Proton leak and CFTR in regulation of Golgi pH in respiratory epithelial cells

Grischa Chandy1, Michael Grabe2, Hsiao-Ping H. Moore1, and Terry E. Machen1

1 Department of Molecular and Cell Biology and 2 Department of Physics, University of California, Berkeley, California 94720-3200


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Work addressing whether cystic fibrosis transmembrane conductance regulator (CFTR) plays a role in regulating organelle pH has remained inconclusive. We engineered a pH-sensitive excitation ratiometric green fluorescent protein (pHERP) and targeted it to the Golgi with sialyltransferase (ST). As determined by ratiometric imaging of cells expressing ST-pHERP, Golgi pH (pHG) of HeLa cells was 6.4, while pHG of mutant (Delta F508) and wild-type CFTR-expressing (WT-CFTR) respiratory epithelia were 6.7-7.0. Comparison of genetically matched Delta F508 and WT-CFTR cells showed that the absence of CFTR statistically increased Golgi acidity by 0.2 pH units, though this small difference was unlikely to be physiologically important. Golgi pH was maintained by a H+ vacuolar (V)-ATPase countered by a H+ leak, which was unaffected by CFTR. To estimate Golgi proton permeability (PH+), we modeled transient changes in pHG induced by inhibiting the V-ATPase and by acidifying the cytosol. This analysis required knowing Golgi buffer capacity, which was pH dependent. Our in vivo estimate is that Golgi PH+ = 7.5 × 10-4 cm/s when pHG = 6.5, and surprisingly, PH+ decreased as pHG decreased.

enhanced yellow fluorescent protein; ultraviolet enhanced green fluorescent protein; trachea; organelle pH; proton permeability; cystic fibrosis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CYSTIC FIBROSIS (CF) is the most common fatal genetic disease among Caucasians. CF is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which is a cAMP/protein kinase A-regulated Cl- channel that is absent or defective in CF. CF patients most commonly succumb to respiratory complications that arise because of colonization of the respiratory tract by Pseudomonas aeruginosa and other opportunistic bacteria. One hypothesis to explain how a reduction of Cl- permeability could lead to colonization of CF lungs by P. aeruginosa is that pH of the Golgi (pHG), trans-Golgi, and trans-Golgi network, which are normally acidic relative to the cytosol, are alkaline in CF. CFTR, which could serve as the counterion conductance, may be required to prevent generation of large lumen-positive membrane voltages during pumping by the electrogenic H+ vacuolar (V)-ATPase (1, 2). The lack of CFTR would lead to an alkaline pHG, which in turn would alter the activities of resident enzymes responsible for proper sialylation, sulfation, and fucosylation [due to sialyltransferase (ST), sulfotransferase, and fucosyltransferase, respectively] of secreted and surface membrane components. These alterations could lead to changes in the chemical properties of membrane and secreted glycoproteins and glycolipids, such as increased asialo-GM1, a hypothesized bacterial binding site on epithelia (5, 11, 25, 63) [see also Schroeder et al. (45) for conflicting opinion].

A number of groups have attempted to test the organelle pH hypothesis. Lukacs et al. (33) demonstrated that CFTR was functional in endosomes of Chinese hamster ovary (CHO) cells heterologously expressing CFTR, but they concluded that factors other than CFTR were the major determinants of endosomal pH. Dunn et al. (12) found that endocytic acidification was independent of CFTR when the CF pancreatic cell line (CFPAC) and CFTR-corrected CFPAC cells were compared. Seksek et al. (48) microinjected liposomes containing pH-sensitive fluid-phase dyes into the Golgi of fibroblasts and epithelial cells and found that pHG was the same in both cell types and also in epithelial cells that normally do (Calu-3) and do not (Madin-Darby canine kidney, SK-MES-1) express CFTR (49).

There were three reasons for performing a rigorous test of the organelle pH hypothesis. First, human airway epithelial cells may be different from fibroblasts, lymphocytes, and non-airway epithelial cells from other species with regard to pHG regulation. Second, none of the previous comparisons of pHG was performed on cells that were genetically matched Delta F508 (deletion of Phe-508 in CFTR) and wild-type CFTR-expressing (WT-CFTR) respiratory cell lines. Third, it was important to measure the pH of Golgi cisternae where ST and the other critical enzymes reside because pH regulatory mechanisms may differ in different organelles (54), and none of the other studies had definitively measured pH in the ST-containing region of the Golgi.

We developed a ratiometric, green fluorescent protein (GFP)-based pH sensor that was targeted to the Golgi with ST in genetically matched Delta F508-CF and CFTR-corrected Delta F508-CF human airway epithelial cells. Various pH-sensitive mutants of GFP (pKa 5-7) have been targeted to the Golgi (as well as mitochondria and endoplasmic reticulum) (28, 32, 55), but artifacts can arise with the use of the single-wavelength intensity changes of these GFPs when the apparent or actual fluorophore concentration changes (e.g., due to bleaching or changes in path length) and pH does not. By fusing ultraviolet enhanced GFP (GFPuv) and enhanced yellow fluorescent protein (EYFP), we created a chimeric protein that has an excitation spectrum with pH-dependent (490 nm) and relatively pH-independent (440 nm) wavelengths. Measurements of pHG in HeLa cells were used to confirm the method, and measurements in CF (Delta F508 CFTR) tracheal (CFT1) and human nasal epithelial (JME) cells and in CFTR-expressing tracheal (CFT1-CFTR) and human bronchial epithelial (HBE) cells were used to determine the role of CFTR in the control of pHG.

It was also expected that pHG would be critically affected by the magnitude of H+ leaks. Because CFTR may control both Na+ conductance (29, 39, 50) and anion (Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) exchange (31) in the plasma membranes of epithelial cells, it seemed possible that CFTR mutations might also affect pHG by altering H+ leak pathways. We therefore compared H+ leaks in CFT1 and CFT1-CFTR cells. We also developed a mathematical model that used measurements of pHG, cytosolic pH [pHC, with the cytosolic dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)], and buffer capacity of the Golgi to make the first in vivo estimates of Golgi membrane H+ permeability (PH+, in cm/s) in CFT1 cells.


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Materials

All salts, glucose, buffers, dibutyryl-cAMP, adenine, transferrin, insulin, chloroquine, DMSO, triiodothyronine, hydrocortisone, cholera toxin, epithelial growth factor (EGF), epinephrine, endothelial cell growth supplement, DEAE dextran, bafilomycin, nigericin, and monensin were obtained from Sigma (St. Louis, MO); forskolin and sometimes bafilomycin were obtained from Calbiochem (San Diego, CA); solvents were from Fisher Scientific (Pittsburgh, PA); and restriction enzymes were from New England Biolabs (Beverly, MA). Tissue culture reagents were obtained from GIBCO-BRL or Cellgro. Fetal bovine serum (FBS) was obtained from Gemini Bio-Products. BCECF-AM and Pluronic F-127 were from Molecular Probes, (Eugene, OR).

Construction of Plasmids

Bacterial expression vector for pHERP. To create a bacterial expression vector for pH-sensitive excitation ratiometric green fluorescent protein (pHERP), EYFP (S65G, S72A, T203Y, H231L) was PCR-amplified with a sense primer (5'-cccaagcttgatggtgagcaagggcgag-3') containing a HindIII restriction site (underlined) and an antisense primer (5'-gacgagctgtacaagggaggaggtctagag-3') that codes for a linker region. An XbaI restriction site (underlined) is present and eliminated the EYFP stop codon. Cloning the product into GFPuv (F99S, M153T, V163A; Clonetech) put EYFP upstream of GFPuv with an intervening linker region having the amino acid sequence GGGLEDPRVPVEK.

ST-pHERP mammalian expression vector. We PCR amplified the ST fragment, amino acids 1-70 containing the cytosolic, transmembrane, and truncated luminal domains, from human 2',6-sialyltransferase (courtesy of Dr. Brian Seed, Harvard Medical School and Massachusetts General Hospital). This portion of ST has been used to target chimeric molecules to the Golgi (60, 61). PCR amplification was performed with primers (5'-cgcgggaagcttgccaccatgattcacaccaacctg-3' and 5'-cgcgggcggatcctgggtgctgcttgagga-3') that allowed cloning of the PCR product into the pcDNA3 vector (Invitrogen, San Diego, CA) with 5' HindIII and 3' BamHI restriction sites. PCR amplification of pHERP was performed with a sense primer (5'-cgcgggagatctagaattcgtgagcaagggcgag-3') that eliminates EYFP's ATG and has a BglII site (underlined) and with an antisense SP6 primer (5'-gatttaggtgacactatag-3'). The EYFP-GFPuv PCR product was subcloned downstream of ST in pcDNA3 with BglII and ApaI. The final construct codes for the chimeric protein with amino acids 1-70 of ST, a 3-amino acid linker (LEF) between ST and EYFP, amino acids 2-239 of EYFP, a 13-amino acid linker (GGGLEDPRVPVEK), and amino acids 1-238 of GFPuv. GT-EGFP [enhanced GFP (EGFP) targeted to Golgi with galactosyltransferase (GT)] was provided by the laboratory of Roger Tsien (Howard Hughes Medical Institute and University of California, San Diego).

