N.D. Sonawane

The “proton sponge hypothesis” postulates enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H+ buffering polyamines by enhanced endosomal Cl- accumulation and osmotic swelling/lysis. To test this hypothesis, we measured endosomal Cl- concentration, pH, and volume after internalization of polyplexes composed of plasmid DNA and polylysine (POL), a non-buffering polyamine, or the strongly buffering polyamines polyethylenimine (PEI) or polyamidoamine (PAM). [Cl-] and pH were measured by ratio imaging of fluorescently labeled polyplexes containing Cl- or pH indicators. [Cl-] increased from 41 to 80 mm over 60 min in endosomes-contained POL-polyplexes, whereas pH decreased from 6.8 to 5.3. Endosomal Cl- accumulation was enhanced (115 mm at 60 min) and acidification was slowed (pH 5.9 at 60 min) for PEI and PAM-polyplexes. Relative endosome volume increased 20% over 75 min for POL-polyplexes versus 140% for PEI-polyplexes. Endosome lysis was seen at >45 min for PEI but not POL-containing endosomes, and PEI-containing endosomes showed increased osmotic fragility in vitro. The slowed endosomal acidification and enhanced Cl- accumulation and swelling/lysis were accounted for by the greater H+ buffering capacity of endosomes containing PEI or PAM versus POL (>90 mm versus 46 H+/pH unit). Our results provide direct support for the proton sponge hypothesis and thus a rational basis for the design of improved non-viral vectors for gene delivery. The “proton sponge hypothesis” postulates enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H+ buffering polyamines by enhanced endosomal Cl- accumulation and osmotic swelling/lysis. To test this hypothesis, we measured endosomal Cl- concentration, pH, and volume after internalization of polyplexes composed of plasmid DNA and polylysine (POL), a non-buffering polyamine, or the strongly buffering polyamines polyethylenimine (PEI) or polyamidoamine (PAM). [Cl-] and pH were measured by ratio imaging of fluorescently labeled polyplexes containing Cl- or pH indicators. [Cl-] increased from 41 to 80 mm over 60 min in endosomes-contained POL-polyplexes, whereas pH decreased from 6.8 to 5.3. Endosomal Cl- accumulation was enhanced (115 mm at 60 min) and acidification was slowed (pH 5.9 at 60 min) for PEI and PAM-polyplexes. Relative endosome volume increased 20% over 75 min for POL-polyplexes versus 140% for PEI-polyplexes. Endosome lysis was seen at >45 min for PEI but not POL-containing endosomes, and PEI-containing endosomes showed increased osmotic fragility in vitro. The slowed endosomal acidification and enhanced Cl- accumulation and swelling/lysis were accounted for by the greater H+ buffering capacity of endosomes containing PEI or PAM versus POL (>90 mm versus 46 H+/pH unit). Our results provide direct support for the proton sponge hypothesis and thus a rational basis for the design of improved non-viral vectors for gene delivery. Although gene delivery using non-viral vectors offers potential advantages over virus-based delivery systems, the relatively low transfection efficiency of non-viral vectors has been their major limitation for in vivo applications (1Midoux P. Monsigny M. Bioconjugate Chem. 1999; 10: 406-411Crossref PubMed Scopus (483) Google Scholar, 2Pack D.W. Putnam D. Langer R. Biotechnol. Bioeng. Symp. 2000; 67: 217-223Crossref Scopus (259) Google Scholar, 3Putnam D. Gentry C.A. Pack D.W. Langer R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1200-1205Crossref PubMed Scopus (466) Google Scholar, 4Abdallah B. Sachs L. Demeneix B.A. Biol. Cell. 1995; 85: 1-7Crossref PubMed Google Scholar). The archetypal non-viral gene delivery system is the cationic polymer-DNA complex (polyplex), 1The abbreviations used are: polyplex, cationic polymer-DNA complex; POL, polylysine; PAM, polyamidoamine; PEI, polyethylenimine; PBS, phosphate-buffered saline; TMR, tetramethylrhodamine; TMR-POL, TMR-labeled polylysine; TMR-PAM, TMR-labeled PAM; TMR-PEI, TMR-labeled PEI; BAC, 10,10′-bis[3-carboxypropyl]-9,9′-biacridinium dinitrate; BAC-dextran-S-S-2Py, BAC-dextran carrying dithian-2-pyridyl linker; FITC, fluorescein isothiocyanate; FITC-PAM-TMR, PAM labeled with FITC and TMR; FITC-PEI-TMR, PEI labeled with FITC and TMR; FITC-POL-TMR, POL labeled with FITC and TMR; N/P, amine-to-phosphate; GFP, green fluorescent protein; CHO, Chinese hamster ovary; BAC-dextran-PAM-TMR, tetramethylrhodamine-labeled polyamidoamine conjugated to BAC-labeled dextran; BAC-dextran-PEI-TMR, tetramethylrhodamine-labeled polyethylenimine conjugated with BAC-labeled dextran; BAC-dextran-POL-TMR, tetramethylrhodamine-labeled polylysine conjugated with BAC-labeled dextran.1The abbreviations used are: polyplex, cationic polymer-DNA complex; POL, polylysine; PAM, polyamidoamine; PEI, polyethylenimine; PBS, phosphate-buffered saline; TMR, tetramethylrhodamine; TMR-POL, TMR-labeled polylysine; TMR-PAM, TMR-labeled PAM; TMR-PEI, TMR-labeled PEI; BAC, 10,10′-bis[3-carboxypropyl]-9,9′-biacridinium dinitrate; BAC-dextran-S-S-2Py, BAC-dextran carrying dithian-2-pyridyl linker; FITC, fluorescein isothiocyanate; FITC-PAM-TMR, PAM labeled with FITC and TMR; FITC-PEI-TMR, PEI labeled with FITC and TMR; FITC-POL-TMR, POL labeled with FITC and TMR; N/P, amine-to-phosphate; GFP, green fluorescent protein; CHO, Chinese hamster ovary; BAC-dextran-PAM-TMR, tetramethylrhodamine-labeled polyamidoamine conjugated to BAC-labeled dextran; BAC-dextran-PEI-TMR, tetramethylrhodamine-labeled polyethylenimine conjugated with BAC-labeled dextran; BAC-dextran-POL-TMR, tetramethylrhodamine-labeled polylysine conjugated with BAC-labeled dextran. in which plasmid DNA and a cationic carrier are condensed into a tight complex suitable for cellular internalization by endocytosis (5Ogris M. Wagner E. Drug Discov. Today. 2002; 7: 479-485Crossref PubMed Scopus (153) Google Scholar, 6Thomas M. Klibanov A.M. Appl. Microbiol. Biotechnol. 2003; 62: 27-34Crossref PubMed Scopus (439) Google Scholar). Transgene delivery to the nucleus is thought to require escape of the polyplex from endosomes, DNA/polymer dissociation, cytoplasmic DNA diffusion, and nuclear uptake (5Ogris M. Wagner E. Drug Discov. Today. 2002; 7: 479-485Crossref PubMed Scopus (153) Google Scholar, 7Xu Y. Szoka F.C. Biochemistry. 