Considering the low plasma membrane density and slow metabolic turnover rate of CFTR in various expression systems (Heda et al., 2001
; Sharma et al., 2004
), it is conceivable that only a small population of secretory and newly formed endocytic vesicles contain the channel. Previous studies, including our own, used pH probes (e.g., cellubrevin-phluorin, synaptobrevin-GFP-phluorin, FITC-transferrin, and FITC-dextran) that labeled organelles in CFTR-independent manner (Barasch et al., 1991
; Lukacs et al., 1992
; Biwersi and Verkman, 1994
; Dunn et al., 1994
; Seksek et al., 1996
; Botelho et al., 2000
; Chandy et al., 2001
; Poschet et al., 2002
; Machen et al., 2003
; Haggie and Verkman, 2007
). Here we introduced two novel approaches to monitor the CFTR-dependent endosomal pH regulation with high specificity. First, we implemented a method that restricted the pH-sensitive probe to CFTR-containing vesicles (A; Sharma et al., 2004
; Glozman et al., 2009
). Second, to control CFTR-independent endosomal pH changes provoked by the activation of cAMP-dependent PKA, a direct regulator of the v-ATPase and Na+
exchanger activity (Marshansky and Futai, 2008
), we utilized the G551D CFTR-3HA, a class IV CF mutation with severely impaired channel activity (Becq et al., 1994
, 2007). In the following experiments, first we validated the use of G551D CFTR-3HA and the pH measurement methodology as well as the counterion and passive proton permeability determination in nonpolarized BHK cells. Subsequent studies were performed on genetically matched CF and non-CF human respiratory epithelia (IB3 and CFBE) as well as cftr−/−
primary mouse macrophages and other heterologous CFTR overexpression systems. These studies not only allowed us to assess the effect of CFTR deficiency in genetically matched respiratory epithelia and alveolar macrophages, but also to determine whether the endogenous counterion permeability can limit the acidification in CFTR-independent cellular models.
Figure 1. Selective labeling of CFTR-3HA–containing endosomes with pH-sensitive probes. (A) Schematic comparison of the subcellular distribution of CFTR and various endosomal pH probes. It is predicted that CFTR has partially overlapping localization with (more ...)
Comparison of the Biosynthetic and Endocytic Membrane Trafficking of wt and G551D CFTR-3HA
To utilize the G551D CFTR-3HA for monitoring the vesicular pH without conferring PKA-dependent chloride permeability to endocytic organelles, first we assessed the membrane trafficking pathway of the G551D CFTR in relation to its wt counterpart. BHK cells were stably transfected with G551D and wt CFTR-3HA. The steady-state expression level of the core- and complex-glycosylated G551D and wt CFTR was comparable, measured by immunoblotting (B). Likewise, the cell surface expression of the wt and G551D CFTR-3HA was similar, determined by anti-HA Ab-binding assay (Supplemental Figure S2A). PKA activation by forskolin, CPT-cAMP, and IBMX failed to activate G551D CFTR contrary to the wt channel, monitored by the iodide efflux assay (C). The biosynthetic processing efficiency, metabolic stability, and internalization rates of the complex-glycosylated wt and G551D CFTR, measured by pulse-chase experiments, and anti-HA Ab uptake assay, respectively, were similar (Supplemental Figure S2, B and C, and data not shown).
To determine the postendocytic fate of the G551D CFTR, internalized channels were colocalized with the Tf receptor (Tf-R), a marker of recycling endosomes (Mukherjee et al., 1997
). CFTR was labeled by in vivo anti-HA Ab capture at 37°C for 1 h. Tf-Rs were visualized by FITC-Tf. Quantitative colocalization of CFTR, on micrographs obtained by fluorescence laser confocal microscopy (FLCM), revealed that 85 ± 2 and 80 ± 4% (mean ± SEM, n = 4 experiments) of internalized Tf was colocalized with G551D and wt CFTR, respectively. Conversely, 52 ± 4% of wt and 62 ± 3% of G551D CFTR were confined to Tf-positive endosomes (Supplemental Figure S3A). Confinement of internalized G551D and wt CFTR to early endosomes was confirmed with their colocalization with rab5 and EEA1 (Supplemental Figure S3B and data not shown) and exclusion from FITC-dextran–loaded lysosomes (D), an observation confirmed on other cells (see below). These results, jointly, indicate that the wt and G551D CFTR have overlapping postendocytic membrane trafficking that was further validated by vesicular pH measurements (see below).