In Vitro Spectra of pHERP

Bacteria expressing the various GFPs were grown overnight in liquid cultures and resuspended in one-tenth the volume of a bacterial lysis solution. Bacterial lysis solution contained (in mM) 10 Tris · HCl (pH 7.4), 100 NaCl, 1 MgCl2, 10 dithiothreitol, and protease inhibitors (0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 20 µg/ml phenylmethylsulfonyl fluoride). Lysed bacteria were diluted into buffer containing (in mM) 50 Na-acetate, 50 glycine, and 50 K2HPO4, which were titrated to the various pH values with HCl or KOH. Excitation spectra were obtained with a fluorometer (Spex Flurolog 1681; Spex Industries, Edison, NJ) containing a 150-W xenon arc lamp.

Cell Culture

All cells were maintained in a 37°C incubator with 5% CO2. All media were supplemented with penicillin, streptomycin, and glutamine. HeLa cells were maintained in DMEM supplemented with 10% FBS. JMEs (obtained from Dr. Douglas Jefferson, Tufts University) were grown in DMEM/F-12 supplemented with 10% FBS, 180 µM adenine, 5 µg/ml insulin, 5 µg /ml transferrin, 30 nM triiodothyronine, 1.1 µM hydrocortisone, 10 µg/ml EGF, and 5.5 µM epinephrine. HBE, CFT1-C2, and CFT1-CFTR cells were obtained from Dr. James Yankaskas (62) and grown in Ham's F-12 supplemented with 10 µg/ml insulin, 1 µM hydrocortisone, 25 ng/ml endothelial cell growth supplement, 10 ng/ml EGF, 30 nM triiodothyronine, 5 µg /ml transferrin, and 10 ng/ml cholera toxin.

Transfections

Cells were transiently transfected with a modified DEAE dextran protocol of Seed and Aruffo (47). Cells were sequentially split first into a tissue culture flask and, on the following day, onto glass coverslips to a confluency of ~30-50%. On the following day, the cells were incubated in a solution containing 5-6 µg/ml plasmid DNA, 100 µg/ml DEAE dextran, and 50 µM chloroquine for 2-4 h. Cells were washed with PBS containing 10% DMSO for 2 min. Medium was then added, and experiments were performed 24-72 h later. Transfection efficiency was low (1-10%) but sufficient for the single-cell experiments. Some transfections were done with a microporator. Cells plated on coverslips were exposed to DNA (2 µg/µl) while three 30-ms pulses of 300 mV/cm were applied (53). This method led to improved transfection efficiency (5-20%).

Solutions

Ringer solution contained (in mM) 141 NaCl, 2 KCl, 1.5 K2HPO4, 1 MgS04, 10 HEPES, 2 CaCl2, and 10 glucose brought to pH 7.4 with NaOH. Na+-free solutions contained (in mM) 141 N-methyl-D-glucamine (NMDG) base, 2 KCl, 1.5 K2HPO4, 1 MgS04, 10 HEPES, 2 CaCl2, and 10 glucose brought to pH 7.4 with HCl. Calibration solutions contained (in mM) 70 NaCl, 70 KCl, 1.5 K2HPO4, 1 MgS04, 10 HEPES, 10 MES, 2 CaCl2, and 10 glucose adjusted to various pH values (5.5, 6.0, 6.5, 7.0, 7.5, or 8.2) with KOH, 0.01 nigericin, and 0.01 monensin. In some experiments in which cells were alkalinized, NH4Cl (30 mM) was substituted for 30 mM NaCl or NMDG where indicated. Buffer capacity experiments were performed in high-K+/0-Na+ solutions with varying amounts of K-acetate or NH4Cl substituting for the KCl. Bafilomycin was used at 100-250 nM. Intracellular cAMP was increased by perfusing cells with solutions containing either 10 µM forskolin alone or 10 µM forskolin plus 500 µM dibutyryl-cAMP.

Fluorescence Ratio Imaging of pHC and pHG

Golgi, labeled with pHERP, and cytosol, labeled with 10 µM BCECF-AM, were monitored in separate experiments with the use of digitally processed fluorescence ratio imaging. Dye-loaded cells were placed in an open perfusion chamber on an inverted IM35 Zeiss microscope. A ×40 oil-immersion objective (1.4 NA; Nikon) was used to collect fluorescence from 1 to 30 cells during each experiment. A lens was used to focus the image through a phototube (Diagnostic Instruments) onto a low-light-level DAGE 68 SIT camera. Emission images of the cells were collected through a 530-nm band-pass filter during sequential excitation at 490 and 440 ± 5 nm (Omega Optical, Brattleboro, VT). Filters were changed with a Lambda 10-2 filter wheel (Sutter Instruments, Novato, CA). Separate images for each wavelength were averaged over eight frames by a digital image processor (Axon Image Lightning; Axon Instruments, Foster City, CA) and subsequently converted pixel by pixel to a ratio image. Data collection rate (1 ratio image every 5-60 s), filter wheel position, and shutter opening/closing were controlled by a 133-MHz Pentium computer (Gateway 2000) running the latest update of version 2.x of Imaging Workbench (Axon Instruments). The ratio images were displayed in pseudocolor.

Data were collected by electronically selecting regions of the image for quantitation. Cytosolic measurements were made from entire cells. When measurements were made on Golgi, only the brightest perinuclear regions were selected. Like FITC and BCECF, the fluorescence of pHERP excited at 490 nm increases with pH, whereas fluorescence at 440 nm is relatively insensitive to pH. Intensities were balanced with neutral density filters. Photobleaching was negligible during our experiments.

Methods describing calibration of cytosolic and organelle pH measurements have been reported previously (54).

Determination of Golgi Buffer Capacity

We made small, stepwise changes in the extracellular concentration of either NH<UP><SUB>4</SUB><SUP>+</SUP></UP> or acetate (OAc-) in the absence of extracellular Na+ to induce defined changes in pHG, which were then used to calculate Golgi buffer capacity (beta G) over a range of pH values. Using measured pHG values and assuming that the Golgi concentration of the uncharged species equaled the external concentration, we calculated [NH4+] or [OAc-] using the Henderson-Hasselbalch equation (Ka = 5.6 × 10-10 for the weak base reaction and 1.8 × 10-5 for the weak acid reaction, where Ka is the equilibrium constant). Golgi buffer capacity was then calculated for the midpoint of the pH change (Delta pH) according to the following equation (41)
&bgr;<SUB>G</SUB><IT>=</IT><FR><NU>−[NH<SUP>4+</SUP>]<SUB>G</SUB></NU><DE>&Dgr;pH</DE></FR><IT>  &bgr;</IT><SUB>G</SUB><IT>=</IT><FR><NU>[OAc<SUP>−</SUP>]<SUB>G</SUB></NU><DE>&Dgr;pH</DE></FR> (1)

Calculation of H+ Permeability of Golgi Membranes

We used the previously described model (18), which accurately describes experimentally determined values of both steady-state and transient acidification of endosomes (56) and Golgi (61), and experimentally determined changes of pHG, pHC (measured in separate but identical experiments), and beta G to calculate PH+ of the Golgi. The model assumes that changes in pHG result from a competition between V-ATPase-mediated H+ pumping and passive H+ leak (which is characterized by PH+) out of the Golgi into the cytosol. It should be noted that the predicted values of active pump numbers and PH+ did not drastically change when the complicated molecular model of the V-ATPase was replaced by a simpler model in which the H+ flux was proportional to the proton motive force working against the pump (18). Thus our calculations of PH+ were insensitive to the specific molecular model of the H+ pump.