1996; 35: 5616-5623Crossref PubMed Scopus (1068) Google Scholar, 8Zuber G. Dauty E. Nothisen M. Belguise P. Behr J.P. Adv. Drug Deliv. Rev. 2001; 52: 245-253Crossref PubMed Scopus (201) Google Scholar). The low efficiency of polyplex escape from endosomes is thought to be an important determinant of the overall efficiency of non-viral gene transfer. Polyamines are useful polycationic macromolecules for non-viral gene transfer because of their high density of positive charges, ease of synthesis, and efficient polyplex formation (8Zuber G. Dauty E. Nothisen M. Belguise P. Behr J.P. Adv. Drug Deliv. Rev. 2001; 52: 245-253Crossref PubMed Scopus (201) Google Scholar, 9Rolland A. Felgner P.L. Adv. Drug Deliv. Rev. 1998; 30: 1-3Crossref PubMed Scopus (24) Google Scholar, 10Luo D. Saltzman W.M. Nat. Biotechnol. 2000; 18: 33-37Crossref PubMed Scopus (370) Google Scholar). Cationic polyamines with fixed, non-titratable charges such as polylysine (POL) are substantially less efficient at gene transfer than polyamines with titratable amines such as polyamidoamine (PAM) (11Haensler J. Szoka F.C. Bioconjugate Chem. 1993; 4: 372-379Crossref PubMed Scopus (1181) Google Scholar) and polyethylenimine (PEI) (12Boussif O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301Crossref PubMed Scopus (5631) Google Scholar, 13Kichler A. Leborgne C. Coeytaux E. Danos O. J. Gene Med. 2001; 3: 135-144Crossref PubMed Scopus (468) Google Scholar). The lower efficiency is not caused by differences in morphology of the complexes or cell association (14Tang M.X. Szoka Jr., F.C. Gene Ther. 1997; 4: 823-832Crossref PubMed Scopus (808) Google Scholar). To explain this observation it has been postulated without direct evidence that the high H+ buffer capacity of polyamines containing titratable amines results in endosomal Cl- accumulation during acidification with presumed osmotic endosome swelling and enhanced polyplex escape (11Haensler J. Szoka F.C. Bioconjugate Chem. 1993; 4: 372-379Crossref PubMed Scopus (1181) Google Scholar, 15Behr J.P. Bioconjugate Chem. 1994; 5: 382-389Crossref PubMed Scopus (395) Google Scholar). This “proton sponge hypothesis” if correct, would have important consequences in defining the barriers to non-viral gene delivery and in the design of improved non-viral vectors. Here, we test the proton sponge hypothesis by comparing the kinetics of acidification, Cl- accumulation, and swelling in endosomes containing polyplexes composed of DNA and fluorescent probe-labeled polyamines (POL, PEI, or PAM). We previously developed long-wavelength, Cl--sensing fluorescent probes for the measurement of [Cl-] in cellular endosomal and Golgi compartments and showed that inward H+ pumping by the bafilomycin-sensitive vacuolar ATPase was coupled quantitatively to Cl- entry in endosomes produced by fluid-phase (16Sonawane N.D. Thiagarajah J.R. Verkman A.S. J. Biol. Chem. 2002; 277: 5506-5513Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) or receptor-mediated (17Sonawane N.D. Verkman A.S. J. Cell Biol. 2003; 160: 1129-1138Crossref PubMed Scopus (63) Google Scholar) endocytosis. We find here that “macropinocytic” endosomes containing DNA polyplexes with titratable amines have remarkably increased buffer capacity, reduced acidification, increased Cl- accumulation, and increased swelling/lysis compared with endosomes containing polyplexes with fixed charges, providing direct evidence in support of the proton sponge hypothesis. Cell Culture—Chinese hamster ovary (CHO)-K1 cells (American Type Culture Collection, Manassas, VA) were grown in Ham's F12K medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured on 18-mm diameter round coverglasses at 37 °C in 95% air/5% CO2 and used just prior to confluence. Fluorescently Labeled Polyamine Conjugates for [Cl-] Measurements—The polymers POL (Aldrich, 25 kDa), PAM (43 kDa, 6th generation fractured dendrimer) (18Tang M. Redemann C.T. Szoka Jr., F.C. Bioconjugate Chem. 1996; 7: 703-714Crossref PubMed Scopus (855) Google Scholar), and PEI (Aldrich, 25 kDa) were purified and characterized as described previously (14Tang M.X. Szoka Jr., F.C. Gene Ther. 1997; 4: 823-832Crossref PubMed Scopus (808) Google Scholar). An aqueous solution of polyamine (POL, PAM, or PEI) (5 μmol in 5 ml of PBS) was stirred slowly with tetramethylrhodamine succinimidyl ester (15 μmol, from Me2SO stock) at room temperature for 1 h (Fig. 1A). Unreacted dye was removed by gel filtration (Sephadex G25, PBS). Using molar absorbance data, TMR/polyamine mol ratios were 3.3:1 (TMR-POL), TMR/polyamidoamine mol ratios were 2.8:1 (TMR-PAM), and TMR/polyethylenimine mol ratios were 4.1:1 (TMR-PEI). Equal volumes of each TMR-polyamine (5 μm in degassed PBS) and iminothiolane (13 μm in PBS containing 5 mm EDTA, pH 8) were incubated for 1 h at room temperature in the dark under N2. Unreacted iminothiolane was removed by gel filtration. Sulfhydryl (SH) group/ligand mol labeling ratios were 0.96:1 (for TMR-POL), 0.93:1 (for TMR-PAM), and 0.95:1 (for TMR-PEI). TMR-polyamine-SH (5 μm) was then incubated with BAC-dextran-S-S-2Py (2 μm in 4 ml of PBS containing 5 mm EDTA, pH 8, synthesized as described in Ref. 16Sonawane N.D. Thiagarajah J.R. Verkman A.S. J. Biol. Chem. 2002; 277: 5506-5513Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) for 18 h at room temperature under N2. Unreacted TMR-polyamine-SH and BAC-dextran-S-S-2Py were removed by gel filtration, and the product was lyophilized and stored at -20 °C. Molecular masses of the conjugates (before and after cell internalization) were determined by column chromatography (Sephacryl 300HR). Eluted fractions were assayed for BAC and TMR fluorescence. In fluorescence-quenching studies, microliter aliquots of NaCl (1 m stock) were added to 3 ml of fluorescent ligand (10 μM, pH 7.4). Stern-Volmer constants (K sv) were calculated from the slope of F0/F - 1 versus [Cl-] plots (F0/F - 1 = K sv [Cl-]), where F0 is BAC fluorescence in absence and F is the presence of Cl-. Fluorescently Labeled Polyamines for pH Measurements—TMR-POL, TMR-PAM, and TMR-PEI (5 μmol) prepared as above were gently stirred for 1 h with fluorescein isothiocyanate (35 μmol from Me2SO stock) in 10 ml of aqueous NaHCO3 (pH 8). Reaction products (FITC-POL-TMR, FITC-PAM-TMR, and FITC-PEI-TMR) were purified by gel filtration chromatography. Thin layer chromatography showed no free dye contamination. FITC/polymer molar labeling ratios were 5.5, 6.0, and 6.8 for POL, PAM, and PEI, respectively. Polyamine-DNA Polyplexes—DNA complexes containing Cl- or pH-sensing fluorescent polyamines (or non-fluorescent