Monitoring the Postendocytic Fate of G551D CFTR by Vesicular pH Determination
Based on the characteristic pH of the endolysosomal compartment (Mukherjee et al., 1997
), the postendocytic sorting of G551D and wt CFTR could be inferred from the luminal pH of internalized CFTR-containing vesicles, as has been shown for a variety of cargo molecules (Barriere et al., 2007
; Kumar et al., 2007
; Duarri et al., 2008
; Varghese et al., 2008
). Cell-surface CFTR-3HA was labeled with primary anti-HA and FITC-conjugated secondary Fab on ice. After the removal of the excess extracellular Ab, internalization was initiated by raising the temperature to 37°C (A). The luminal pH of individual endocytic vesicles was measured by FRIA, using 450- and 490-nm excitation wavelengths with an in situ calibration technique, and was plotted as their frequency distribution (, A–C, Barriere et al., 2007
; Barriere and Lukacs, 2008
; Glozman et al., 2009
). Although the cell-surface–bound probe (0-h chase) was exposed to the extracellular medium pH (~7.4), more than 90% of vesicles acidified to pH ~6.3–6.5 after a 0.5–1-h chase at 37°C (A). Comparable results were obtained by continuous labeling of CFTR at 37°C for 1 h to enhance the signal-to-noise ratio (B). Importantly, G551D, similar to the wt CFTR, was targeted to mildly acidic recycling endosomes after 1-h chase (pH ~ 6.52, C). Considering that the recycling endosomes mean pH is 6.4–6.5, measured in FITC-Tf–loaded BHK cells (Sharma et al., 2004
), these results indicate that G551D like its wt counterpart recycles back to the cell surface and largely avoids lysosomal delivery. This conclusion is also in line with the limited (<8%) colocalization of the wt and G551D CFTR with Lamp2- and dextran-loaded lysosomes, determined by the Volocity program (see D and C and Supplemental Figure S3D). CFTR was also confined to recycling endosomes after 4-h chase (data not shown). No significant dissociation of Ab from CFTR was observed at pH 5 (see Materials and Methods
). Furthermore, the metabolic stability of the Ab-bound CFTR complex remained unaltered (Sharma et al., 2004
), consistent with the notion that Ab binding did not provoke premature lysosomal degradation of the channel. Thus the G551D CFTR could serve as a negative control for evaluating the contribution of wt CFTR activation to the endosomal pH regulation.
Figure 3. Wt and G551D CFTR expression, function, and postendocytic localization in CF respiratory epithelia and HeLa cells. (A) CFTR expression was probed by immunoblot analysis in mock, wt, and G551D CFTR-3HA expressing CFBE and IB3 respiratory epithelia, as (more ...)
The Effect of CFTR Activation on the Counterion Conductance and the Steady-State pH of Endosomes In Situ
To assess the relative magnitude of CFTR-dependent counterion permeability and the passive proton leak of individual endosomes, determinants of the endosomal pH, we modified a technique developed to measure these parameters in cell suspension of mouse peritoneal macrophages and Chinese hamster ovary (CHO) cells (Lukacs et al., 1990
). The assay is based on the assumption that the dissipation rate of vesicular pH gradient by high concentration of protonophore (20 μM FCCP or CCCP) is rate-limited by the endosomal counterion conductance in the presence of the H+
-ATPase inhibitor, Baf (Lukacs et al., 1990
). Under these conditions, activation of CFTR should enhance the initial rate of Baf+CCCP–induced H+
-efflux by the provision of additional Cl−
conductance and dissipation of the inside negative endosomal membrane potential generated by the H+
efflux (D, right panel). This was a reasonable assumption, considering that the luminal Cl−
concentration of newly formed endosomes was rapidly reduced from 150 to 10–50 mM (Hara-Chikuma et al., 2005b
The pH of individual, CFTR-3HA–containing endosomes labeled by primary anti-HA– and FITC-conjugated secondary Fab, was determined by FRIA as a function of time. After the inhibition of the vacuolar H+-ATPase activity by Baf, slow endosomal alkalinization, caused by the passive H+ efflux along the proton electrochemical gradient, was detected (D). The observation that saturating concentration of protonophore CCCP (20 μM) accelerated the Baf-induced pH dissipation rate by nearly 10-fold (D) suggests that early endosomes have a relatively small passive proton and large counterion conductance.