One experimental protocol was to add the H+ V-ATPase inhibitor bafilomycin to cells and model measured rates of alkalization of pHG while pHC remained constant (see Fig. 8A). The second approach was to acidify both cytosol and Golgi cells with an NH4Cl pulse and then to model changes of pHG while pHC also changed (see Figs. 6 and 8B). Without loss of generality, the equation that describes transient changes in pHG during the above-mentioned experimental manipulations is
<FR><NU>dpH</NU><DE>d<IT>t</IT></DE></FR><IT>=</IT><FR><NU>1</NU><DE><IT>&bgr;</IT><SUB>G</SUB></DE></FR><IT>·</IT><FR><NU><IT>S</IT></NU><DE>V</DE></FR><IT>·</IT>(<IT>J</IT><SUB>leak</SUB><IT>−&rgr;</IT><SUB>pump</SUB><IT>·J</IT><SUB>pump</SUB>) (2)
where Jpump is the H+ flux of an individual V-ATPase, rho pump is the density of H+ pumps in the Golgi, and Jleak is the passive flux of H+ across a unit area of Golgi membrane; the term in parentheses is the total flux of H+ across a unit area of membrane. These fluxes depend on the Golgi-cytosol [H+] gradient, the change in pH defined as pHG - pHC, and the membrane potential (Psi G). S and V are the Golgi surface area and volume, which were assumed to be 8 × 10-6 cm2 and 6 × 10-12 cm3, respectively (30). beta G is the buffering capacity of the Golgi (determined as in Fig. 7).

Using the previously described model of the V-ATPase (19), we found computed values for Jpump to be relatively constant over moderate luminal pH ranges but sensitive to changes in membrane potential (calculated as shown in Eq. 4), consistent with previous current-voltage data (7). In the present work, calculated Jpump changed by only 1-10% over the time course of any experiment.

Although the true nature of the proton leak, Jleak, is not known, we modeled this transport as simple, passive diffusion (23)
J<SUB>i</SUB><IT>=P</IT><SUB>i</SUB><IT>·</IT><FR><NU><IT>z</IT><SUB>i</SUB><IT>·U·</IT>([C<SUB>i</SUB>]<SUB>G</SUB><IT>−</IT>[C<SUB>i</SUB>]<SUB>C</SUB><IT>·e</IT><SUP><IT>−z</IT><SUB>i</SUB><IT>U</IT></SUP>)</NU><DE>1<IT>−e</IT><SUP><IT>−z</IT><SUB>i</SUB><IT>U</IT></SUP></DE></FR> (3)
where i denotes the ionic species, Ji is the ionic flux density, Pi is the permeability of the membrane to ion i, [Ci]C and [Ci]G are the concentrations of the ion in the cytoplasm and Golgi, respectively, zi is the valance of the ion, and U = Psi GF/(RT), where U is reduced voltage, Psi G is the Golgi membrane potential, and F, R, and T have their usual meanings.

Because Psi G will affect both Jpump and Jleak, we included this effect by writing an explicit form for Psi G in terms of the excess charge inside the Golgi, the membrane of which was treated as a parallel plate capacitor, and by assuming that the dominant counterions were K+ and Cl-
&PSgr;<SUB>G</SUB><IT>=</IT><FR><NU><IT>F·</IT>V</NU><DE><IT>C</IT><SUB>m</SUB></DE></FR><IT>·</IT><FENCE>[K<SUP>+</SUP>]<SUB>G</SUB><IT>−</IT>[Cl<SUP>−</SUP>]<SUB>G</SUB><IT>+</IT><LIM><OP>∫</OP><LL>pH<SUB>C</SUB></LL><UL>pH<SUB>G</SUB></UL></LIM><IT> &bgr;</IT><SUB>G</SUB><IT>·</IT>dpH<IT>−B</IT></FENCE> (4)
where Cm is the total capacitance of the membrane, [K+]G and [Cl-]G are the molar concentrations of K+ and Cl- in the Golgi, the integral term represents the total amount of H+ in the Golgi lumen (buffered plus free), and B (a constant) is the molar concentration of charged species that are trapped in the Golgi. When the concentration of trapped protein, B, is balanced by the net sum of all ionic species in the lumen, Psi G is zero.

When K+ and Cl- permeabilities were assumed to be 10-5 cm/s (22), Psi G was calculated (from Eqs. 2 and 3) to be <10 mV. This result was consistent with recent experiments showing that the Golgi and trans-Golgi network had relatively large conductances to both K+ and Cl- and that Psi G was likely to be an unimportant determinant of either Jpump or Jleak (43, 61). Schapiro and Grinstein (43) arrived at this conclusion by finding that [K+]G, measured to be 107 mM using a null point method, was similar to the cytosolic [K+] ([K+]C).

Equation 2 together with equations describing the passive fluxes of K+ and Cl- (from Eq. 3) forms a set of three ordinary differential equations that are coupled by the algebraic constraint of Eq. 4 and that uniquely determine the time course of changes of ionic concentrations in the Golgi given an initial set of conditions. We assumed [K+]C = 130 mM and [Cl-]C = 20 mM throughout each experimental run, and initial [K+]G and [Cl-]G were chosen to keep the Golgi close to electroneutral (consistent with Psi G < 10 mV).

pHC and transient changes of pHG (similar to those shown in Fig. 8, A or B) were measured in separate experiments, and then the model was used (Eq. 3) to predict the changes in Golgi acidification. This was done by varying PH+, rho pump, and the concentration of fixed negative charge in the lumen (B) until a best fit to pHG was obtained. When results were modeled from experiments using V-ATPase inhibitors (similar to Fig. 8A), rho pump was set to zero and pHC was held constant to match experimental conditions. The Na+-free, acid-loaded Golgi experiments (see Fig. 8B) were more complex because pHC and pHG both varied. However, this procedure was advantageous because it allowed us to acidify the Golgi and keep pHC relatively constant (see Figs. 6 and 8B). For these experiments, we reported the average of six predicted PH+ values, for each pHG experiment, where each pHG run was fit against a different pHC run (see Possible errors in predicting PH+). All searches were performed with a Nelder-Mead algorithm, and the ordinary differential equations were solved with a stiff method in both Matlab and Berkeley Madonna software (38).

Although Eqs. 2-4 yielded estimates of PH+, B, and the number of active H+ V-ATPases (NOP = rho pump · S), we have reported only predicted values for PH+. The model yielded average values of B approx  140 mM and NOP approx  2,000. Within a particular data set, these two parameters had counteracting effects: a decrease in rho pump together with an increase in B sometimes resulted in very similar pH fits. This made it very difficult for the search algorithm to find a unique, best fit, and the same data set with different initial search conditions would yield very different values for B and rho pump. In contrast, estimates of PH+ were more robust to the initial conditions of the search algorithm, and the same best value for PH+ was usually found regardless of the values of B and rho pump. As mentioned above, Jpump changed little for any given run. Thus the parameter PH+ had the strongest influence over the shape of the predicted pH curves.

It should be noted that in the limit of Psi  = 0 mV, Eqs. 2-4 can be replaced by an intuitive and simple expression for the PH+ of the Golgi in terms of the instantaneous rate of change of the Golgi, the H+ gradient, and a few physical parameters
<FR><NU>dpH<SUB>G</SUB></NU><DE>d<IT>t</IT></DE></FR><IT>=</IT><FR><NU>1</NU><DE><IT>&bgr;</IT><SUB>G</SUB></DE></FR><IT>·</IT><FR><NU><IT>S</IT></NU><DE>V</DE></FR><IT>·P</IT><SUB>H<SUP>+</SUP></SUB><IT>·</IT>([H<SUP>+</SUP>]<SUB>G</SUB><IT>−</IT>[H<SUP>+</SUP>]<SUB>C</SUB>) (5)

⇒ P<SUB>H<SUP>+</SUP></SUB><IT>=</IT><FR><NU>dpH<SUB>G</SUB></NU><DE>d<IT>t</IT></DE></FR><IT>·</IT><FR><NU>V</NU><DE><IT>S</IT></DE></FR><IT>·</IT><FR><NU><IT>&bgr;</IT><SUB>G</SUB></NU><DE>([H<SUP>+</SUP>]<SUB>G</SUB><IT>−</IT>[H<SUP>+</SUP>]<SUB>C</SUB>)</DE></FR>
Thus PH+ could also be determined from initial rates of alkalinization after treatment with bafilomycin to block the H+ pumps without appealing to the more complicated model. For the bafilomycin-treated Golgi, this method predicted PH+ values that were within factors of 2-4 of the values determined using the full model.

Possible Errors in Predicting PH+

There are several uncertain parameters and assumptions that affect our estimates of PH+. We explored how errors in each of the following affected the predicted value of PH+.

Surface-to-volume ratio. We arbitrarily chose the value of 1.33 × 10-6 cm-1 for the surface-to-volume ratio (S/V) of the Golgi obtained from rat kidney cells (30). Golgi S/V in terminal tubule and acinar cells of the rat submandibular gland (51) is an order of magnitude smaller. If the latter estimates were correct, our PH+ values would be 10-fold larger; thus our calculations provide a lower estimate. This S/V parameter is most likely the largest source of error in our study.

Buffering. If the true buffering capacity is 20% larger or smaller than the value measured, which is consistent with our errors, then the true PH+ will be 20% larger or smaller than predicted.