polyamines) were prepared by mixing plasmid DNA (pCDNA3 encoding GFP, 3.8 kb; 80 μg/ml in 10 mm HEPES, 10 mm NaCl, 10% glucose) and polyamine (in distilled water) at specified primary amine/phosphate (N/P) ratios (2.2, 5.5, and 6.0 for PEI, PAM, and POL, respectively). Polyplexes were dispersed in PBS for endosomal uptake. For measurements of transfection efficiency, CHO cells were incubated with polyplexes at different N/P in 96-well plates for 30 min in serum-free medium and then for 36 h in serum-containing medium. GFP fluorescence was quantified by a fluorescence plate reader. Endosome Labeling, [Cl-], and pH Measurements—Cells were incubated in serum-free medium for 15 min at 37 °C and then pulse-labeled by incubation with DNA polyplex (50 μg/ml) in PBS for 5 min at 4 °C. Coverslips were washed twice at 4 °C with PBS containing dextran sulfate (10 kDa, 1 mg/ml) and washed once in PBS, then transferred to a pre-cooled perfusion chamber. Sets of BAC and TMR images (for Cl-) or FITC and TMR images (for pH) were acquired at specified times after perfusion with PBS at 37 °C. In some experiments, the perfusate contained NH4Cl (5-40 mm) or chloroquine (100 μm). For in vivo calibration of BAC/TMR fluorescence ratio versus [Cl-], perfusate and endosomal [Cl-] were equalized by the incubation of cells for 15-20 min at 37 °Cin 120 mm KCl/KNO3 (0-120 mm Cl-), 20 mm NaCl/NaNO3, 1 mm CaCl2, 1 mm MgCl2, and 10 mm HEPES (pH 7.4) containing the ionophores nigericin, valinomycin, carbonyl cyanide p-chlorophenylhydrazone, monensin (all 10 μm), and bafilomycin (200 nm). For calibrations of TMR/FITC fluorescence ratio versus pH, cells were incubated with high K+ solutions containing nigericin, valinomycin, and bafilomycin (pH adjusted to 4-8). Endosomal Buffer Capacity—Buffer capacity (β) was determined from the rapid increase in endosomal pH in response to the addition of 5-40 mm NH4Cl to the perfusate: β = ([NH4Cl]/ΔpH)·10(pHout-pHfinal), where ΔpH is the pH increase just after NH4Cl addition, pHout is perfusate pH, and pHfinal is endosomal pH after the addition of NH4Cl. Perfusate [NH4Cl] was chosen to increase endosome pH from ∼6.1 to 6.7. Fluorescence Microscopy—Cells were imaged using a Leitz upright epifluorescence microscope equipped with Nipkow-wheel confocal attachment and a 14-bit cooled (-30 °C) charge-coupled device camera (19Zen K. Biwersi J. Periasamy N. Verkman A.S. J. Cell Biol. 1992; PubMed Scopus Google Scholar). Fluorescence was using a = a BAC = 5 = = 20 and TMR and FITC was using to (16Sonawane N.D. Thiagarajah J.R. Verkman A.S. J. Biol. Chem. 2002; 277: 5506-5513Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Endosome volumes were determined from of endosomes In Endosome 30 or min after internalization of polyplex and 37 °C cells were in mm K 10 mm NaCl, 1 mm and MgCl2, 15 mm HEPES pH low for 10 the containing endosomes was added to of to an osmotic The of polyplex was determined by from the versus increase in FITC fluorescence after the addition of 1 m stock) to increase pH to of polyamines PEI and PAM titratable and amines in the pH whereas POL not (Fig. 1A). PEI, POL, and PAM were labeled with TMR and conjugated with BAC-dextran using a (Fig. BAC fluorescence is green and whereas TMR fluorescence is and the absence of DNA gel in at the ratios used to the was an of fluorescent polymers the (Fig. fluorescent as is in transfection filtration chromatography of polyamines and after cellular internalization of Cl- showed at masses of and for PAM, PEI, and POL, The BAC and TMR fluorescence and no fluorescence was seen at lower of the fluorescent polyamine conjugates in Fluorescence of polyplexes showed at and and at for for TMR, was at and was at BAC fluorescence in the polyplexes was by Cl- by a with a Stern-Volmer of 36 whereas TMR fluorescence was not BAC fluorescence was not by polyamine and DNA of the polyplexes were measured to N/P for Cells were incubated for 30 min at 37 °C with polyplexes containing or fluorescently labeled efficiency, measured as cellular GFP fluorescence at 36 was times for PAM versus POL polyplexes (Fig. transfection and N/P were for versus fluorescent polyamines and the transfection are with results (12Boussif O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301Crossref PubMed Scopus (5631) Google Scholar, M.X. Szoka Jr., F.C. Gene Ther. 1997; 4: 823-832Crossref PubMed Scopus (808) Google Scholar). Cl- and pH in fluorescence of cells labeled m with polyplexes containing and The cell was after incubation for 5 min at 4 °C labeled at which the green BAC fluorescence was relatively because of the high Polyplexes were to 37 °C. labeled with polyplexes containing PAM and PEI, but not POL, substantially over of fluorescence versus [Cl-] were by cells with endosomes with a high K+ buffer containing ionophores and versus [Cl-] for in aqueous solution and in cells with a Stern-Volmer of 37 constants of were determined for polyplexes containing and The versus [Cl-] calibration of [Cl-] in endosomes by ratio as in the in which was for images at min after calibration of fluorescence versus pH for the pH-sensing polyamine was in cells using high K+ buffer containing ionophores and bafilomycin (Fig. versus pH was in cells and solution a results were for and pH in endosomes at min after at 4 °C and rapid to 37 [Cl-] in endosomes containing PAM polyplexes increased from to mm during the and then decreased to mm at 75 min (Fig. the in endosomal [Cl-] results from of a of [Cl-] kinetics were measured in endosomes containing PEI containing POL polyplexes less Cl- mm at Endosomal acidification was measured in (Fig. pH in endosomes containing PAM and PEI polyplexes decreased slowly from to over 60 to at 75 of endosomes containing POL polyplexes was relatively with pH to 5.9 in 15 Cl- accumulation and acidification in endosomes containing each of the polyplexes We postulated that the increased Cl- accumulation and slowed acidification in endosomes containing PAM or PEI versus POL polyplexes was caused by their greater H+ buffer capacity Using the β (in the pH was and for endosomes containing PAM and PEI which was greater than that of 46 for endosomes containing POL polyplexes (Fig. The increased buffer capacity in endosomes containing PAM or PEI versus POL polyplexes for the increased Cl- accumulation 80 versus mm over 60 min) and slowed acidification versus 1 over 15 and of greater volume of endosomes compared with