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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Department of Pediatrics, University of Iowa Children's Hospital, University of Iowa, Iowa City, Iowa
Submitted 31 July 2007 ; accepted in final form 27 October 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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chloride channel; chloride-proton exchanger; swelling-activated chloride channel
B by cytokines (33). Unfortunately, a lack of consensus as to the biophysical characteristics of ClC-3 has hampered interpretation of these complex phenotypes. Electrophysiological characterizations of heterologously expressed ClC-3 currents have yielded discrepant results (reviewed in Ref. 28). ClC-3 was first expressed in Xenopus oocyte cells where it produced an outwardly rectifying anion current that was I– > Cl– selective, noninactivating at positive potentials, and inhibited by protein kinase C (PKC; see Ref. 29). Expression of ClC-3 in NIH 3T3 cells yielded an enhancement of swelling-activated, outwardly rectifying anion currents with time-dependent inactivation that was also inhibited by PKC (10). Other studies have shown that anti-ClC-3 antibodies block the swelling-induced anion current (IClswell; see Refs. 11 and 55), suggesting that ClC-3 may be an IClswell. This hypothesis is also supported by a recent study which showed that NH2-terminal deletion mutants of ClC-3 yielded constitutively active IClswell-like currents that no longer responded to PKC (42). Other investigators have either been unable to functionally express ClC-3 currents (16, 25, 54) or have observed currents that were insensitive to cell volume changes (24, 30, 37, 46, 54). Furthermore, cells from ClC-3 null mice still exhibit normal-appearing whole cell (17, 48) and single channel (50) IClswell currents. The regulation of these currents, however, was altered in ClC-3 null cells, and the possibility was raised that some other protein can substitute for the loss of ClC-3 (55). Alternatively, it seems plausible that ClC-3 is required for a signaling process involved in activation of IClswell. Further studies identified a calcium-calmodulin kinase II (CamKII)-dependent Cl- current that was dependent upon the expression of ClC-3 (24). These currents exhibited voltage and time dependence and ion selectivity (I– > Cl–) typical of IClswell but they were not responsive to changes in cell volume. These currents were absent in smooth muscle cells (41) and hippocampal neurons from ClC-3 null mice (52).
Most of the currents that have been attributed to ClC-3 have shown some degree of time-dependent inactivation similar to that of IClswell, with two notable exceptions. Li et al. (31) used plasmids to overexpress ClC-3 in CHO-K1 cells and noted small, sharply outwardly rectifying currents that displayed virtually no time dependence. Mutation of an extracellular glutamate (E224A) altered the voltage dependence of the expressed current in a manner similar to that observed with a similar mutation in ClC-4 and ClC-5 (16). The analogous mutation E148A in ClCec1 (2), E224A in ClC-4 (39, 44), and E211A in ClC-5 (44) abolished proton but not Cl- transport and helped to demonstrate that these proteins function as Cl–/H+ exchangers or antiporters over the physiological pH range, rather than as anion channels. ClC-3, by virtue of its close sequence homology to ClC-4 and ClC-5, was also proposed to act as an antiporter (39); however, no direct evidence for this has ever been provided.
The ClC-3, -4, and -5 branches of the ClC family are all primarily expressed in intracellular organelles where they have been proposed to provide shunt conductances for current generated by the vacuolar (V-type) H+-ATPase (V-ATPase; see Ref. 27). This concept is supported by data which suggests that ClC-3 contributes to the acidification of synaptic vesicles (48), insulin granules (4), lysosomes (31), and endosomes (19). This acidification process leads to intraorganellar pH values ranging from 5.9 to 6.2 for early endosomes to 5.0 to 6.0 for late endosomes and 5.0 to 5.5 for lysosomes (15). Although 94% of transfected ClC-3 was located intracellularly in COS-7 cells, the protein cycles through the plasma membrane via clathrin-mediated endocytosis with a half-life on the membrane of 
9 min (57).
The purpose of the present study was to characterize the biophysical properties of ClC-3 expressed in HEK293T cells using both plasmids and adenovirus. The behavior of ClC-3 is more consistent with the function of a Cl–/H+ exchanger than an anion channel. Currents induced by ClC-3 expression are clearly distinct from IClswell.
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METHODS |
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Cells were infected with adenovirus in serum-free DMEM for 16 h before being returned to their standard serum concentration. Transfections with pacAd5 plasmids (1 µg/well in 6-well plates containing 106 cells/well) were performed using lipofectamine 2000 (5 µl/ml) in 2% serum. Adenovirus was allowed to express for 48 h before experimentation, whereas plasmids were allowed to express for 48–96 h.