H+ gradients. In many of the bafilomycin-induced alkalization experiments, pHC was assumed to be 7.55 (the average value). Varying the assumed pHC by ±0.2 pH units for a typical run had a ±25% effect on the estimated PH+. For the experiments where the Golgi has been acidified, each pHG run (see examples in Figs. 6 and 8B) was fit against six pHC runs that represented the entire set of measured cytoplasmic recovery experiments. Most of these reported PH+ values had SD of 2 × 10-4, although one had a SD of 2 × 10-3.

Membrane potential. The movement of counterions influences H+ movement through effects on Psi G as shown in Eq. 4. When the Golgi membrane was assumed to have K+ and Cl- permeabilities >5 × 10-9 cm/s, Psi G was calculated (from Eqs. 2 and 3) to be <10 mV. When permeabilities to K+ and Cl- were <5 × 10-9 cm/s, K+ and Cl- movements became quite slow, and Psi G began to affect pHG. The present PH+ predictions remain unchanged for K+ and Cl- permeabilities >5 × 10-9 cm/s. Below this value, movements of counterions resulted in transient changes of Psi G that affected calculations of PH+. When Psi G was arbitrarily varied from +7 mV to +50 mV and held constant over the time course of the simulation, the predicted PH+ values varied from +50% to -50% of the original value determined with our model of Psi G. Therefore, the PH+ values determined with Eqs. 2 and 3 were relatively insensitive to Psi G.

Obtaining Initial Alkalization Rates and PH+ From the Literature

To compare our data with those from previous work, we scanned published figures as PICT files and used Data Thief (Computer Systems Group of the Nuclear Physics Section at the National Institute for Nuclear Physics and High Energy Physics, Amsterdam, The Netherlands) to extract data points of pHG vs. time. These data points were then used to obtain initial rates or were used with the model (Eqs. 2-4) to determine PH+ (see Table 2).

Statistics

Unless otherwise specified, data have been presented as means ± SD. Experimental data were compared using unpaired Student's t-test (two-tailed). Differences were considered significant if P < 0.01.

As an objective measure of the quality of our fits, the conventional root mean square (RMS) value was computed. When a particular run did not have an RMS value <0.175 pH units, it was dropped from the analysis. The average RMS value for all runs fit by the model was 0.06 ± 0.03 pH units (mean ± SD).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

pH Sensitivity and Golgi Targeting of pHERP

The ST-containing compartment was targeted with pHERP. We first created a chimeric protein of two commercially available mutant GFPs by placing GFPuv NH2-terminal to EYFP. The chimeric protein retains the dominant excitation peaks of the individual molecules at 397 nm (GFPuv) and ~500 nm (EYFP) when emission intensity is measured at 520 nm (Fig. 1). The peak at 500 nm was extremely sensitive to pH, whereas the trough at ~440 nm was relatively insensitive to pH, indicating that this molecule could be used as an excitation ratiometric (490/440) pH indicator. By leaving the luminal and transmembrane amino acids (1-70) of ST intact and replacing its cytosolic domain with pHERP, we were able to target the pH sensor to the Golgi lumen (Figs. 1 and 2). ST was used to target pHERP because it is one of the trans-Golgi enzymes (52) whose activity has been proposed to be altered in CF (2). Cells transiently expressing ST-pHERP displayed the characteristic perinuclear staining pattern typical of Golgi. A typical result for JME cells is shown in Fig. 2, and similar staining patterns were observed in HeLa, HBE, CFT1, and CFT1-CFTR cells (not shown). An in vivo calibration was performed at the end of every experiment (Fig. 1) by perfusing solutions containing nigercin (K+/H+ exchanger) and monensin (Na+/H+ exchanger) with different pH values onto the cells. The pKa of the ratio 490/440 was ~6.5 and thus was optimal for measuring pHG (Fig. 1).


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Fig. 1.   Construction of sialyltransferase (ST)-pH-sensitive excitation ratiometric green fluorescent protein (pHERP). A: in vitro excitation spectrum of green fluorescent protein (GFP) constructs measured at 520 nm. Spectra for enhanced yellow fluorescent protein (EYFP) and ultraviolet enhanced GFP (GFPuv) were obtained at pH 7.5. EYFP and GFPuv were fused together, separated by a 13-amino acid linker to create pHERP. The spectrum of pHERP is extremely sensitive to pH at ~490 nm and relatively insensitive at 440 nm, allowing for ratiometric imaging (ex, excitation). B: schematic of ST-pHERP. Amino acids 1-70 of ST were used to target pHERP to the Golgi. N, NH2 terminal; C, COOH terminal. C: representative in vivo calibration of ST-pHERP (performed at the end of each experiment). Golgi pH was allowed to equilibrate with solutions of various pH values in the presence of 10 µM each of nigericin and monensin.



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Fig. 2.   ST-pHERP morphology. Human nasal epithelial (JME) cells were transiently transfected with ST-pHERP and mounted on the stage of the microscope. Fluorescence image overlaid on the bright-field image shows that pHERP exhibit a perinuclear staining pattern typical of Golgi.

pHG Measured With pHERP

We performed control experiments on HeLa cells to compare measurements of pHG obtained using ST-pHERP with previous measurements of HeLa cell pHG obtained using other methods. As shown in the typical individual experiment and in the summarized data (Fig. 3), steady-state pHG was 6.4, in good agreement with previous results of 6.4-6.6 obtained using Golgi-targeted fluorescein and GFP-based pH sensors (26, 32, 61). When HeLa cells were pulsed with a solution containing 30 mM NH4Cl, pHG instantly alkalinized because of the entry of the weak base NH3. With removal of the NH4Cl, the cells acidified below basal levels and rapidly recovered (Fig. 3). These results were in good agreement with results obtained in Vero cells using FITC-verotoxin (27, 43) and in CHO cells using a mutant GFP (28).


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Fig. 3.   ST-pHERP reports Golgi pH (pHG) in HeLa cells. A: a pulse of 30 mM NH4Cl alkalinized the Golgi due to NH3 entry. The slower NH<UP><SUB>4</SUB><SUP>+</SUP></UP> entry led to a slight acidification, which was apparent when the NH4Cl was washed away. Golgi pH rapidly recovered from the acidification. B: histogram of steady-state pHG measured with ST-pHERP at the beginning of each experiment.

ST-pHERP consists of ST fused to the relatively pH-insensitive GFPuv and pH-sensitive EYFP. EYFP has recently been reported also to be sensitive to [Cl-], with decreases in [Cl-] causing increases in fluorescence (57). Although the changes in EYFP fluorescence due to changes in [Cl-] are smaller than those due to pH (32, 57), we were concerned that changes in ST-pHERP fluorescence ratio in vivo would be larger than predicted from calibrations in which pH was varied but [Cl-] was held constant. We therefore compared measurements of pHG made with ST-pHERP with those obtained with GT-EGFP (13, 28, 32). EGFP is insensitive to [Cl-] but sensitive to pH, with a pKa of 6.4 (32, 57). When expressed in both CFT1 and CFT1-CFTR cells, GT-EGFP and ST-pHERP yielded similar results when pHG was perturbed. When cells expressing either ST-pHERP (see Fig. 6) or GT-EGFP (data not shown) cells were pulsed with NH4Cl followed by removal and then Na+-free treatment, pHG acidified and recovered partially. Full recovery was completed only when Na+ was added back to the cells. These results support the conclusion that ST-pHERP was accurately reporting pHG. The fact that ST-pHERP was more easily calibrated in terms of pHG made it preferable to GT-EGFP.

CFTR and Steady-State pHG

As summarized in Table 1, pHG in respiratory epithelial cells was more alkaline (6.7-7.0) than in HeLa cells (6.4; see Fig. 3). Airway epithelial cells from Delta F508-CF patients (CFT1 and JME) had pHG values of 6.7 and 7.0, respectively, while those from WT-CFTR cells (CFT1-CFTR and HBE) had pHG values of 6.9 and 6.7, respectively. When the genetically matched CFT1 cells were compared with CFT1-CFTR cells, the Delta F508-expressing CFT1 cells were 0.2 pH units more acidic (P < 0.01). There was no significant difference in pHC (7.4-7.6) among the various cells.

                              
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Table 1.   Steady-state pHG and pHC

The activity of CFTR is increased by cellular cAMP concentration, and the effects of cAMP on pHG have been controversial (32, 48). We therefore tested the effects of cAMP in CFT1 and CFT1-CFTR cells. In both cell types, there was little change in pHG in response to cAMP whether experiments were performed in the presence of 25 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/5% CO2 or in nominally HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions (Fig. 4).