endosomes by fluid-phase or receptor-mediated of their volume by that the volume of fluorescent endosomes containing PAM polyplexes increased with after with a increase for POL The chloroquine increased volume and Cl- accumulation in endosomes containing POL polyplexes (Fig. and providing evidence Cl- accumulation and To endosome swelling is with the kinetics of pH in endosomes was that endosome or lysis would in endosome or pH in of endosomes containing PAM polyplexes at min after some PAM endosomes as as 30 min after as a of lysis and polyamine (Fig. In under the no POL endosomes or strongly (Fig. that endosomes containing PAM or PEI polyplexes be to osmotic lysis than endosomes containing POL Cell containing labeled endosomes, prepared at min after cellular internalization of fluorescent were to osmotic and the of polyamine was from the versus fluorescence increase after addition (Fig. remarkably greater in endosomes containing PAM versus POL polyplexes after osmotic as The here were to test the proton sponge hypothesis, which postulates that enhanced transgene delivery by polyplexes containing titratable amines is caused by increased endosomal Cl- accumulation and swelling/lysis. Cl- accumulation was to be remarkably greater in endosomes containing PEI and PAM versus POL was swelling was and buffer capacity was and lysis were seen at min in PAM and PEI but not POL endosomes, and osmotic fragility was POL endosomes be to PAM endosomes by in the solution of a that in We that increased polyplex buffer capacity results in increased endosomal Cl- entry because of osmotic swelling and endosome results provide direct evidence in support of the proton sponge hypothesis. Our fluorescence labeling of Cl- and to polyamines for with plasmid of of the BAC direct to the Cl- of BAC-labeled dextran with TMR-labeled of fluorescent polyplexes in in and in vivo Cl- and pH absence of and transfection of fluorescent and containing DNA polyplexes are remarkably greater in than endosomes relatively fluid-phase such as fluorescent or high such as and M. Klibanov A.M. Appl. Microbiol. Biotechnol. 2003; 62: 27-34Crossref PubMed Scopus (439) Google Scholar, Rev. Cell Biol. 1996; PubMed Scopus Google Scholar). of DNA polyplexes into endosomes Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar) and thus to be a different than fluid-phase or receptor-mediated endocytosis. gene transfer in cell a of the cationic such that with We as for free was and that endosomes containing free acidification kinetics and accumulation as endosomes containing DNA polyplexes is not the and of endosomes are different from of endosomes by fluid-phase or receptor-mediated results that their endosomes a H+ ATPase and Cl- Our of PAM and PEI polyplexes gene transfer in cultured cells (11Haensler J. Szoka F.C. Bioconjugate Chem. 1993; 4: 372-379Crossref PubMed Scopus (1181) Google Scholar, O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301Crossref PubMed Scopus (5631) Google Scholar). A. Leborgne C. Coeytaux E. Danos O. J. Gene Med. 2001; 3: 135-144Crossref PubMed Scopus (468) Google Scholar) showed that of cells to PEI polyplexes with bafilomycin in a in gene that the transfection efficiency of PEI is because of proton during endosomal acidification and that gene transfer 4 h of polyplex the of endosomal acidification and the proton sponge hypothesis have been M.A. P. J. 2000; PubMed Scopus Google Scholar) that gene delivery not by of the endosomal and S. A. J. 2002; PubMed Scopus Google Scholar) that gene transfer from the and not the The in the measurements here to in a that 75 min of to the are with the of gene because gene is 4 h after cell to DNA polyplexes and at h after A. Leborgne C. Coeytaux E. Danos O. J. Gene Med. 2001; 3: 135-144Crossref PubMed Scopus (468) Google Scholar). Cl- and acidification in the 60 min of uptake and are with endosomal The of fluorescence from but not POL-containing endosomes that the endosome has in polyplex into the because DNA into the not from the of D. L. M. D. R. J. C. N. J. Cell Biol. 2000; PubMed Scopus Google Scholar, D. Gene Ther. 2002; PubMed Scopus Google Scholar), the of fluorescence that the fluorescent has from the DNA and the it that a polymer-DNA complex is from the of into the nucleus J. D. J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100 PubMed Scopus Google Scholar). Our results provide the direct that endosomal pH, and endosomes that are to osmotic The that the fluorescence has an for the design of non-viral vectors for efficient transgene the to into the polyplex to the transfer of the DNA into the

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated epithelial Cl- channel that, when defective, causes cystic fibrosis. Screening of a collection of 100,000 diverse small molecules revealed four novel chemical classes of CFTR inhibitors with Ki < 10 microM, one of which (glycine hydrazides) had many active structural analogues. Analysis of a series of synthesized glycine hydrazide analogues revealed maximal inhibitory potency for N-(2-naphthalenyl) and 3,5-dibromo-2,4-dihydroxyphenyl substituents. The compound N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101) reversibly inhibited CFTR Cl- conductance in <1 min. Whole-cell current measurements revealed voltage-dependent CFTR block by GlyH-101 with strong inward rectification, producing an increase in apparent inhibitory constant Ki from 1.4 microM at +60 mV to 5.6 microM at -60 mV. Apparent potency was reduced by lowering extracellular Cl- concentration. Patch-clamp experiments indicated fast channel closures within bursts of channel openings, reducing mean channel open time from 264 to 13 ms (-60 mV holding potential, 5 microM GlyH-101). GlyH-101 inhibitory potency was independent of pH from 6.5-8.0, where it exists predominantly as a monovalent anion with solubility approximately 1 mM in water. Topical GlyH-101 (10 microM) in mice rapidly and reversibly inhibited forskolin-induced hyperpolarization in nasal potential differences. In a closed-loop model of cholera, intraluminal GlyH-101 (2.5 microg) reduced by approximately 80% cholera toxin-induced intestinal fluid secretion. Compared with the thiazolidinone CFTR inhibitor CFTR(inh)-172, GlyH-101 has substantially greater water solubility and rapidity of action, and a novel inhibition mechanism involving occlusion near the external pore entrance. Glycine hydrazides may be useful as probes of CFTR pore structure, in creating animal models of CF, and as antidiarrheals in enterotoxic-mediated secretory diarrheas.

