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Determinants of [Cl-] in recycling and late endosomes and Golgi complex measured using fluorescent ligands.

Sonawane ND, Verkman AS - J. Cell Biol. (2003)

Bottom Line: In pulse-chase experiments, [Cl-] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over approximately 10 min in parallel to a drop in pH from 6.91 to 6.05.The low [Cl-] just after internalization (compared with 137 mM solution [Cl-]) was prevented by reducing the interior-negative Donnan potential. [Cl-] in alpha2-macroglobulin-labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1-45 min after internalization, whereas pH decreased from 6.85 to 5.20.Cl- accumulation was prevented by bafilomycin but restored by valinomycin.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
Chloride concentration ([Cl-]) was measured in defined organellar compartments using fluorescently labeled transferrin, alpha2-macroglobulin, and cholera toxin B-subunit conjugated with Cl--sensitive and -insensitive dyes. In pulse-chase experiments, [Cl-] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over approximately 10 min in parallel to a drop in pH from 6.91 to 6.05. The low [Cl-] just after internalization (compared with 137 mM solution [Cl-]) was prevented by reducing the interior-negative Donnan potential. [Cl-] in alpha2-macroglobulin-labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1-45 min after internalization, whereas pH decreased from 6.85 to 5.20. Cl- accumulation was prevented by bafilomycin but restored by valinomycin. A Cl- channel inhibitor slowed endosomal acidification and Cl- accumulation by approximately 2.5-fold. [Cl-] was 49 mM and pH was 6.42 in cholera toxin B subunit-labeled Golgi complex in Vero cells; Golgi compartment Cl- accumulation and acidification were reversed by bafilomycin. Our experiments provide evidence that Cl- is the principal counter ion accompanying endosomal and Golgi compartment acidification, and that an interior-negative Donnan potential is responsible for low endosomal [Cl-] early after internalization. We propose that reduced [Cl-] and volume in early endosomes permits endosomal acidification and [Cl-] accumulation without lysis.

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Role of Cl− conductance in endosomal acidification. (A) Inhibition of endosomal Cl− conductance by NPPB. Kinetics of endosomal [Cl−] was determined as in Fig. 4 A (right) in PBS (control) and 100 μM PBS-containing NPPB (n = 4 sets of experiments). (B) Kinetics of endosomal acidification as in Fig. 4 B (right; n = 4). Where indicated, 100 μM NPPB was present in the incubation solution and perfusate. Where indicated, Cl− was replaced by gluconate for 2 h before experiments and during measurements. (C) Time course of endosomal [Cl−] was determined as in Fig. 4 A (right), where 10 μM valinomycin and/or 200 nM bafilomycin were present in the incubation solution and perfusate. (D) Representative measurements of endosomal buffer capacity showing the prompt increase in endosomal pH after addition of NH4Cl to the perfusate.
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fig6: Role of Cl− conductance in endosomal acidification. (A) Inhibition of endosomal Cl− conductance by NPPB. Kinetics of endosomal [Cl−] was determined as in Fig. 4 A (right) in PBS (control) and 100 μM PBS-containing NPPB (n = 4 sets of experiments). (B) Kinetics of endosomal acidification as in Fig. 4 B (right; n = 4). Where indicated, 100 μM NPPB was present in the incubation solution and perfusate. Where indicated, Cl− was replaced by gluconate for 2 h before experiments and during measurements. (C) Time course of endosomal [Cl−] was determined as in Fig. 4 A (right), where 10 μM valinomycin and/or 200 nM bafilomycin were present in the incubation solution and perfusate. (D) Representative measurements of endosomal buffer capacity showing the prompt increase in endosomal pH after addition of NH4Cl to the perfusate.