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Fig. 4.   Increasing cAMP concentration does not alter pHG. Left: experiments were conducted in the presence of 5% CO2 and 25 mM HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Intracellular cAMP concentration was increased by application of 10 µM forskolin (arrow). Right: experiments were conducted in HEPES-buffered nominally HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free conditions. cAMP concentration was increased by application of 10 µM forskolin and 500 µM dibutyryl-cAMP (arrow). Traces are from Golgi of single cells. CFTR, cystic fibrosis transmembrane conductance regulator; CFT1(CFTR), CFTR-expressing tracheal cells.

Golgi H+ "Leak" and Potential Effect of CFTR

We determined the role of CFTR in controlling H+ leak across the Golgi membrane into the cytosol in CFT1 and CFT1-CFTR cells that had been treated with bafilomycin. Once the bafilomycin effect had reached a steady state with pHG and pHC approximately equal, cells were treated with an NH4Cl pulse followed by the Na+-free condition. This caused both the Golgi and the cytosol to acidify. We then added back extracellular Na+, which allowed both pHG and pHC to realkalinize. Rates of alkalinization of the Golgi lumen (using ST-pHERP) and cytosol (using BCECF) were measured in separate experiments. It was expected that if the H+ leak in the Golgi were large, removal of H+ from the cytosol by the Na+/H+ exchanger (NHE) in the plasma membrane would cause a similarly rapid alkalinization of pHG because H+ in the Golgi lumen would rapidly leak into the cytosol to be pumped out of the cell across the plasma membrane by the NHE. By comparing recovery rates of pHC and pHG measured using the same experimental protocol, we found that the Golgi in CFT1 cells alkalinized at a rate of 2.2 ± 0.7 × 10-2 pH units/s (n = 9), while the cytosol alkalinized at a rate of 5.0 ± 1.6 × 10-2 pH units/s (n = 33). Experiments on CFT1-CFTR cells showed that pHG and pHC alkalinized at rates similar to those in CFT1 cells. These data implied that the Golgi membrane had a large PH+ that was unaffected by the presence or absence of CFTR.

Golgi-to-Cytosol [H+] Gradient

We compared pHC and pHG from experiments in which the CFT1 cells were acidified with a 5-min pulse of 30 mM NH4+ followed by Na+-free Ringer. Removal of extracellular Na+ prevented the NHE in the plasma membrane from pumping out the accumulated H+. On average, the steady-state pHC of CFT1 cells was 7.5 ± 0.2 (n = 68), while under the conditions used to acidify the cells, pHC dropped to 6.4 ± 0.2 (n = 55). Under the same conditions, average pHG dropped from 6.7 ± 0.2 (n = 45) to 5.9 ± 0.3 (n = 31) and then partially recovered with a single-exponential time course (see Fig. 6C). The average [H+] gradient (Golgi to cytosol) returned to 2 × 10-7, close to the value exhibited under control conditions (1.7 × 10-7) after ~7 min (Fig. 6).

Golgi H+ Permeability and Buffer Capacity

When the H+ V-ATPase was inhibited by bafilomycin, the Golgi rapidly alkalinized with an initial rate of 8.6 ± 7.8 × 10-3 pH units/s (n = 9; Fig. 5). This result was similar to those in previously published experiments (summarized in Table 2) on HeLa, Vero, and CHO cells obtained by using a variety of methods and indicated that the Golgi had a large H+ leak that ordinarily counters the H+ pump (13, 26, 32, 42, 43, 61). These observations led to the qualitative conclusion that PH+ was large. Data from these experiments can also be used to calculate PH+, but the Golgi buffer capacity must be known.


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Fig. 5.   The Golgi H+ leak is large. A: a representative trace of cytosolic pH (pHC). Cells were exposed to a pulse of 30 mM NH4Cl for 5 min, and Na+ was then replaced with N-methyl-D-glucamine (NMDG) 2.5 min into the NH4Cl pulse. When NH<UP><SUB>4</SUB><SUP>+</SUP></UP> was removed, the cells acidified and remained acidic. Reapplication of Na+ allowed pHC to recover to control levels. B: a representative trace of pHG during an experiment similar to that performed on the cytosol in A, although the cell was first exposed to bafilomycin (250 nM), a specific inhibitor of the H+-ATPase. C: cytosolic and Golgi pH alkalization curves from A and B are superimposed and the time scale is magnified. Slope at pH 7 was taken for a number of similar experiments. D: model of the likely transporters and channels that are regulating pH during the experiment.


                              
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Table 2.   Summary of Golgi PH+

It is well established that the cytosolic buffering capacity varies with pH (58, 59), which is a function of the pKa and concentration of titratable groups (41). Previous measurements of Golgi buffer capacity have reported only one value (42, 61) or claimed that beta G was constant between pH 6 and 7 (13). We felt that it was necessary to measure beta G over a wide pH range to calculate PH+ of the Golgi at the various pH values observed in our experiments. Values for beta G were obtained by first treating the cells with bafilomycin (250 nM) and then titrating in various amounts of weak base (NH3) or weak acid (HOAc). We measured beta G at pH < 7 by following the bafilomycin treatment with an acidification step (NH4Cl prepulse followed by incubation in Na+-free solution) and then adding either NH4Cl or HOAc to the Na+-free solutions. A typical experiment in which beta G was measured is shown in Fig. 7A. Data summarizing measurements of beta G as a function of pH are summarized in Fig. 7B. Buffer capacities were grouped into 0.2-pH unit buffering domains for simplicity. At pH 6.9 ± 0.02 (mean ± SE), beta G = 17.2 ± 4 mM per pH unit (mean ± SE), in good agreement with previous results of 10-40 mM per pH unit (13, 42, 61). At higher and lower pH values, beta G varied, and the variation was well fit by a single exponential (Fig. 7B), which allowed us to extrapolate to acidic values that were attained in some measurements of PH+.

By using the experimentally determined beta G and a model of pHG (Eqs. 2-4), we were able to estimate the PH+ of bafilomycin-treated cells when pH > 6.4 (Fig. 8, A and C). Additionally, the model allowed estimates of PH+ under various conditions where organelle and pHC varied over a considerable range: recovery of both pHG and pHC were measured after acidification (using an NH<UP><SUB>4</SUB><SUP>+</SUP></UP> prepulse) and treatment with Na+-free solution (see example in Fig. 8, B and C). These data provided PH+ when pHG < 6.4. These calculations showed that the log of Golgi PH+ was inversely correlated with the pHG (Fig. 8C). The data were well fit by a linear equation, and the slope of this line was significantly different from the slope of 0 (P < <0.01).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ST-pHERP for Measuring pHG

By fusing EYFP and GFPuv, each having different excitation spectra and different pH dependencies, we created the genetically targeted ratiometric pH indicator pHERP (Figs. 1 and 2). A variety of other techniques have been used to measure pHG, but each has limitations that our technique attempted to improve. Isolated Golgi vesicles are rarely pure and can be damaged and lose soluble regulatory factors during preparation. Electron microscopic methods (2) allow only imprecise quantitation at single time points. Microinjection of dye-filled liposomes (48, 49) is laborious and invasive, and since the dyes are delivered to the Golgi relatively slowly and then removed relatively rapidly at 37°C, measurements can be performed only during short time periods. FITC-labeled beta -subunits of toxins (27, 42-44) can be used only in cells that express the toxin receptors. Genetic targeting of "receptors" and then adding membrane-permeant, pH-sensitive, fluorescent "ligands" to label the receptors (13, 61) requires complicated chemical syntheses and loading procedures.

GFPs have many advantages as organelle pH sensors: any organelle can be targeted, a variety of mutants with a wide range of pKa values (5.5-7.0) is available (28, 32, 55), and changes of pH can be measured with little background signal, no diffusive loss of fluorophore, minimal bleaching, and no requirement for exogenous dyes that require hydrolysis and might lead to cytotoxicity. Despite these many advantages, calibrating the single-wavelength intensity changes of these GFPs is problematic, because artifacts can arise when apparent fluorophore concentration changes and pH does not. Miesenbock et al. (34) overcame this problem by developing a GFP with a spectrum that shifted with pH, thereby providing an excitation ratiometric pH indicator. Our development of pHERP allows an alternative approach that has many of the same advantages as pHlorin for measuring pHG.