Incomplete inhibition of the H+
-ATPase cannot account for the slow pH dissipation rate, because the Baf dose–response curve indicated that the v-ATPase was fully inhibited at 0.5 μM Baf concentration in vivo (data not shown; Lukacs et al., 1990
). These observations also imply that a relatively slow H+
-ATPase activity can maintain the endosomal pH gradient at steady state. Similar results were observed in parental BHK cells, ruling out the possibility that CFTR expression is responsible for the high constitutive counterion conductance and limited proton leak of endosomes (see Supplemental Figure S3E).
Activation of wt CFTR by PKA stimulation increased the CCCP+Baf–induced endosomal pH dissipation rate by threefold (, E and F). In sharp contrast, the pH dissipation remained unaltered in G551D CFTR-expressing cells (, E and F, and B). These results confirmed that wt, but not the G551D CFTR, is susceptible to PKA activation in endosomes (Becq et al., 1994
To determine the consequence of CFTR activation on the steady-state endosomal pH, the luminal pH values were plotted before and after 3-min stimulation by PKA agonists. The endosomal pH of PKA-stimulated cells remained unaltered, regardless whether wt or G551D CFTR was expressed (, F, right panel, and G).
A small fraction of internalized CFTR was confined to vesicles with pH < 6 and pH > 6.6 after 1-h chase, likely representing channels in late endosomes en route to lysosomes and in endocytic carrier vesicles, respectively (, A and B; Sharma et al., 2004
). Analysis of the luminal pH of these vesicles revealed that PKA-dependent CFTR activation was unable to influence the steady-state pH of late endosomes/lysosomes and endocytic carrier vesicles (G).
Endosomal pH Regulation Is Not Influenced by CFTR Ablation or Overexpression in CF Epithelia and Heterologous Cell Models, Respectively
The inability of CFTR functional overexpression to hyperacidify BHK endosomes suggested that the relatively high endogenous counterion permeability in the presence of a small passive proton leak cannot limit the proton accumulation by the v-ATPase in cells that have no endogenous CFTR. This inference was tested by overexpressing wt CFTR in HeLa cells and polarized MDCK epithelia.
The loss of endogenous CFTR on the endosomal pH homeostasis was examined using genetically matched CF and non-CF respiratory epithelia. To this end, wt and G551D CFTR-3HA were stably expressed in IB3 and CFBE cells, widely used models of CF respiratory epithelia lacking functional CFTR (Gruenert et al., 1995
; Bruscia et al., 2002
). IB3 and CFBE cells were derived from the bronchial epithelia of CF patients with ΔF508/W1282X
CFTR genotypes, respectively (Zeitlin et al., 1991
; Cozens et al., 1994
) and have no detectable CFTR expression by immunoblotting (A). Although IB3 cells are nonpolarized, both CFBE and MDCK cells were differentiated into polarized monolayers to ensure selective labeling of apical endosomes (see Materials and Methods
Heterologous expression of wt and G551D CFTR-3HA was verified by immunoblotting (A) and cell surface anti-HA Ab-binding assay (Supplemental Figure 2A). Iodide efflux assay revealed that wt but not G551D CFTR expression conferred PKA-stimulated plasma membrane halide conductance in HeLa, IB3, and CFBE cells (B). None of the parental cells had detectable endogenous CFTR- and PKA-activated halide conductance (, A and B, and data not shown).