RNAi directed against the 5'-untranslated region of ClC-3 (Dicer duplex system; IDT, Coralville, IA) was used to suppress endogenous ClC-3 protein levels. The targeted sequences in Clcn3 were 5'-CAUCUGUUUCAAACCUAGAACCUAGCU-3' or 5'-GAGUAAAGUAGGAUGGCUUUCAACCCA-3'. These sequences do not appear in the pacAd5 clones, thus allowing ClC-3 plasmid expression in the setting of reduced levels of native ClC-3. A scrambled control RNAi duplex was provided by the manufacturer. Cells growing in 2% serum were exposed to RNAi duplex at 25–50 nM concentrations in the presence of oligofectamine (5 µl/ml) and were studied 72–96 h later.
Electrophysiology.
Cl– currents were measured at room temperature (22°C) using either standard whole cell voltage-clamp techniques (18) or perforated-patch recording (23) performed with an Axopatch 200B patch-clamp amplifier driven by pClamp 9 software (Molecular Devices, Sunnyvale, CA). Pipette resistances were 3–5 M
. Pipette and whole cell capacitance and series resistance compensations were done before recording. Currents were elicited from a holding potential of –40 mV to test potentials from –100 to +100 mV in 20-mV increments. Test pulses were 1 s in duration delivered at 3-s intervals. Currents were sampled at 5 kHz and filtered at 1 kHz.
Standard bath solution contained (in mM): 120 NaCl, 2.5 MgCl2, 2.5 CaCl2, 10 HEPES, and 5.5 glucose, pH 7.2 with NaOH. Ion substitution experiments were done by replacing NaCl with eqimolar NaI or NaBr. The 435 mM Cl bath solution contained (in mM): 425 NaCl, 10 HEPES, 2.5 MgCl2, 2.5 CaCl2, and 5.5 glucose. The 42 mM Cl– bath solution contained (in mM): 32 NaCl, 2.5 CaCl2, 2.5 MgCl2, 10 HEPES, and 240 glucose. The 13 mM Cl– bath solution contained (in mM): 3 NaCl, 2.5 CaCl2, 2.5 MgCl2, 10 HEPES, and 270 glucose 270. Osmolality of all solutions was determined using a micro OSMETTE osmometer, and all extracellular solutions (except 435 mM Cl) were titrated to 300 mosmol/kgH2O 1 M mannitol. Hypotonic solution (240 mosmol/kgH2O) was identical to the standard bath solution except for the exclusion of mannitol. Liquid junction potentials were minimized by using 3 M KCl agar bridges and were calculated using pClamp 9.0 to be 5.0, 5.3, and 5.1 mV for the Cl–-, Br–-, and I–-containing solutions, respectively, and 8.4, 2.4, and –0.4 mV for 435, 42, and 13 mM Cl– buffers, respectively. Pipette solutions for standard whole cell recordings contained (in mM): 120 CsCl, 4 TEA-Cl, 5 EGTA, 1.187 CaCl2, 2 MgCl2, 5 Na-ATP, 0.5 Na-GTP, and 10 HEPES, pH 7.2 with CsOH, osmolality 290 mosmol/kgH2O, free Ca2+ concentration = 55 nM (calculated using WEBMAXC http://www.stanford.edu/cpatton/webmaxc/webmaxcS.htm). HEPES (pKa 7.5) was used as the buffer in all experiments. Pipette solution for perforated-patch recording contained (in mM): 120 CsCl, 2.5 MgCl2, and 10 HEPES, pH 7.2 with CsOH (liquid junction potential 4.4 mV using standard bath solution). Amphotericin was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 60 mg/ml, and then 20 µl of this solution were mixed with 5 ml of pipette solution by vortexing. Currents were normalized to cell membrane capacitance and expressed as current density (pA/pF). Identification of GFP-positive cells was done immediately before cell selection using a fluorescence-equipped inverted microscope (Zeiss Axiovert 25). Phloretin and tamoxifen were dissolved in DMSO for stock solution concentrations of 30 and 10 mM, respectively, and then mixed with the bath solution at a 1:1,000 dilution. All chemicals were obtained from Sigma (St. Louis, MO).