CFTR, Counterion Conductance, and Membrane Potential in Determining pHG

As measured with pHERP, HeLa cells had an average pHG of 6.4 (Fig. 3), which was nearly identical to previous measurements obtained using a variety of other methods in HeLa, fibroblast, and Vero cells (27, 32, 42, 43, 61). In contrast, the Golgi of airway epithelial cells was more alkaline (pHG = 6.7-7.0) than in HeLa cells. Although, there was no clear correlation between the presence of CFTR and pHG when all the airway epithelial cells were compared, there was a significant difference when the genetically matched CFT1 cells were compared (Table 1). Cells expressing Delta F508-CFTR had a pHG of 6.7 (CFT1) and 7.0 (JME), while those expressing WT-CFTR had a pHG of 6.9 (CFT1-CFTR) and 6.7 (HBE). The finding that Delta F508-CFTR-expressing CFT1 cells were more acidic (0.2 pH units) than the WT-CFTR-expressing CFT1-CFTR cells is a result directly opposite the trend measured by Barasch et al. (2). The pH dependence of ST can be quite variable depending on the particular isoform and cell type involved (3, 4, 16, 36), so the potential significance of this small difference in Golgi acidity in CF remains to be determined. It seems likely that the results of Barasch et al. (2) were influenced by the technical problems associated with isolating purified Golgi and the relative insensitivity of the electron microscopic dinitrophenol-3-(2,4-dinitroanilino)-3'- amino-N-methyldipropylamine (DAMP) method. Our data (also see Refs. 12 and 49) therefore showed that the chronic lung infection of CF is not due to an alkaline pHG. Indeed, the presence of CFTR in CFT1 cells was correlated with significant alkalinization, not acidification, of pHG.

Al-Awqati and colleagues (1, 2) suggested that the Golgi Cl- permeability was able to limit H+ flux by dissipating the membrane potential. Subsequent data in the literature indicated that conductances to both K+ and Cl- in the Golgi and trans-Golgi network are so high that membrane voltage is small and unimportant in determining organelle pH (10, 43, 61). This is consistent with observations that neither ouabain nor its membrane-permeant analog acetyltrophanthidin have any effect on pHG in HeLa cells, which have no CFTR (Wu M and Machen T, unpublished observations), implying that the Na+-K+-ATPase activity was low or nonexistent. Our model confirmed and extended the conclusions regarding the small Golgi membrane potential and also allowed us to estimate the number of counterion channels necessary to achieve this goal. Our model showed that K+ and Cl- permeability could be decreased to as low as 5 × 10-9 cm/s before membrane potential became a factor in fitting the pH transients (see METHODS and Fig. 8). For a Golgi with a surface area of 8×10-6 cm2 (30), these very low permeabilities corresponded to K+ and Cl- conductances <20 pS for the entire Golgi, which could be achieved with between one and two K+ or Cl- channels. Calculation of K+ conductance was performed by using the Goldman-Hodgkin-Katz (GHK) current equation (23) after compensating for the assumed surface area of the Golgi. Conductance was calculated as the slope of the current with respect to voltage at 0 mV, assuming [K+]G = 110 mM (43) and [K+]C =130 mM. In this circumstance, it is likely that eliminating CFTR has no effect on pHG, because other K+ and Cl- channels can provide sufficient counterion conductance to ensure that membrane potential in the Golgi is small and therefore has little effect on H+ pumps or leaks. Two other studies have provided evidence for the presence of Cl- channels in the Golgi: Nordeen et al. (35) found an anion channel in enriched Golgi fractions, and Schwappach et al. (46) found that Gef1p (the only yeast ClC Cl- channel) localizes to the Golgi. Additionally, elimination of Gef1p from yeast does result in altered cation homeostasis (17) but does not effect Kar2p secretion or glycosylation of invertase, both dependent on an acidic Golgi (46).

It might be argued that since our experiments were performed on single cells, they are not comparable to those performed on confluent monolayers. We attempted to make pHG measurements on confluent cells grown on filters, but the background fluorescence contributed by the filter exceeded the specific fluorescence of pHERP when excited at 440 and 490 nm under acidic conditions. Therefore, it was impossible to perform experiments when cells were grown on filters. Because of technical difficulties, we were able to obtain only one pHG measurement from a CFT1-CFTR cell in a confluent patch. Results from this experiment clustered with most of the other CFT1-CFTR data (Fig. 8C). We also note that CFTR transits through the Golgi and becomes functional in the plasma membrane of a variety of cells grown on glass either as single cells or in confluent patches (14, 15). We therefore believe, but have not proven, that pHG experiments performed on isolated cells grown on glass reflect those of confluent cells.

Roles of H+ Pump, pH-Dependent Leak, and pHC in Determining pHG

It has been proposed that pH of the Golgi and other organelles in the secretory pathway is determined primarily by a balance between the active accumulation of protons by the H+ V-ATPase and the passive loss of H+ through leaks (10, 61) and that the flux of protons is balanced at pHG (13). The previous work has not accurately distinguished between effects of the pH gradient and the PH+ in determining H+ fluxes across the Golgi. Farinas and Verkman (13) showed that as pHG decreased, the passive H+ flux out of the Golgi increased. Regardless of the true nature of the H+ leak pathway, this is not a surprising finding because as the luminal [H+] is increased, the efflux rate will most probably increase. The present work has extended these conclusions by estimating PH+ and showing that this permeability decreased as pH decreased. We also showed that Golgi buffer capacity was approximately equal to, and showed a pH dependency similar to, that of the cytosol (see Refs. 58 and 59), which supports the assumption that beta G is pH sensitive (42).

As summarized in Fig. 8C, PH+ in Golgi of CFT1 cells was ~7.5 × 10-4 cm/s when pHG was 6.5, and there was little difference to CFTR-corrected CFT1 cells. Similar values for Golgi PH+ of HeLa, Vero, and CHO cells were calculated using our model (Eqs. 2-4), and bafilomycin-induced alkalinization data were obtained from the literature (Table 2). These PH+ values are large compared with typical permeability values for Na+, K+, and Cl-: 10-12 cm/s for a membrane without channels (37) and up to 10-5 cm/s for a membrane with channels (22, 24). However, our estimates of PH+ compare favorably with PH+ in lipid bilayers and liposomes: 10-7-10-2 cm/s (8, 21, 37, 40). Similar to the data presented for PH+ of the Golgi, artificial lipid bilayers also have pH-dependent PH+ (8, 21). It therefore seems possible that PH+ of the Golgi might be solely due to simple H+ diffusion through membranes, and variations in PH+ could therefore be due to differences in fatty acids (which act as weak acid shuttles) and/or lipids (which can form water "wires") (8, 20, 21) in the Golgi of different cells. In addition, H+ channels may also provide leak pathways for H+ in the Golgi (43). However, estimates of PH+ > 5 × 10-1 cm/s for plasma membranes of lung alveolar cells with H+ channels [using GHK, reported pH gradients, and measured current densities (6, 9)] was 100 times larger than the largest PH+ observed in the present experiments. Finally, Na+/H+ exchange in the Golgi might contribute to the H+ permeability. However, Schapiro and Grinstein (43) showed that NHEs play no role in the efflux of H+ from the Golgi. Also, we have found that treatment of CFT1 cells with hexamethylene amiloride (membrane-permeant analog of amiloride) had no effect on steady-state pHG (data not shown).

A consequence of the Golgi having a relatively large, pH-dependent PH+ is that pHG will be a complicated function of pHC, PH+, and H+ pumping. The role of pHC in determining pHG is shown by experiments in which cells were treated with NH3/NH<UP><SUB>4</SUB><SUP>+</SUP></UP> and both cytosol and Golgi were first alkalinized and then secondarily acidified (Figs. 3 and 6). Because this secondary acidification also occurred in the presence of bafilomycin (Fig. 5) and thus was not due to the H+ V-ATPase (as proposed in Refs. 27 and 42), it may have been due to H+ (or NH<UP><SUB>4</SUB><SUP>+</SUP></UP>) accumulation across the plasma membrane into the cytosol, which then equilibrated with the Golgi. When extracellular Na+ was added back to acidified cells, the Golgi alkalinized at a rate that was only 2.5-fold slower than that of the cytosol, likely due to a delayed leak of H+ from the Golgi into the cytosol, which was being alkalinized due to active H+ extrusion by NHE in the plasma membrane.


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Fig. 6.   Golgi recovery from an acid load is partially dependent on extracellular Na+. A: the same representative pHC trace from Fig. 5. B: representative trace of pHG during the same protocol performed on the cell in A. C: model of the likely transporter and channels regulating pH during the experiment.

The role of H+ pumps in setting pHG was suggested by the fact that HeLa cells had a more acidic Golgi than respiratory epithelial cells (Table 1). This was not solely due to the more acidic pHC in HeLa cells because the Golgi-to-cytosol [H+] gradient was larger in HeLa (3.6 × 10-7 M) than in respiratory epithelial cells (0.75-1.7 × 10-7 M) (Table 1). Given that PH+ of HeLa and other cells was similar in the pH 6.4-6.6 range (Fig. 8C), the most likely explanation is a difference in H+-pump activity between HeLa cells and CFT1 cells. This explanation would also apply to the difference between pHG of CFT1 and CFT1-CFTR cells, although why or how various pump activities would arise in different cells is not clear.