Internalized wt and G551D CFTR were primarily targeted to early endosomes and excluded from lysosomes in IB3, CFBE, and HeLa cells, visualized by colocalization with FITC-Tf, EEA1, or rab5 and exclusion from dextran- or Lamp2-containing lysosome (C, Supplemental Figure S3, C and D, and data not shown). These results indicate that the recycling propensity of endocytosed G551D CFTR is independent of the cellular expression system, as observed previously for wt CFTR (Sharma et al., 2004
; Gentzsch et al., 2007
; Varga et al., 2008
The PKA-induced acceleration of the pH dissipation rate in the presence of CCCP+Baf showed that wt CFTR can function as a chloride conductive efflux pathway to facilitate H+
egress from early endosomes of CFBE, IB3, HeLa, and MDCK cells (, A and B). This conclusion was confirmed by the inability of PKA to activate H+
efflux from endosomes of G551D CFTR-expressing cells (B). Importantly, despite full activation of wt CFTR by the agonist cocktail in 1.5–2 min at room temperature (see Supplemental Figure S4B), no significant change in the steady-state pH of wt CFTR-expressing endosomes, including the CFBE and IB3 respiratory epithelia, was observed relative to that in G551D CFTR-expressing cells (C). Likewise, we were unable to detect significant changes in the initial acidification rate of early endosomes, after the synchronized internalization of Ab-labeled CFTR from the cell surface (, E and F). These data strongly suggest that the CFTR-independent counterion permeability is sufficient to ensure unrestricted proton accumulation during the acidification and at steady state by the v-ATPase. Finally, the HeLa and MDCK data suggest that the endosomal acidification is not limited by their endogenous counterion permeability, because provision of exogenous CFTR chloride conductance at an inwardly directed electrochemical chloride gradient (Sonawane and Verkman, 2003
) could not hyperacidify endosomes.
CFTR Effect on the pH Homeostasis of Recycling Endosomes
Although the anti-HA and FITC-Fab labeling of CFTR enabled us to restrict the pH measurements to CFTR-containing vesicles, we wanted to establish whether the Ab binding interferes with the channel function. To assess the potential effect of Ab labeling on CFTR transport activity, CFTR-3HA–expressing BHK cells were incubated with saturating concentration of anti-HA Ab (37°C for 30 min). The mean PKA-dependent whole cell current density was reduced by 42% (from 35.1 ± 8.0 to 20.5 ± 6.7 pA/pF) in the presence of anti-HA Ab measured by the patch-clamp technique (Supplemental Figure S4A). The activation kinetics of CFTR by PKA agonists remained unaltered (Supplemental Figure S4B). Although these observations suggested that the CFTR-Ab complex retains significant activity, we sought an alternative assay to evaluate the CFTR-dependent endosomal pH regulation in the presence of fully functional channels.
On the basis of the overlapping subcellular distribution of FITC-Tf with wt and G551D CFTR (D and Supplemental Figure S3A), we followed the endosomal pH after labeling the recycling endosomes with FITC-Tf. The Baf+CCCP–induced pH dissipation rates of recycling endosomes were determined in IB3 respiratory epithelia, as well as in BHK and HeLa cells. The endosomal counterion conductance was stimulated by about twofold with PKA agonists in wt, but not in G551D CFTR or parental cells (Supplemental Figure S3E). This suggests that the anti-HA Ab binding did not significantly limit the CFTR-dependent counterion flux in early or recycling endosomes. The steady-state endosomal pH was not, or only marginally affected by the wt CFTR activation relative to that of the G551D CFTR (D), supporting our conclusion that neither ablation nor overexpression of CFTR influences the endosomal pH regulation, presumably due to the relatively high CFTR-independent counterion conductance.
