/Ostm1 described here have rece
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