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Fig. 7.   Golgi buffer capacity (beta G) is pH dependent. A: representative trace of 1 cell for which Golgi buffer capacity was obtained. The cell was first acid-loaded with an NH4Cl prepulse and then exposed to varying amounts of NH4Cl under Na+-free/high-K+ conditions. B: summary of all Golgi buffer capacity experiments performed with NH4Cl and KHOAc. Buffer capacities were grouped by 0.2-pH unit increments. Data are means ± SE. The smooth line is an exponential fit through the data. (beta G = 10.4 × 104 e-1.3 pH units).



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Fig. 8.   PH+ is pH dependent. A: representative trace showing that when cells were treated with bafilomycin, pHG rapidly alkalinized. Data from the average of 6 cells are represented by points. The smooth line is the best fit of the data obtained from Eqs. 2-4 with the pumps turned off. B: superimposition of pHG (Golgi) and pHC (cytosol) traces after compartments were acid loaded (as in Fig. 6, A and B). Notice that on this time scale pHC was relatively constant, while pHG recovered. Golgi pH (data points) was fit using Eqs. 2-4 (smooth line). At ~7 min, the average gradient returned to 2.0 × 10-7 M. C: log PH+ plotted against pHG. Filled diamonds represent data from bafilomycin-treated CFT1 cells as described in Fig. 7A. Asterisks represent data from bafilomycin-treated CFT1-CFTR cells (1 CFT1-CFTR in the cluster of 5 was a cell from a confluent patch). Open diamonds represent acid-loaded cells that were not treated with bafilomycin, as in Fig. 7B. Open circles (a-d) represent values extrapolated from the literature (see Table 2). The smooth line is a linear regression through the CFT1 data where log PH+ = 0.53 pH - 6.50. The slope of the line is significantly different from a line with a slope of zero (P < 0.01). Data from the literature and CFT1-CFTR cells were not included in the linear regression.

A potential regulatory role for the H+ pump has been considered previously by Kim et al. (27), who found that pHG was constant in the face of altered pHC. In contrast, the present data show that when pHC was acidified from an average of 7.5 to 6.5, pHG acidified on average from 6.5 to 6.2. These averages predict that the Golgi-to-cytosol [H+] gradient is constant in the face of an apparently decreasing PH+ and constant H+ pump. It will be necessary to measure pHC and pHG in the same cells to determine whether H+ pump and leak may be regulated. It also will be important to determine the roles of pHC, H+ pump, and pH-dependent H+ leak in generating the wide range of pH values found in other acidic organelles [e.g., endosomes (pH 6.0-6.5), lysosomes (pH 4-5), and secretory granules (pH 4-5)] in different cells.


    ACKNOWLEDGEMENTS

We thank Minnie Wu for useful discussions and for providing measurements of average pHC for HeLa cells. We thank Eric Wunderlich for providing measurements of average pHC for JME cells.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-51799 (to T. Machen), and grants from Cystic Fibrosis Research, Inc. (to G. Chandy and H.-P. H. Moore). M. Grabe was supported by National Science Foundation Grant DMS9220719 (to George Oster, whom we thank for useful discussions).

Present address of G. Chandy: Dept. of Molecular Pharmacology, Stanford, CA 94305-5175.

Address for reprint requests and other correspondence: T. E. Machen, 231 LSA, Univ. of California, Berkeley, CA 94720-3200 (E-mail: machen{at}socrates.berkeley.edu).

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.

Received 26 October 2000; accepted in final form 16 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Al-Awqati, Q, Barasch J, and Landry D. Chloride channels of intracellular organelles and their potential role in cystic fibrosis. J Exp Biol 172: 245-266, 1992[Abstract/Free Full Text].

2.   Barasch, J, Kiss B, Prince A, Saiman L, Gruenert D, and al-Awqati Q. Defective acidification of intracellular organelles in cystic fibrosis. Nature 352: 70-73, 1991[ISI][Medline].

3.   Breen, KC, and Regan CM. Developmental control of N-CAM sialylation state by Golgi sialyltransferase isoforms. Development 104: 147-154, 1988[Abstract].

4.   Busam, K, and Decker K. Ganglioside biosynthesis in rat liver. Characterization of three sialyltransferases. Eur J Biochem 160: 23-30, 1986[Abstract].

5.   Cheng, PW, Boat TF, Cranfill K, Yankaskas JR, and Boucher RC. Increased sulfation of glycoconjugates by cultured nasal epithelial cells from patients with cystic fibrosis. J Clin Invest 84: 68-72, 1989[ISI][Medline].

6.   Cherny, VV, Markin VS, and DeCoursey TE. The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient. J Gen Physiol 105: 861-896, 1995[Abstract].

7.   Davies, JM, Hunt I, and Sanders D. Vacuolar H+-pumping ATPase variable transport coupling ratio controlled by pH. Proc Natl Acad Sci USA 91: 8547-8551, 1994[Abstract].

8.   Deamer, DW. Proton permeation of lipid bilayers. J Bioenerg Biomembr 19: 457-479, 1987[ISI][Medline].

9.   DeCoursey, TE, and Cherny VV. Voltage-activated hydrogen ion currents. J Membr Biol 141: 203-223, 1994[ISI][Medline].

10.   Demaurex, N, Furuya W, D'Souza S, Bonifacino JS, and Grinstein S. Mechanism of acidification of the trans-Golgi network (TGN). In situ measurements of pH using retrieval of TGN38 and furin from the cell surface. J Biol Chem 273: 2044-2051, 1998[Abstract/Free Full Text].

11.   Dosanjh, A, Lencer W, Brown D, Ausiello DA, and Stow JL. Heterologous expression of Delta F508 CFTR results in decreased sialylation of membrane glycoconjugates. Am J Physiol Cell Physiol 266: C360-C366, 1994[Abstract/Free Full Text].

12.   Dunn, KW, Park J, Semrad CE, Gelman DL, Shevell T, and McGraw TE. Regulation of endocytic trafficking and acidification are independent of the cystic fibrosis transmembrane regulator. J Biol Chem 269: 5336-5345, 1994[Abstract/Free Full Text].

13.   Farinas, J, and Verkman AS. Receptor-mediated targeting of fluorescent probes in living cells. J Biol Chem 274: 7603-7606, 1999[Abstract/Free Full Text].

14.   Fischer, H, Illek B, and Machen T. Regulation of CFTR by protein phosphatase 2B and protein kinase C. Pflügers Arch 436: 175-181, 1998[ISI][Medline].

15.   Fischer, H, Kreusel K, Illek B, Machen T, Hegel U, and Clauss W. The outwardly rectifying Cl- channel is not involved in cAMP-mediated Cl. Pflügers Arch 422: 159-167, 1992[ISI][Medline].

16.   Freischutz, B, Saito M, Rahmann H, and Yu RK. Characterization of sialyltransferase-IV activity and its involvement in the c-pathway of brain ganglioside metabolism. J Neurochem 64: 385-393, 1995[ISI][Medline].

17.   Gaxiola, R, Yuan D, Klausner R, and Fink G. The yeast CLC chloride channel functions in cation homeostasis. Proc Natl Acad Sci USA 95: 4046-4050, 1998[Abstract/Free Full Text].

18.   Grabe, M, and Oster G. Regulation of organelle acidity. J Gen Physiol 117: 329-344, 2001[Abstract/Free Full Text].

19.   Grabe, M, Wang H, and Oster G. The mechanochemistry of V-ATPase proton pumps. Biophys J 78: 2798-2813, 2000[Abstract/Free Full Text].

20.   Gutknecht, J. Proton conductance caused by long-chain fatty acids in phospholipid bilayer membranes. J Membr Biol 106: 83-93, 1988[ISI][Medline].

21.   Gutknecht, J. Proton conductance through phospholipid bilayers: water wires or weak acids? J Bioenerg Biomembr 19: 427-442, 1987[ISI][Medline].

22.   Hartmann, T, and Verkman AS. Model of ion transport regulation in chloride-secreting airway epithelial cells. Integrated description of electrical, chemical, and fluorescence measurements. Biophys J 58: 391-401, 1990[Abstract].

23.   Hille, B. Ionic channels of excitable membranes. Sunderland, MA: Sinauer, 1992.

24.   Huflejt, ME, Blum RA, Miller SG, Moore HP, and Machen TE. Regulated Cl transport, K and Cl permeability, and exocytosis in T84 cells. J Clin Invest 93: 1900-1910, 1994[ISI][Medline].

25.   Imundo, L, Barasch J, Prince A, and Al-Awqati Q. Cystic fibrosis epithelial cells have a receptor for pathogenic bacteria on their apical surface Proc Natl Acad Sci USA 92: 3019-3023, 1995[Abstract/Free Full Text]. [Corrigenda. Proc Natl Acad Sci USA 92: November 1995, p. 11322.]