A novel long wavelength fluorescent Cl− indicator was used to test whether endosomal Cl− conductance provides the principal electrical shunt to permit endosomal acidification. The green fluorescent Cl−-sensitive chromophore 10,10′-bis[3-carboxypropyl]-9,9′-biacridinium dinitrate (BAC) was conjugated to aminodextran together with the red fluorescent Cl−-insensitive chromophore tetramethylrhodamine (TMR). BAC fluorescence is pH-insensitive and quenched by Cl−with a Stern-Volmer constant of 36 m−1. Endosomes in J774 and Chinese hamster ovary (CHO) cells were pulse-labeled with BAC-TMR-dextran by fluid-phase endocytosis. Endosomal [Cl−] increased over 45 min from 17 to 53 mm in J774 cells and from 28 to 73 mm in CHO cells, during which time endosomal pH decreased from 6.95 to 5.30 (J774) and 6.92 to 5.60 (CHO). The acidification and increased [Cl−] were blocked by bafilomycin. Together with ion substitution and buffer capacity measurements, we conclude that Cl− transport accounts quantitatively for the electrical shunt during vacuolar acidification. Measurements of relative endosomal volume by a novel ratio imaging method involving fluorescence self-quenching indicated a 2.5-fold increase in volume during early acidification and Cl− accumulation, which was blocked by bafilomycin. These experiments provide the first direct measurement of endosomal [Cl−] and indicate that endosomal acidification is accompanied by significant Cl− entry and volume increase. A novel long wavelength fluorescent Cl− indicator was used to test whether endosomal Cl− conductance provides the principal electrical shunt to permit endosomal acidification. The green fluorescent Cl−-sensitive chromophore 10,10′-bis[3-carboxypropyl]-9,9′-biacridinium dinitrate (BAC) was conjugated to aminodextran together with the red fluorescent Cl−-insensitive chromophore tetramethylrhodamine (TMR). BAC fluorescence is pH-insensitive and quenched by Cl−with a Stern-Volmer constant of 36 m−1. Endosomes in J774 and Chinese hamster ovary (CHO) cells were pulse-labeled with BAC-TMR-dextran by fluid-phase endocytosis. Endosomal [Cl−] increased over 45 min from 17 to 53 mm in J774 cells and from 28 to 73 mm in CHO cells, during which time endosomal pH decreased from 6.95 to 5.30 (J774) and 6.92 to 5.60 (CHO). The acidification and increased [Cl−] were blocked by bafilomycin. Together with ion substitution and buffer capacity measurements, we conclude that Cl− transport accounts quantitatively for the electrical shunt during vacuolar acidification. Measurements of relative endosomal volume by a novel ratio imaging method involving fluorescence self-quenching indicated a 2.5-fold increase in volume during early acidification and Cl− accumulation, which was blocked by bafilomycin. These experiments provide the first direct measurement of endosomal [Cl−] and indicate that endosomal acidification is accompanied by significant Cl− entry and volume increase. Progressive acidification of vesicles in the endosomal pathway is important for receptor and ligand sorting and vesicular fusion (1Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1338) Google Scholar, 2Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. 1997; 77: 759-803Crossref PubMed Scopus (1310) Google Scholar). Endosomal acidification is driven by a vacuolar-type H+pump that is present in the endosomal-limiting membrane. To maintain electroneutrality, H+ entry into the endosomal aqueous lumen must be accompanied by anion entry and/or cation exit. The principal transportable intracellular anion is Cl− and cation is K+. Studies of organellar pH in living cells and isolated vesicles have provided evidence that Cl− entry may be the rate-limiting passive conductance in permitting active H+ entry in endosomes (3Van Dyke R.W. J. Biol. Chem. 1988; 263: 2603-2611Abstract Full Text PDF PubMed Google Scholar, 4Barasch J. Gershon M.D. Nunez E.A. Tamir H. Al-Awqati Q. J. Cell Biol. 1988; 1070: 2137-2147Crossref Scopus (71) Google Scholar, 5Schmid A. Burckhardt G. Gogelein H. J. Membr. Biol. 1989; 111: 265-275Crossref PubMed Scopus (35) Google Scholar, 6Blair H.C. Teitelbaum S.L. Tan H.L. Koziol C.M. Schlesinger P.H. Am. J. Physiol. 1991; 260: C1315-C1324Crossref PubMed Google Scholar, 7Forgac M. J. Bioenerg. Biomembr. 1999; 31: 57-65Crossref PubMed Scopus (38) Google Scholar, 8Barasch J. Kiss B. Prince A. Saiman L. Gruenert D. Al-Awqati Q. Nature. 1991; 352: 70-73Crossref PubMed Scopus (425) Google Scholar, 9Al-Awqati Q. Curr. Opin. Cell Biol. 1995; 7: 504-508Crossref PubMed Scopus (96) Google Scholar). In isolated endocytic vesicles from kidney proximal tubule (10Bae H.R. Verkman A.S. Nature. 1990; 348: 637-639Crossref PubMed Scopus (103) Google Scholar) and liposomes reconstituted with proteins from clathrin-coated vesicles (11Mulberg A.E. Tulk B.M. Forgac M. J. Biol. Chem. 1991; 266: 20590-20593Abstract Full Text PDF PubMed Google Scholar), a protein kinase A-activated Cl− conductance was characterized, and it was proposed that activation of Cl− channels might regulate endosomal acidification by providing a shunt to dissipate the interior-positive potential produce by the H+ pump. There is also evidence that Na+/K+ pump activity in early endosomes may alter the driving force for H+ entry and thus regulate acidification (12Cain C.C. Sipe D.M. Murphy R.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 544-548Crossref PubMed Scopus (169) Google Scholar, 13Fuchs R. Male P. Mellman I. J. Biol. Chem. 1989; 264: 2212-2220Abstract Full Text PDF PubMed Google Scholar, 14Fuchs R. Schmid S. Mellman I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 539-543Crossref PubMed Scopus (161) Google Scholar). In Golgi, there is evidence that both Cl− and K+ conductances may contribute to acidification (15Glickman J. Croen K. Kelly S. Al-Awqati Q. J. Cell Biol. 1983; 97: 1303-1308Crossref PubMed Scopus (187) Google Scholar, 16Schapiro F.B. Grinstein S. J. Biol. Chem. 2000; 275: 21025-21032Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 17Wu M.M. Grabe M. Adams S. Tsien R.Y. Moore H.P. Machen T.E. J. Biol. Chem. 2001; 276: 33027-33035Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), whereas acidification of secretory granules in synaptic vesicles appears to require the expression of a specific Cl− channel (18Stobrawa S.M. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A.A. Bosl M.R. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T.J. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). Although measurements of endosomal pH have been reported utilizing ratioable pH-sensitive fluorescent indicators (19Geisow M.J. Evans W.H. Exp. Cell Res. 1984; 150: 36-46Crossref PubMed Scopus (137) Google Scholar, 20Yamashiro D.J. Maxfield F.R. J. Cell Biol. 1987; 105: 2713-2721Crossref PubMed Scopus (74) Google Scholar, 21Zen K. Biwersi J. Periasamy N. Verkman A.S. J. Cell Biol. 1992; 119: 99-110Crossref PubMed Scopus (73) Google Scholar, 22Overly C.C. Lee K.D. Berthiaume E. Hollenbeck P.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3156-3160Crossref PubMed Scopus (191) Google Scholar), there have been no measurements of ion concentrations in the endosomal lumen. Physico-chemical considerations indicate that endosomal [Cl−] and pH should depend on the activity of the vacuolar H+ pump, the magnitude of endosomal cation (K+, Na+, and H+) and anion (Cl− and HCO3−) conductances, endosomal membrane potential, buffer capacity, Donnan potential, and cytoplasmic pH and ion concentrations. Although attempts have been made to model endosomal/organelle acidification mathematically (23Rybak S.L. Lanni F. Murphy R.F. Biophys. J. 1997; 73: 674-687Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 24Grabe M. Oster G. J. Gen. Physiol. 2001; 117: 329-344Crossref PubMed Scopus (241) Google Scholar), the paucity of information about key endosomal parameters precludes meaningful predictions about endosomal regulatory processes. Taken in reference to endosomal pH and cytoplasmic pH/[Cl−], endosomal [Cl−] is a particularly important parameter because of its implications for relative endosomal ion conductances and membrane potential. If Cl− conductance is the rate-limiting ion conductance in endosomal acidification, then the interior-positive endosomal electrical potential should produce marked Cl− accumulation in the endosomal aqueous lumen during acidification. The purpose of this study is to develop and apply methodology to measure endosomal [Cl−] quantitatively in living cells. For these measurements, we synthesized a ratioable long wavelength fluorescent Cl− indicator that is brightly fluorescent, pH-insensitive, sensitive to [Cl−] from 0 to >100 mm, biochemically stable, and membrane-impermeant. The endosomal aqueous lumen in cultured cells was stained with Cl− and pH indicators by fluid-phase endocytosis, and the kinetics of endosomal [Cl−] and pH were measured by ratio image analysis. Pulse labeling, inhibitor addition, and ion substitution maneuvers established quantitatively the role of Cl− conductance in endosomal acidification. An unexpectedly low [Cl−] early after endocytosis led us to postulate that endosomal volume increases substantially during acidification, which was supported experimentally using a novel ratio imaging strategy to measure relative endosomal volume. All chemicals for synthesis were purchased from Aldrich. Aminodextran (M r 40,000), 5-(and 6)-carboxyfluorescein (CF) 1The abbreviations used are:CF5-(and 6)-carboxyfluoresceinBAC10,10′-bis[3-carboxypropyl]-9,9′-biacridinium dinitrateTMRtetramethylrhodamineSPQ6-methoxy-N-[3-sulfopropyl] quinoliniumCHOChinese hamster ovaryBCECF2′,7′-bis-(2-carboxyethyl)-CF succinimidylester, 5-(and 6)-carboxytetramethylrhodamine succinimidylester (TMR-SE), CF-carboxytetramethyl-rhodamine (TMR)-dextran (CF-TMR-dextran), calcein, sulforhodamine B, 6-methoxy-N-[3-sulfopropyl] quinolinium (SPQ), and 2′,7′-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) were obtained from Molecular Probes (Eugene, OR). Nigericin, bafilomycin, valinomycin, monensin, and carbonyl cyanidem-chlorophenylhydrazone (CCCP) were obtained from Sigma. 5-(and 6)-carboxyfluorescein 10,10′-bis[3-carboxypropyl]-9,9′-biacridinium dinitrate tetramethylrhodamine 6-methoxy-N-[3-sulfopropyl] quinolinium Chinese hamster ovary 2′,7′-bis-(2-carboxyethyl)-CF J774.1 macrophages (ATCC no. TIB-67) were obtained from American Type Cell Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagles medium DME-H21 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. CHO-K1 cells (ATCC no. CCL-61) were also obtained from the ATCC and grown in Ham's F12K medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured on 18-mm diameter round glass coverslips at 37 °C in a 95% air, 5% CO2 incubator and used just prior to confluence. To a stirred suspension of 10-[3-carboxypropyl]-9(10H)-acridone (Fig. 1 A,compound 1 ; prepared according to Ref. 25Litt M.H. Kim J. Rodriguez P.J. J. Polym. Sci., Polym. Chem. Ed. 1985; 23: 1307-1319Crossref Google Scholar) (2.8 g, 10 mmol) in acetone (80 ml) was added zinc dust (13.3 g, 201 gram atom). The mixture was stirred for 20 min at 30–40 °C. The flask was in and g, mmol) was added over at 10 °C. The mixture was stirred at and of was A was by with and in of a 5% aqueous The mixture was and the was with to a The was with and from to of a A suspension of g, 10 mmol) in ml) was for 1 at °C of the was and the was with and from to of a and and A of g, mmol) in ml) was g, mmol) g, mmol) at for The mixture was with ml) and stirred for 1 and the was by in the was on a and the was added into a stirred mixture of The was by and the was with The was by in and in to of a and and For synthesis of 5-(and ester mmol) was stirred with g, r in aqueous pH at for The was by for and then for 36 at °C. The was ratio For synthesis of g, mmol) was with g, mmol) in aqueous pH at for The mixture was to of BAC-TMR-dextran with to ratio and were measured by using a measurements were at and of were added to of in at pH Stern-Volmer were from the of [Cl−] [Cl−] is BAC fluorescence in the of in the of Endosomes were by of cells on coverslips with BAC-TMR-dextran for min in mm mm 1 mm 1 mm 10 at 37 °C. were then in bovine and in a at 37 °C in a Cells were with In experiments the 10 For experiments buffer Cl− was by For in Cl− fluorescence ratio and endosomal [Cl−] were by of cells for to 1 at 37 °C in 20 mm 1 mm 1 mm and 10 mm pH with [Cl−] from mm the and the H+ pump inhibitor pH fluorescence cells were with 1 1 mm 10 mm 10 mm mm 20 mm and the mixture with pH to K. Biwersi J. Periasamy N. Verkman A.S. J. Cell Biol. 1992; 119: 99-110Crossref PubMed Scopus (73) Google Scholar). pH was measured using by ratio imaging and T.J. Tsien R.Y. T. J. Cell Biol. PubMed Scopus