Western blotting. To demonstrate adenoviral-mediated overexpression of ClC-3, HEK293T cells were grown in T150 flasks and infected with GFP or bicistronic GFP and ClC-3-expressing adenoviruses for 48 h. To demonstrate the efficacy of RNAi directed at ClC-3, RNAi-treated cells and treated cells transfected with ClC-3 plasmids as described above were harvested after 96 h. All cells were washed two times with PBS and then lysed in 0.75 ml of 1x lysis buffer (E397A; Promega). After homogenization, the lysates were cleared by centrifugation at 12,000 rpm at 4°C for 5 min. Protein was quantified using the Micro-BCA kit (Bio-Rad). Protein (5 mg) was immunoprecipitated using a custom rabbit anti-ClC-3 antibody raised against a mixture of two NH2-terminal peptides, TYDDFHTIDWVREKC and CKDRERHRRINSKKKES (Research Genetics), and covalently linked to protein G-Sepharose. Immunoprecipitated proteins were washed three times with PBS and eluted using SDS Laemmli buffer. They were heated to 60°C for 30 min, and one-half of the eluted protein was loaded on a precast Criterion 10% SDS polyacrylamide gel (Bio-Rad). Proteins were resolved for 80 min and then transferred to a nylon membrane (Bio-Rad) using a liquid Transblot apparatus (300 mA for 3 h; Bio-Rad). Blots were blocked with Odyssey blocking reagent and incubated overnight with a 1:2,000 dilution of affinity-purified rabbit anti-ClC-3 antibody (C9602; Sigma) and then washed three times with PBS containing 0.1% Tween 20. The secondary antibody (goat anti-rabbit conjugated to Cy5) was used at a 1:3,000 dilution and incubated for 2 h. The signal was detected by scanning the membranes using the Li-COR, Odyssey infrared detection system.
Microscopy. Subcellular localization of ClC-3 was done in HEK293T cells transfected with the short ClC-3-eGFP fusion protein construct. GFP was imaged in live cells 48 h after transfection using a Zeiss inverted confocal microscope. Images were edited offline using Adobe Photoshop software.
Data analysis and statistics.
Unless otherwise indicated, steady-state currents (measured 5 ms before the end of the depolarizing pulse) were used to calculate current-voltage (I-V) relationships using pClamp 9 software. Reversal potentials were obtained from each individual I-V relationship by fitting a straight line (y = mx + b) between consecutive data points negative to and positive to zero current density and extrapolating to the x-intercept. Reversal potential estimates were corrected for liquid junction potentials. Permeability coefficients were determined using the Goldman-Hodgkin-Katz equation: PX/PCl = [Cl–]i/{[X]oexp(
ErevF/RT)} – [Cl–]o/[X]o, where Erev is the difference between the reversal potential with the test anion X and that observed with symmetrical Cl–, and F, R, and T have their normal thermodynamic meanings. The activation time courses were best fit using two exponentials, and time constants of activation were determined using the Clampfit module of pClamp 9. Results are expressed as means ± SE. Unpaired Student's t-tests with a Bonferroni correction were used to determine statistical significance.
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RESULTS |
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40 mV) were significantly larger for the ClC-3-expressing cells compared with eGFP-expressing cells, reflecting the contribution of ClC-3 to the overall outward current.
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Mutations in ClC-3 alter rectification and pH sensitivity but not anion selectivity. Previous studies have demonstrated that mutation of a critical extracellular glutamate in ClC-4 or ClC-5 (39, 44) and ClCec1 (1) alters sensitivity to protons. In addition, this mutation has been reported to alter rectification of ClC-3, -4, and -5 currents (31, 39, 44). We expressed ClC-3 protein containing this mutation (E224A) and analyzed the pH and extracellular Cl– dependence of the resulting currents. These cells were pretreated with RNAi to inhibit endogenous ClC-3 expression. This method was used to avoid the formation of wild-type mutant heterodimers that may have altered function. Figure 7A shows a Western blot for ClC-3 protein in cells treated with control scrambled RNAi, RNAi directed at ClC-3, or RNAi against ClC-3 plus E224A plasmid. It demonstrates both gene-specific RNAi-mediated suppression of endogenous ClC-3 protein and reconstitution of ClC-3 protein expression by mutant plasmid. Figure 7B shows currents induced by the expression of E224A ClC-3 in the indicated bath Cl– concentrations. Larger inward currents could be seen at all Cl– concentrations compared with wild-type currents. As a result, the I-V relationships (Fig. 7C) displayed substantially less outward rectification. There were also significant changes in reversal potential (Fig. 7C, inset). Linear regression of the reversal potentials obtained in 42, 130, and 430 mM Cl– yielded a slope of –60.0 ± 3.4 mV/decade change in Cl– concentration, very close to that predicted for a Cl–-selective anion channel (–58.4 mV/decade at 22°C). Table 1 provides the observed and predicted reversal potentials for the E224A currents at the various extracellular Cl– concentrations.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ClC-3 overexpression produced stable and novel currents at neutral pH. These currents exhibited very steep outward rectification, reversed near the Cl– equilibrium potential, were inhibited by removal of extracellular Cl–, and displayed slow time-dependent activation. These basic properties were strikingly similar to those of published ClC-4 (16) and ClC-5 (16, 53) currents. Over the physiological pH range, changes in current amplitude and reversal potential in response to altered extracellular Cl– and H+ concentrations were consistent with ClC-3 acting as a proton-anion exchanger, similar to ClC-4 and -5 (39, 44). These results deviated significantly from predictions based on ClC-3 acting as an anion channel.
