Chloride Concentration in Endosomes Measured Using a Ratioable Fluorescent Cl− Indicator

Abstract

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. Bosl M.R. Jentsch T.J. Nature. 2000; PubMed Scopus Google Scholar), and 8Barasch J. Kiss B. Prince A. Saiman L. Gruenert D. Al-Awqati Q. Nature. 1991; 352: 70-73Crossref PubMed Scopus (425) Google and J. Verkman A.S. Am. J. Physiol. 266: PubMed Google Scholar). The F. M.D. D. B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar) for by to increase buffer capacity and Cl− accumulation and should be to direct it should also be to measure [Cl−] in endosomal of in and in of the secretory have been to fluorescent to organellar transport of Grinstein S. J. Cell Biol. 1996; PubMed Scopus Google Scholar), of J. Verkman A.S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), of N. S. Grinstein S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), of 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), and by Adams Tsien R.Y. PubMed Scopus Google Scholar). Grinstein for