Internalized CFTR Traverses the Immature Phagosome
Although there is no direct evidence available for the functional expression of CFTR in phagosomes, recent observations suggested that the phagolysosomal acidification of CFTR-deficient alveolar macrophages was severely compromised (Di et al., 2006
). These results could not be confirmed by Haggie and coworkers (Haggie and Verkman, 2007
). Considering that the phagosomal membrane undergoes substantial compositional change during maturation (Steinberg and Grinstein, 2007
), it was plausible to assume that CFTR may traverse the limiting membrane of phagosomes and facilitate acidification by provision of chloride as a counterion. To assess CFTR localization and impact on the phagosomal proton and counterion permeability, first we used transiently transfected RAW264.7 mouse peritoneal macrophages. We were unable to detect endogenous CFTR by immunoblotting and iodide efflux assay in RAW cells (, A and B, and data not shown). Transient expression of CFTR was verified by immunoblotting and functional assay (, A and B). Immunostaining showed that the internalized channel was targeted to Tf-labeled recycling endosomes (~90% colocalization) and largely excluded from dextran-labeled lysosomes (~10% colocalization) after the labeling of endocytosed CFTR by anti-HA Ab capture in vivo (C).
Figure 5. Expression, activity, and membrane trafficking of CFTR-3HA in RAW macrophages. (A) CFTR plasma membrane channel activity was measured by the iodide efflux assay in transiently transfected RAW cells as described in C. Data are means of triplicate (more ...)
To assess whether the activation of the phagocytic signaling cascade influences the subcellular targeting of CFTR, the postendocytic fate of the channel was determined in cells ingesting fluorophore-labeled P. aeruginosa
(PAO1). Control experiments showed that after the engulfment of the FITC-labeled P. aeruginosa
, newly formed phagosomes matured into phagolysosomes in ~20 min, as shown by their fusion with TRITC-dextran loaded lysosomes (C, bottom panels). Colocalization of bacteria with labeled Tf-R confirmed that the assay can reproduce the transient recruitment of Tf-receptors into immature phagosomes (C, bottom panels) as reported earlier (Botelho et al., 2000
CFTR recruitment to phagosomes was followed after labeling the endocytic CFTR-3HA pool with anti-HA Ab for 1–2 h at 37°C. P. aeruginosa was then bound to the plasma membrane at 4°C, and phagocytosis was initiated by raising the temperature to 37°C. Limited colocalization of CFTR with newly formed phagosomes was observed after 5 min of incubation by FLCM (D). In sharp contrast, CFTR was largely undetectable in mature phagosomes 15–30 min after P. aeruginosa ingestion (D). Similar results were obtained upon labeling CFTR on ice for 1 h before phagocytosis and initiating both internalization and phagocytosis simultaneously (data not shown). These results suggest that CFTR and ingested bacteria have only transiently overlapping postendocytic trafficking pathways and CFTR is virtually eliminated from mature phagosomes in RAW macrophages. Another possibility is that the anti-HA Ab complex is susceptible to rapid degradation in the proteolytically active phagosomes. This is unlikely to be the case because the labeled CFTR-Ab complex remained detectable after 90-min chase with comparable staining intensity, and inhibition of phagolysosomal proteases did not prevent the removal of Ab-labeled CFTR from phagosomes (see F and data not shown). To further support the observation that CFTR is only transiently confined to immature phagosomes, we compared the luminal pH of CFTR-containing vesicles in phagocytosing RAW cells.
Figure 6. The phagosomal acidification is CFTR-independent in RAW macrophages. (A) The pH distribution profile of wt CFTR-containing endosomes in transiently transfect RAW macrophages. FRIA of internalized CFTR-3HA was performed as described in B. (B) The (more ...)
CFTR-independent Endosomal and Phagosomal Acidification in RAW Macrophages
The postendocytic trafficking of transiently expressed CFTR-3HA was determined by FRIA in RAW macrophages. The pH distribution profile of internalized wt CFTR-3HA indicated that the channels were primarily targeted to recycling endosomes in accord with immunolocalization data (A). The PKA-dependent phosphorylation augmented the Baf+ CCCP–induced endosomal pH dissipation by threefold, confirming that the channel is functional in endosomes (, B and C). Remarkably, preincubating the cells with the water-soluble MalH2, a specific inhibitor of CFTR (Muanprasat et al., 2004
; Sonawane et al., 2008
), fully suppressed the PKA-dependent pH dissipation of recycling endosomes (, B and C).