ClC-3-mediated currents are not very halide selective based on reversal potential measurements with anion substitution. We observed a tendency toward larger Cl– currents compared with I– or Br– only at very positive test potentials, and slightly higher I– and Br– permeability based on reversal potential measurements. These results are similar to previous work (46) that also suggested an I– > Cl– selectivity for ClC-3 according to reversal potential and a Cl– > I– preference based on current magnitudes at strongly positive voltages (30). ClC-4 and ClC-5 currents have been suggested to display a small degree of selectivity for Cl– over I– (16, 21, 47); however, these statements were based solely based on the magnitude of currents at positive potentials. In a detailed analysis of ClC-4 permeability, Hebeisen et al. (21) showed an anion permeability coefficient of 1.1 for I–/Cl–, which closely approximates our results with ClC-3. Very importantly, they also demonstrated via noise analysis that ClC-4 is open throughout the experimental voltage range (–100 to 100 mV), allowing for valid reversal potential measurements despite the sharp outward rectification.
Is ClC-3 a Cl–/H+ exchanger? As our experiments were configured, there were limited charge carriers available to produce the currents observed in response to overexpression of ClC-3. The currents reversed close to 0 mV. The dominant intracellular cation was cesium, which has very low permeability through potassium channels, which were also blocked by 4 mM intracellular TEA. The intracellular sodium concentration was very low (5.5 mM), far out of balance with the extracellular concentration of sodium (120 mM). This made inward movement of anions the most likely primary source of the current. The only extracellular anions present were Cl– and OH–. Lowering extracellular Cl– 10-fold drastically reduced the size of the current, consistent with this anion accounting for the bulk of charge movement. The concentrations of both intracellular H+ and extracellular OH– were so low that their permeabilities would have to be orders of magnitude greater than that of Cl– for them to selectively produce the current by moving through channels. Interpretation of the data as simple Cl– channel activity only became problematic when the shifts in reversal potential induced by altering ion gradients were carefully examined.
The changes in reversal potential observed with manipulation of extracellular Cl– and H+ concentrations did not fit with ClC-3 acting as an anion-selective channel, even if it were proton activated. The observation of significant changes in reversal potential in response to changes in extracellular pH can only be explained by protons actually contributing to charge movement. The most reliable previous estimates of the stoichiometric ratio of Cl–/H+ antiport by ClC-ec1 have been based on either reversal potential (2) or H+ flux determination by pH measurement (36). These efforts have yielded an estimated coupling ratio of 2:1. The combined slope that describes the additive relationship between Cl– and H+ concentrations and reversal potential (48.3 + 10.2 = 58.5 mV/decade) accounts very well for total charge movement (predicted change of 58.4 mV/decade at 22°C). However, these data do not fit a coupling ratio of 2:1 but rather are consistent with a ratio very close to 4:1. Unfortunately, the precision of estimation of reversal potential is subject to certain limitations. Analysis of the phloretin subtraction currents suggested that the values were not significantly impacted by leak current, since no significant differences were observed between values obtained before and after leak subtraction. Even though inward currents were very small at voltages negative to the reversal potential, we were readily able to define a point where outward currents become significant. That this point changes in a predictable fashion with changes in pH or Cl– concentration suggests that it is a valid representation of reversal potential. In addition, the interventions employed to alter the coupling of Cl– and H+ movement (E224A mutation and acidic pipette pH) yielded currents that did behave in a Nernstian manner. This was observed despite the persistence of strong outward rectification in the case of pH 5.0 pipette solution. Looking across all of the estimates of reversal potential, regardless of condition, there was a small (6–8 mV) offset from predicted values, always in the negative direction. Reversal potential calculations are subject to systematic error at a number of levels, including buffer composition, amplifier function, assumptions related to calculation of liquid junction potentials, and unrecognized current from other sources. If the observed offset is coming from some other current, it is a very small one and is constant across all of the experimental conditions. The offset was still observed in the estimates of reversal potential obtained for the E224A mutant, suggesting that it was not an artifact induced by strong rectification. Fortunately, the presence of a small offset did not impact the calculation of slopes for the change in reversal potential across multiple ion concentrations. However, given the overall limitations of the current data, it seems best to simply conclude that, while Cl– and H+ movement through wild-type ClC-3 clearly appears to be coupled, determination of the precise ratio of this coupling must be determined by other techniques.