Google Scholar). [Cl−] was measured using Verkman A.S. Biophys. J. 1989; Full Text PDF PubMed Scopus Google Scholar). fluid-phase with BAC-TMR-dextran and the cells were with in the and then with at 37 °C. of BAC and and were at and 45 In experiments was added to the from the of the 45 min after capacity was from the increase in endosomal pH in to of to the mm mm mm 1 1 mm and 10 mm pH was from the is the pH increase just after addition, is and is endosomal pH after A. Physiol. Rev. PubMed Scopus Google Scholar, M. S. 1995; PubMed Scopus Google Scholar). Endosomes were pulse-labeled with the mixture of self-quenching and sulforhodamine low in to for 1 min by with The was to the A of and sulforhodamine were obtained at increased fluorescence because of volume in sulforhodamine In experiments was added to the An in of fluorescence was obtained by of of coverslips and glass of and sulforhodamine after with were using a fluorescence with a and a with a (19Geisow M.J. Evans W.H. Exp. Cell Res. 1984; 150: 36-46Crossref PubMed Scopus (137) Google Scholar). Cell fluorescence was with a with a of mm, The was a using Cells were by using red and was were obtained using for the calcein, sulforhodamine and model in the and were used for of BAC fluorescence 20 and fluorescence image indicated BAC image and for In time endosomes in cells were for time was in to In of cells endosomes were by and for of the were for The were in the BAC image and because of The was for from the of the of the were from in of were for time The was used with for pH for relative volume measurements were by over endosomes in green and red were from endosomes the were for endosomes in image and at image were time The ratioable fluorescent Cl− indicator BAC-TMR-dextran was synthesized by the chromophore BAC in from and and the Cl−-insensitive chromophore to in 1 BAC fluorescence was sensitive to Cl− in the 0 to mm with a Stern-Volmer constant of 36 1 B, BAC fluorescence was to pH in the for measurements B, and to and and The BAC chromophore fluorescence at and and a with at (Fig. 1 B, The chromophore is sensitive to pH ion concentrations that the ratio of red fluorescence to green BAC fluorescence A fluorescence of the Cl−-sensitive green BAC fluorescence and the Cl−-insensitive red For with were used macrophages and CHO a of BAC-TMR-dextran fluorescence ratio in aqueous and J774 cells Cl− was by in the For measurements, endosomes were by fluid-phase endocytosis with a with in in in endosomes were on a The fluorescent with to from early endosomes to to The red fluorescence in with the green BAC was cells were for min with BAC-TMR-dextran at °C of 37 °C of [Cl−] in endosomes was by cells with a K+ buffer mixture to and endosomal [Cl−] in and was measured in endosomes from cells in that [Cl−] in endosomes is from that in that BAC-TMR-dextran fluorescence is and thus to present in the lumen. The [Cl−] was used to endosomal [Cl−] A and BAC of J774 cells at and 45 min after together with a ratio image with a [Cl−] Endosomal [Cl−] was in image and increased with the kinetics of endosomal [Cl−] in J774 and CHO cells a of time at 37 °C. Endosomal [Cl−] increased and after of these in of driving of endosomal pH cytoplasmic pH and [Cl−] that Cl− accumulation in endosomes was by the of the vacuolar H+ pump inhibitor the was added after the These that H+ pump activity Cl− accumulation in Endosomal pH was measured the used in endosomal [Cl−] The ratioable pH indicator was used in which green fluorescence is pH-sensitive and red fluorescence is pH-insensitive K. Biwersi J. Periasamy N. Verkman A.S. J. Cell Biol. 1992; 119: 99-110Crossref PubMed Scopus (73) Google Scholar). A a of fluorescence ratio pH in The and to and endosomal was sensitive to pH in the for endosomal the time of endosomal pH in J774 and CHO cells a of Endosomes were with for min for the [Cl−] measurements using reported in (19Geisow M.J. Evans W.H. Exp. Cell Res. 1984; 150: 36-46Crossref PubMed Scopus (137) Google Scholar, 20Yamashiro D.J. Maxfield F.R. J. Cell Biol. 1987; 105: 2713-2721Crossref PubMed Scopus (74) Google Scholar, 21Zen K. Biwersi J. Periasamy N. Verkman A.S. J. Cell Biol. 1992; 119: 99-110Crossref PubMed Scopus (73) Google Scholar) endosomes to the the endosomal lumen A. K. Proc. Natl. Acad. Sci. U. S. A. 1988; PubMed Scopus Google Scholar). To in [Cl−] and pH buffer capacity was capacity was from the pH increase of 10 to the (Fig. F.R. J. Cell Biol. PubMed Scopus Google Scholar). entry of which on the of entry and endosomal buffer The endosomal buffer of 36 1 for J774 cells and for CHO cells (Fig. to that of measured in kidney cells M. S. 1995; PubMed Scopus Google Scholar). the of the endosomal-limiting membrane. If Cl− from endosomes is the of to maintain electroneutrality, then it is that should be accompanied by Cl− exit. that after a BAC-TMR-dextran labeling, in Cl− whereas by endosomal in cells also the in B, that BAC-TMR-dextran fluorescence is The time of endosomal acidification (Fig. and [Cl−] increase (Fig. that the entry of to H+ during endosomal acidification be for by the entry by For a in pH from to buffer capacity measurements in J774 cells H+ of mm 36 pH to the measured increase in [Cl−] of a test of the that Cl− conductance is the endosomal the time of endosomal [Cl−] was measured in the of in the after (Fig. Endosomal [Cl−] was 10 mm that there was a [Cl−] driving Cl− entry [Cl−] mm, A significant conductance to K+ have increase in also the time of endosomal [Cl−] after min of with BAC-TMR-dextran in a Cl− Endosomal [Cl−] increases in B, that [Cl−] obtained BAC-TMR-dextran is in the pH was measured by ratio imaging using the fluorescent pH indicator that cytoplasmic pH was in J774 cells and in CHO cells. [Cl−] was measured using the Cl− indicator Cells were with by and fluorescence was measured Cells were with the buffer used for the endosomal [Cl−] and pH measurements and then with a of and [Cl−] [Cl−] was mm in J774 cells and mm in CHO cells. The low [Cl−] just after BAC-TMR-dextran was because endosomes the [Cl−] of it that a should for the of Cl− of endosomes just after we that for the low may for endosomes in a low that the Donnan of membrane proteins might potential that Cl− during the H+ and Cl− entry in and decreased of there is evidence that volume increases after P. J. J. Cell Sci. 1992; PubMed Google Scholar, G. R. M. J. Cell Biol. 1989; PubMed Scopus Google Scholar). To test the that endosomes after we a ratio imaging to measure in S. Verkman A.S. J. Gen. Physiol. 