Next, the PKA sensitivity of the endogenous counterion permeability of Tf-labeled recycling endosomes was measured. The Baf+CCCP–induced pH dissipation assay revealed that recycling endosomes have PKA-stimulated and MalH2-sensitive endogenous counterion conductance (B). This observation suggests that RAW macrophages express a low level of endogenous CFTR that is below the detection limit of immunoblotting (B). Neither PKA-activation nor MalH2 inhibition caused any significant change in the early endosomal pH of parental and CFTR-expressing RAW cells (D).
It has been accepted that the rapid acidification of newly formed phagosomes is mediated by the vacuolar proton ATPase in macrophages (Lukacs et al., 1990
; Hackam et al., 1997
; Yates et al., 2005
) and precedes the phagolysosomal fusion (Geisow et al., 1981
). CFTR-dependent chloride uptake may promote proton accumulation by shunting the membrane potential of immature phagosomes. To address this scenario, macrophages were allowed to engulf FITC-labeled P. aeruginosa
synchronously, in parental and CFTR overexpressing RAW cells, and the phagosomal pH was monitored by FRIA. CFTR expressors were identified by anti-HA Ab and TRITC-Fab staining. Neither overexpression nor inhibition of CFTR by MalH2 influenced the rapid, early phase of the Baf-sensitive acidification of newly formed phagosomes (E). After 30 min of bacterial engulfment, the phagosomal H+
concentration reached pH ~5 regardless of CFTR activity (E).
The possible influence of phagocytosis on the postendocytic CFTR sorting next was assessed using FRIA. Both the cell surface binding of P. aeruginosa and CFTR labeling by anti-HA and FITC-Fab was performed on ice. Internalization and phagocytosis was initiated by shifting the temperature to 37°C. After transient confinement of wt CFTR to vesicles with luminal pH ~6.0, likely representing sorting endosomes and immature phagosomes, the channel was transferred into recycling endosomes, characterized by more alkaline luminal pH (~6.3–6.5; F). The CFTR transfer to recycling endosomes coincided with the progressive acidification of phagosomes and the formation of the mature phagolysosomes with luminal pH ~5.0, determined in separate samples (E). These observations underscore the distinct trafficking route of CFTR and bacteria in RAW cells. The phagosomal acidification was blocked by Baf and reversed by NH4Cl (, E and G). CFTR activation had no measurable effect on the steady-state pH of CFTR-containing endosomes and early or mature phagosomes (, G and H). On the basis of the immunochemical localization and pH measurements, however, we could not rule out that a small fraction of CFTR reached the lysosomes. Next, we assessed CFTR function in lysosomal pH regulation.
Acidification of Endolysosomes and Phagosomes Is CFTR-independent in Primary Alveolar and Peritoneal Macrophages
CFTR was proposed to be indispensable for the normal acidification and bactericidal effect of phagolysosomal compartment in alveolar macrophages (Di et al., 2006
). Functional expression of CFTR was measured by the pH dissipation assay in primary alveolar and peritoneal macrophages harvested from cftr+/+
mice. The pH dissipation rate of FITC-Tf–labeled recycling endosomes revealed that only cftr+/+
, but not cftr−/−
macrophages possessed PKA-activated and MalH2-sensitive counterion conductance (, A and B), providing strong evidence for CFTR expression in endosomes in line with the electrophysiological detection of the channel at the plasma membrane (Di et al., 2006
Figure 7. Acidification of phagosomes in CFTR-deficient primary alveolar (AM) and peritoneal (PM) macrophages is preserved. (A and B) CFTR is active in early endosomes, but not in lysosomes, of primary alveolar and peritoneal mouse macrophages from cftr+/+ mice. (more ...)
Remarkably, immature phagosomes also displayed PKA-activated counterion permeability after 5-min ingestion of FITC-conjugated P. aeruginosa in cftr+/+ alveolar and peritoneal macrophages (, C and D). Because this counterion permeability was inhibited by the MalH2 and was undetectable in cftr−/− cells, we concluded that functional CFTR is associated with immature phagosomes in primary macrophages (, C and D). Maturation of phagosomes, however, led to the loss of PKA-stimulated counterion permeability, measured after 30 min of phagosome formation (, C and D). The assay pH sensitivity was preserved, as demonstrated by the rapid alkalinization of phagosomes by NH4Cl (data not shown).