Activation by extracellular protons is a property of ClC-0 and ClC-1 channel-type ClC currents (7, 43). Low pH enhances steady-state current by enhancing the opening rate of the "fast gate" by a mechanism that is distinct from the ability of Cl– to activate the gate. Fast gating of ClC pores has been proposed to be mediated by an extracellular glutamate (E224 of ClC-3, E232 of ClC-1; see Ref. 12). The side chain of this amino acid blocks access of Cl– to its extracellular binding site. Mutation of E232 of ClC-1 (E232C) enhances steady-state current but seems to do so largely via a reduction in time-dependent inactivation (45). In exchanger-type ClC proteins, mutation of this glutamate renders them pH insensitive and uncouples them (1, 13, 16). Mutation of the fast gate also has been shown to alter rectification of both channel (45) and exchanger-type ClCs (1, 13, 16), including E224A ClC-3 mutants (31). We confirmed the previously reported loss of rectification in E224A mutants expressed in HEK293T cells. In addition, we show that the mutation yields a channel protein that is pH insensitive, and anion and proton transport are uncoupled.
ClC-3 has already been predicted to function as a Cl–/H+ antiporter based upon homology to ClC-4 and ClC-5 (39, 44). These predictions are supported by the observation that all of the ClC proteins thought to function as exchangers, including ClC-3 through -7 and the prokaryotic ClCs (ClC-ec1, ClC-st1, and GEF1), have a glutamate residue at the intracellular position of E203 in ClC-ec1 (E281 of ClC-3). This glutamate may facilitate partitioning of protons into the exchanger from the cytoplasm. All of the channel-type ClCs have valine in this position (3). We were unable to study the E281Q mutant directly because of the lack of significant current. However, acidification of pipette pH to 5.0 resulted in an effect that was similar to that predicted for this mutation; Cl– and H+ transport became uncoupled. While proof that this effect was exerted by a change in the protonation state of E281 remains a topic for further experimentation, these experiments did provide a nice control by which to rule out unanticipated effects of altering extracellular pH. They also demonstrate that the shifts in reversal potential observed in the wild-type ClC-3 currents with altered pH were not artifactual. In addition, we were able to measure a Nernstian shift in the reversal potential of these uncoupled currents in response to changes in extracellular Cl– concentration despite the persistence of sharp outward rectification. Although all of our data are consistent with ClC-3 functioning as a Cl–/H+ antiporter, definitive proof of this will need to come from future demonstration of ClC-3-mediated transport of either Cl– or H+ against its concentration gradient, driven by a gradient for the other ion.
ClC-3 currents are distinct from IClswell. Several of our findings suggest that ClC-3 is not the protein responsible for IClswell. The slowly activating time course clearly distinguishes ClC-3 currents from IClswell. Furthermore, Ad-ClC-3-induced currents did not exhibit the well-established IClswell permeability sequence with a strong preference for I– over Cl– (see Ref. 5 for a review), and there were no significant differences in the hypotonic-induced IClswell between control and ClC-3-overexpressing HEK293 cells. Last, tamoxifen blocked IClswell but not the current induced by ClC-3 overexpression.
Other investigators have also found no difference in IClswell between ClC-3 wild-type and null cells (17, 48, 50). This, however, contradicts studies showing that ClC-3 antibodies (9, 11, 55) and antisense oligonucleotides (22, 51) inhibit IClswell n a variety of cell types. Recently, an NH2-terminus deletion mutant of ClC-3 was expressed in NIH-3T3 cells, which affected the PKC regulation and biophysical properties of IClswell (42), yielding a constitutively active current that was unresponsive to PKC agonists or changes in cell volume.