2001; 117: PubMed Scopus Google Scholar). Endosomes were pulse-labeled with a mixture of at self-quenching and sulforhodamine at low The ratio of green fluorescence to sulforhodamine red fluorescence provides a measure of relative volume. A the of on of the increase in because of decreased fluorescence at 0 and min after with the The green fluorescence to red sulforhodamine was at self-quenching and increased volume. of in increase in relative endosomal volume over after The increase in volume was blocked by of acidification by bafilomycin. The purpose of this study was to measure endosomal Cl− to whether endosomal acidification is accompanied by Cl− in the Cl− is the intracellular and its transport organellar been proposed to regulate endosomal acidification. is to endosomal [Cl−] a because of its endosomal H+ pump ion buffer capacity, membrane and Donnan and For endosomal [Cl−] be driven its Cl− is the principal ion that active H+ provided that endosomal [Cl−] is and/or buffer capacity is To measure endosomal [Cl−] we synthesized and a fluorescent fluid-phase of endocytosis for ratio image analysis. The indicator was to measure the kinetics of endosomal [Cl−] in and the were by of ion endosomal volume and cytoplasmic [Cl−] and There were a of for the ratioable Cl− and long wavelength fluorescence was to fluorescence from endosomes on Cl− which have been used to measure cytoplasmic [Cl−] Biwersi J. Verkman A.S. 1999; PubMed Scopus Google Scholar), were for measurement of endosomal [Cl−] because of the the for and the Cl− of the indicator to pH and to [Cl−] in the mm was for measurement of endosomal [Cl−] in the endosomal fluorescent were because of the pH and low Cl− S. P. Verkman A.S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The synthesized the long wavelength pH Cl− and The was the of the chromophore to which was that for be obtained with using concentrations during and using low for endosomal [Cl−] ratio imaging of vesicles in cells. An important of the measurement was the sensitive imaging of with and low to and a for of fluorescence made the of endosomal and relative volume by ratio Endosomal acidification in J774 and CHO cells was accompanied by accumulation of Cl− entry was blocked by bafilomycin, a Cl− was The of a significant conductance H+) have Cl− endosomal acidification and Cl− accumulation, by in Cl− the that is for Cl− and H+ The of Cl− entry was with that of H+ entry from measurements of endosomal acidification and buffer were obtained was with endosomal [Cl−] was that was in the increases in [Cl−] were these indicate that Cl− is the conductance in endosomes of J774 and CHO cells and that Cl− transport H+ a from K+ conductance H+ the during endosomal (Fig. In addition, these provide the first on endosomal If Cl− is the principal endosomal the endosomal cytoplasmic [Cl−] at 45 min after endocytosis a interior-positive endosomal membrane potential in the Measurements of endosomal membrane potential and to experimentally the driving to a of endosomal transport The low [Cl−] of 17 mm (J774) and 28 mm (CHO) just after endocytosis was because in we that endosomal [Cl−] should be low at the time of from the because of the Donnan potential of a acidification accompanied by Cl− entry is to be accompanied by a volume increase. is to a the magnitude of the volume increase because of the the of membrane fusion of endosomes by the that endosomal volume increases early after P. J. J. Cell Sci. 1992; PubMed Google Scholar, G. R. M. J. Cell Biol. 1989; PubMed Scopus Google Scholar), from measurements it is to time to in relative endosomal volume on the self-quenching of the fluid-phase fluorescent indicator J. L. 1995; PubMed Scopus Google Scholar). was that volume increased over min after and that the volume increase was blocked by of endosomal acidification by bafilomycin. Endosomal acidification is thus accompanied by active Cl− accumulation and In for the first time a ratio imaging method to measure endosomal The measurement of endosomal [Cl−] should have in the of intracellular Cl− channels (18Stobrawa S.M. Breiderhoff T. Takamori S. Engel D. Schweizer M. Zdebik A.A. Bosl M.R. Ruether K. Jahn H. Draguhn A. Jahn R. Jentsch T.J. Neuron. 2001; 29: 185-196Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, N. N. R.F. R. J. PubMed Scopus Google Scholar), A. F. A. Jentsch T.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, N. M. 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We investigated the involvement of ClC-3 chloride channels in endosomal acidification by measurement of endosomal pH and chloride concentration [Cl-] in control versus ClC-3-deficient hepatocytes and in control versus ClC-3-transfected Chinese hamster ovary cells. Endosomes were labeled with pH or [Cl-]-sensing fluorescent transferrin (Tf), which targets to early/recycling endosomes, or alpha2-macroglobulin (alpha2M), which targets to late endosomes. In pulse label-chase experiments, [Cl-] was 19 mM just after internalization in alpha2M-labeled endosomes in primary cultures of hepatocytes from wild-type mice, increasing to 58 mM over 45 min, whereas pH decreased from 7.1 to 5.4. Endosomal acidification and [Cl-] accumulation were significantly impaired in hepatocytes from ClC-3 knock-out mice, with [Cl-] increasing from 16 to 43 mM and pH decreasing from 7.1 to 6.0. Acidification and Cl- accumulation were blocked by bafilomycin. In Tf-labeled endosomes, [Cl-] was 46 mM in wild-type versus 35 mM in ClC-3-deficient hepatocytes at 15 min after internalization, with corresponding pH of 6.1 versus 6.5. Approximately 4-fold increased Cl- conductance was found in alpha2M-labeled endosomes isolated from hepatocytes of wild-type versus ClC-3 null mice. In contrast, Golgi acidification was not impaired in ClC-3-deficient hepatocytes. In transfected Chinese hamster ovary cells expressing ClC-3A, endosomal acidification and [Cl-] accumulation were enhanced. [Cl-] in alpha2M-labeled endosomes was 42 mM (control) versus 53 mM (ClC-3A) at 45 min, with corresponding pH 5.8 versus 5.2; [Cl-] in Tf-labeled endosomes at 15 min was 37 mM (control) versus 49 mM (ClC-3A) with pH 6.3 versus 5.9. Our results provide direct evidence for involvement of ClC-3 in endosomal acidification by Cl- shunting of the interior-positive membrane potential created by the vacuolar H+ pump.