Neither the initial rate of acidification nor the steady-state pH of mature phagosomes was influenced by CFTR expression in alveolar and peritoneal macrophages (, E and F). Furthermore, no significant change in the steady-state pH of immature and mature phagosomes was detected upon PKA activation in cftr+/+ and cftr−/− macrophages (E, insert).
Because lysosomal fusion was proposed to play a pivotal role in phagosomal maturation (Di et al., 2006
), CFTR contribution to the lysosomal pH homeostasis was also assessed using FITC-dextran– and Oregon488-dextran–labeled lysosomes. No lysosomal CFTR activity was measurable by the Baf+CCCP–induced pH dissipation assay (, A and B). This could be attributed to the channel efficient recycling, inactivation, and degradation in lysosomes or a combination of these processes. Finally, no difference in the lysosomal pH of control or PKA-stimulated primary macrophages isolated from cftr−/−
mice could be detected (Supplemental Figure S6A). These observations support the notion that the phagosomal and lysosomal acidifications are CFTR-independent processes in primary macrophages.
The Endogenous Counterion Permeability of Endophagocytic Compartments Does Not Restrict the Proton Pump Activity, the Role of Passive Proton Permeability
The counterion flux required for the dissipation of the vacuolar H+
-ATPase–induced membrane potential is determined by the proton accumulation rate. To achieve the set point of organellar pH, the proton accumulation rate should be adjusted according to the buffer capacity and the proton leak (Wu et al., 2001
). Considering that CFTR overexpression, activation, and ablation failed to change the organellar acidification in endolysosomes and phagosomes, we hypothesized that the endogenous, CFTR-independent counterion permeability is sufficiently high to dissipate the membrane potential (Steinberg et al., 2007b
) and thereby cannot impede the proton translocation rate. This assumption implies that the passive proton permeability of endocytic organelles is small in comparison with their counterion permeability. To support this prediction, we assessed the counterion and passive proton permeabilities of endocytic organelles in respiratory epithelia, macrophages, and other heterologous expression systems, assuming that the passive pH dissipation rate is proportional with the proton permeabilities.
The Baf-induced proton efflux reflects the passive proton leak that is compensated by the v-ATPase activity at the steady-state pH. Comparison of the Baf- and Baf+CCCP–induced pH dissipation rates revealed that the CFTR-independent counterion permeability was ~5–10 times higher than the passive proton permeability both in recycling endosomes and lysosomes of IB3 and CFBE epithelia (G). Similar data were obtained in primary macrophages (H). These results strongly suggest that the counterion permeability is sufficiently high to support the acidification of organelles in CFTR-deficient respiratory epithelia and primary macrophages.
Decreasing passive proton permeability along the secretory pathway was proposed as a critical determinant of the progressively increasing luminal acidification (Wu et al., 2001
). An analogous role of the passive proton permeability may prevail along the endocytic pathway. To determine the passive proton permeability of endocytic organelles, first the buffer capacity of endosomes, lysosomes, and phagosomes was measured, using the ammonium chloride pulse technique (Roos and Boron, 1981
; , A and B). The passive proton permeability was calculated based on the Baf-induced proton efflux rate and the assumption that the predominant driving force is the transmembrane pH gradient at a constant cytoplasmic pH 7.3 as described in Materials and Methods.
The passive proton permeability of lysosomes was at least twofold lower than in recycling endosomes in BHK, HeLa, IB3, and CFBE cells (C), supporting the notion that down-regulation of the passive proton leak may contribute to the progressive acidification along the endocytic pathway. For comparison, the passive proton permeability of the ER and Golgi compartment was also plotted, as determined in previous publications (Llopis et al., 1998
; Chandy et al., 2001
; Wu et al., 2001
; Weisz, 2003b
Figure 8. Determination of the passive proton permeability of endocytic organelles. (A) Measurement of the buffer capacity of endocytic organelles. The lysosomal compartment of BHK cells were loaded with dextran as described in Materials and Methods, and the organellar (more ...)