26.   Kim, JH, Johannes L, Goud B, Antony C, Lingwood CA, Daneman R, and Grinstein S. Noninvasive measurement of the pH of the endoplasmic reticulum at rest and during calcium release. Proc Natl Acad Sci USA 95: 2997-3002, 1998[Abstract/Free Full Text].

27.   Kim, JH, Lingwood CA, Williams DB, Furuya W, Manolson MF, and Grinstein S. Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor. J Cell Biol 134: 1387-1399, 1996[Abstract].

28.   Kneen, M, Farinas J, Li Y, and Verkman AS. Green fluorescent protein as a noninvasive intracellular pH indicator. Biophys J 74: 1591-1599, 1998[Abstract/Free Full Text].

29.   Kunzelmann, K, Kathofer S, and Greger R. Na+ and Cl- conductances in airway epithelial cells: increased Na+ conductance in cystic fibrosis. Pflügers Arch 431: 1-9, 1995[ISI][Medline].

30.   Ladinsky, MS, Mastronarde DN, McIntosh JR, Howell KE, and Staehelin LA. Golgi structure in three dimensions: functional insights from the normal rat kidney cell. J Cell Biol 144: 1135-1149, 1999[Abstract/Free Full Text].

31.   Lee, MG, Choi JY, Luo X, Strickland E, Thomas PJ, and Muallem S. Cystic fibrosis transmembrane conductance regulator regulates luminal Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in mouse submandibular and pancreatic ducts. J Biol Chem 274: 14670-14677, 1999[Abstract/Free Full Text].

32.   Llopis, J, McCaffery JM, Miyawaki A, Farquhar MG, and Tsien RY. Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc Natl Acad Sci USA 95: 6803-6808, 1998[Abstract/Free Full Text].

33.   Lukacs, GL, Chang XB, Kartner N, Rotstein OD, Riordan JR, and Grinstein S. The cystic fibrosis transmembrane regulator is present and functional in endosomes. Role as a determinant of endosomal pH. J Biol Chem 267: 14568-14572, 1992[Abstract/Free Full Text].

34.   Miesenbock, G, De Angelis DA, and Rothman JE. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394: 192-195, 1998[ISI][Medline].

35.   Nordeen, M, Jones S, Howell K, and Caldwell J. GOLAC: an endogenous anion channel of the Golgi complex. Biophys J 78: 2918-2928, 2000[Abstract/Free Full Text].

36.   Ostrander, GK, and Holmes EH. Characterization of a CMPNeuAc: lactosylceramide alpha2----3sialyltransferase from rainbow trout hepatoma (RTH-149) cells. Comp Biochem Physiol B 98: 87-95, 1991[ISI][Medline].

37.   Paula, S, Volkov AG, Van Hoek AN, Haines TH, and Deamer DW. Permeation of protons, potassium ions, and small polar molecules through phospholipid bilayers as a function of membrane thickness. Biophys J 70: 339-348, 1996[Abstract].

38.   Press, WH. Numerical recipes in C: the art of scientific computing. Cambridge, UK: Cambridge Univ. Press, 1997.

39.   Reddy, MM, Light MJ, and Quinton PM. Activation of the epithelial Na+ channel (ENaC) requires CFTR Cl- channel function. Nature 402: 301-304, 1999[ISI][Medline].

40.   Rivers, R, Blanchard A, Eladari D, Leviel F, Paillard M, Podevin RA, and Zeidel ML. Water and solute permeabilities of medullary thick ascending limb apical and basolateral membranes. Am J Physiol Renal Physiol 274: F453-F462, 1998[Abstract/Free Full Text].

41.   Roos, A, and Boron WF. Intracellular pH. Physiol Rev 61: 296-434, 1981[Free Full Text].

42.   Schapiro, F, Sparkowski J, Adduci A, Suprynowicz F, Schlegel R, and Grinstein S. Golgi alkalinization by the papillomavirus E5 oncoprotein. J Cell Biol 148: 305-315, 2000[Abstract/Free Full Text].

43.   Schapiro, FB, and Grinstein S. Determinants of the pH of the Golgi complex. J Biol Chem 275: 21025-21032, 2000[Abstract/Free Full Text].

44.   Schapiro, FB, Lingwood C, Furuya W, and Grinstein S. pH-independent retrograde targeting of glycolipids to the Golgi complex. Am J Physiol Cell Physiol 274: C319-C332, 1998[Abstract/Free Full Text].

45.   Schroeder, TH, Zaidi T, and Pier GB. Lack of adherence of clinical isolates of Pseudomonas aeruginosa to Asialo-GM(1) on epithelial cells. Infect Immun 69: 719-729, 2001[Abstract/Free Full Text].

46.   Schwappach, B, Stobrawa S, Hechenberger M, Steinmeyer K, and Jentsch T. Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p. J Biol Chem 273: 15110-15118, 1998[Abstract/Free Full Text].

47.   Seed, B, and Aruffo A. Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc Natl Acad Sci USA 84: 3365-3369, 1987[Abstract].

48.   Seksek, O, Biwersi J, and Verkman AS. Direct measurement of trans-Golgi pH in living cells and regulation by second messengers. J Biol Chem 270: 4967-4970, 1995[Abstract/Free Full Text].

49.   Seksek, O, Biwersi J, and Verkman AS. Evidence against defective trans-Golgi acidification in cystic fibrosis. J Biol Chem 271: 15542-15548, 1996[Abstract/Free Full Text].

50.   Stutts, MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC, and Boucher RC. CFTR as a cAMP-dependent regulator of sodium channels. Science 269: 847-850, 1995[ISI][Medline].

51.   Taga, R, Alvares EP, and Sesso A. Morphometric studies on terminal tubule and acinar cells in developing submandibular gland of the rat. Arch Histol Cytol 56: 517-523, 1993[ISI][Medline].

52.   Tang, BL, Low SH, Wong SH, and Hong W. Cell type differences in Golgi retention signals for transmembrane proteins. Eur J Cell Biol 66: 365-374, 1995[ISI][Medline].

53.   Teruel, MN, Blanpied TA, Shen K, Augustine GJ, and Meyer T. A versatile microporation technique for the transfection of cultured CNS neurons. J Neurosci Methods 93: 37-48, 1999[ISI][Medline].

54.   Teter, K, Chandy G, Quinones B, Pereyra K, Machen T, and Moore HP. Cellubrevin-targeted fluorescence uncovers heterogeneity in the recycling endosomes. J Biol Chem 273: 19625-19633, 1998[Abstract/Free Full Text].

55.   Tsien, RY. The green fluorescent protein. Annu Rev Biochem 67: 509-544, 1998[ISI][Medline].

56.   Van Dyke, R, and Belcher J. Acidification of three types of liver endocytic vesicles: similarities and differences. Am J Physiol Cell Physiol 266: C81-C94, 1994[Abstract/Free Full Text].

57.   Wachter, RM, and Remington SJ. Sensitivity of the yellow variant of green fluorescent protein to halides and nitrate. Curr Biol 9: R628-R629, 1999[ISI][Medline].

58.   Weintraub, WH, and Machen TE. pH regulation in hepatoma cells: roles for Na-H exchange, Cl-HCO3 exchange, and Na-HCO3 cotransport. Am J Physiol Gastrointest Liver Physiol 257: G317-G327, 1989[Abstract/Free Full Text].

59.   Wenzl, E, and Machen TE. Intracellular pH dependence of buffer capacity and anion exchange in the parietal cell. Am J Physiol Gastrointest Liver Physiol 257: G741-G747, 1989[Abstract/Free Full Text].

60.   Wong, SH, Low SH, and Hong W. The 17-residue transmembrane domain of beta-galactoside alpha 2,6-sialyltransferase is sufficient for Golgi retention. J Cell Biol 117: 245-258, 1992[Abstract].

61.   Wu, MM, Llopis J, Adams S, McCaffery JM, Kulomaa MS, Machen TE, Moore HPH, and Tsien RY. Organelle pH studies using targeted avidin and fluorescein-biotin. Chem Biol 7: 197-209, 2000[ISI][Medline].

62.   Yankaskas, JR, Haizlip JE, Conrad M, Koval D, Lazarowski E, Paradiso AM, Rinehart CA, Jr, Sarkadi B, Schlegel R, and Boucher RC. Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype. Am J Physiol Cell Physiol 264: C1219-C1230, 1993[Abstract/Free Full Text].

63.   Zar, H, Saiman L, Quittell L, and Prince A. Binding of Pseudomonas aeruginosa to respiratory epithelial cells from patients with various mutations in the cystic fibrosis transmembrane regulator. J Pediatr 126: 230-233, 1995[ISI][Medline].


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