It is challenging to explain how specific ClC-3 mutations can cause alterations in IClswell behavior without ClC-3 actually being the protein directly responsible for IClswell. However, our data clearly suggest that ClC-3 and IClswell are distinct currents in HEK293 cells. These data might be reconciled if ClC-3 is part of a signaling pathway that is required for activation of IClswell in response to certain stimuli. We have demonstrated previously that ClC-3 modifies reactive oxygen production by the Nox2 NADPH oxidase in neutrophils (34). We have also shown that ClC-3 is required for the Nox1-mediated production of reactive oxygen species and activation of NF-B by cytokines in vascular smooth muscle cells (33). Both stretch and epidermal growth factor-mediated activation of IClswell require production of H2O2 in HeLa cells (49), and ANG II-induced activation of IClswell is also linked to H2O2 production in ventricular myocytes (6). However, stretch must also be able to activate IClswell independent of H2O2 in some cell types where no difference in IClswell is observed in ClC-3 null cells (55). We hypothesize that ClC-3-dependent reactive oxygen production can functionally link these two anion conductances. Thus mutations and manipulation of regulatory pathways that alter ClC-3 activity can modify IClswell currents indirectly. Future experiments may explore the ability of other stimuli to activate IClswell in the absence of ClC-3.
It is also difficult to explain the ability of constitutively active CamKII to activate a current that is superficially very similar to IClswell (outwardly rectifying, time-dependent inactivation and I– > Cl– selectivity) and is absent in ClC-3 null cells (41, 52). However, if ClC-3 is CamKII-activated, a constitutively active enzyme could enhance H2O2 production and promote activation of IClswell. CamKII has been implicated in the production of reactive oxygen species by lymphocytes (38), and both H2O2 and CamKII have been linked to activation of IClswell in neurons (20). The absence of an IClCamKII in ClC-3 null cells might therefore be related to impaired production of H2O2. Future experiments will be required to explore the relationship between ClC-3, reactive oxygen production, and IClswell.
Functional significance. ClC-3, ClC-4, and ClC-5 have all been hypothesized to provide shunt conductances in the membranes of intracellular vesicular organelles that permit intraluminal acidification by the V-ATPase (26). This hypothesis is supported by a study showing impaired endosomal acidification in ClC-3 deficient mice (19). The properties of ClC-3 described herein seem poorly suited to this task. ClC-3 currents rectify strongly, such that the protein very preferentially conducts Cl– to the cytoplasm from the extracellular space. Endocytosis of this channel would place the extracellular face of ClC-3 within the endosome. The strong outward rectification of ClC-3 current would therefore favor movement of Cl– out of, rather than into, endosomes. Furthermore, immediately after endocytosis, when luminal pH of the early endosome is near neutral, for ClC-3 to act as a H+/Cl– antiporter and move current in the direction required to neutralize the V-ATPase (Cl– in, H+ out of the endosome), each cycle would require the removal of a proton from the endosome. Although the net charge movement of ClC-3 could still provide the needed shunt current, this process would be energetically unfavorable. The V-ATPase would need to consume more net energy to move each net proton into the endosome compared with a process where charge neutralization was provided by an anion channel. The degree of inefficiency would be related to the stoichiometry of Cl–/H+ exchange, with a lower coupling ratio such (2:1) requiring more V-ATPase activity to achieve the same degree of acidification as a less tightly coupled exchanger.
Consistent with our previous observations that both Nox1 (33) and Nox2 (34) require ClC-3 to make reactive oxygen intracellularly, ClC-3 seems better suited to neutralize the movement of negative charge into vesicles (Fig. 12). Nox2 generates a rapid depolarization of >100 mV across the plasma membrane of polymorphonuclear neutrophils and this effect may be even larger across phagosomal membranes (35). The positive voltages required to activate ClC-3 may therefore exist in the confined space of the phagosome or endosome upon activation of the NADPH oxidase. In addition, once an active NADPH oxidase is incorporated into an electrophysiological model of an intracellular vesicle, change neutralization of the V-ATPase is no longer an issue. The negative intraluminal charge that is generated by electron flow through the oxidase obviates the need for inward movement of another anion. The dependence of NADPH oxidase activity on ClC-3 therefore suggests that ClC-3 indeed contributes to the acidification of vesicles, but this effect may be a combination of the direct inward movement of protons through ClC-3 and indirect facilitation of V-ATPase activity by allowing the oxidase to move electrons. Future studies may be focused on the precise role of ClC-3 in reactive oxygen species production and redox-dependent signaling.
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ACKNOWLEDGMENTS |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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