Mentions: Several complementary approaches were used to examine the role of Cl− conductance in endosomal acidification. First, endosomal Cl− conductance was partially inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) at a high concentration of 100 μM used previously to study the role of Cl− conductance in acidification of isolated endosomes (Schmid et al., 1989; Busch et al., 1996; Marshansky and Vinay, 1996). Endosomal Cl− accumulation (Fig. 6 A) and acidification (Fig. 6 B) were inhibited partially by NPPB (similar results were obtained using 300 μM NPPB). NPPB did not induce H+ leak under the conditions of these experiments (not depicted). These results support the conclusion that endosomal Cl− conductance plays an important role in endosomal acidification; however, the data do not provide quantitative information about the role of endosomal Cl− versus other conductances because of the uncertain percent inhibition of endosomal Cl− channels by NPPB. Fig. 6 B shows that Cl− substitution by gluconate produced a more substantial inhibition of endosomal acidification, providing additional evidence for a role of Cl−. In this experiment, cytosolic [Cl−] was depleted to <5 mM as shown by SPQ fluorescence measurements. However, it is not possible to deduce quantitative information because of the nonzero permeability of gluconate in the small endosomes (high surface/volume ratio) as well as other phenomena such as endosomal fusion events.


Determinants of [Cl-] in recycling and late endosomes and Golgi complex measured using fluorescent ligands.

Sonawane ND, Verkman AS - J. Cell Biol. (2003)

Role of Cl− conductance in endosomal acidification. (A) Inhibition of endosomal Cl− conductance by NPPB. Kinetics of endosomal [Cl−] was determined as in Fig. 4 A (right) in PBS (control) and 100 μM PBS-containing NPPB (n = 4 sets of experiments). (B) Kinetics of endosomal acidification as in Fig. 4 B (right; n = 4). Where indicated, 100 μM NPPB was present in the incubation solution and perfusate. Where indicated, Cl− was replaced by gluconate for 2 h before experiments and during measurements. (C) Time course of endosomal [Cl−] was determined as in Fig. 4 A (right), where 10 μM valinomycin and/or 200 nM bafilomycin were present in the incubation solution and perfusate. (D) Representative measurements of endosomal buffer capacity showing the prompt increase in endosomal pH after addition of NH4Cl to the perfusate.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172765&req=5

fig6: Role of Cl− conductance in endosomal acidification. (A) Inhibition of endosomal Cl− conductance by NPPB. Kinetics of endosomal [Cl−] was determined as in Fig. 4 A (right) in PBS (control) and 100 μM PBS-containing NPPB (n = 4 sets of experiments). (B) Kinetics of endosomal acidification as in Fig. 4 B (right; n = 4). Where indicated, 100 μM NPPB was present in the incubation solution and perfusate. Where indicated, Cl− was replaced by gluconate for 2 h before experiments and during measurements. (C) Time course of endosomal [Cl−] was determined as in Fig. 4 A (right), where 10 μM valinomycin and/or 200 nM bafilomycin were present in the incubation solution and perfusate. (D) Representative measurements of endosomal buffer capacity showing the prompt increase in endosomal pH after addition of NH4Cl to the perfusate.
Mentions: Several complementary approaches were used to examine the role of Cl− conductance in endosomal acidification. First, endosomal Cl− conductance was partially inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) at a high concentration of 100 μM used previously to study the role of Cl− conductance in acidification of isolated endosomes (Schmid et al., 1989; Busch et al., 1996; Marshansky and Vinay, 1996). Endosomal Cl− accumulation (Fig. 6 A) and acidification (Fig. 6 B) were inhibited partially by NPPB (similar results were obtained using 300 μM NPPB). NPPB did not induce H+ leak under the conditions of these experiments (not depicted). These results support the conclusion that endosomal Cl− conductance plays an important role in endosomal acidification; however, the data do not provide quantitative information about the role of endosomal Cl− versus other conductances because of the uncertain percent inhibition of endosomal Cl− channels by NPPB. Fig. 6 B shows that Cl− substitution by gluconate produced a more substantial inhibition of endosomal acidification, providing additional evidence for a role of Cl−. In this experiment, cytosolic [Cl−] was depleted to <5 mM as shown by SPQ fluorescence measurements. However, it is not possible to deduce quantitative information because of the nonzero permeability of gluconate in the small endosomes (high surface/volume ratio) as well as other phenomena such as endosomal fusion events.

Bottom Line: In pulse-chase experiments, [Cl-] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over approximately 10 min in parallel to a drop in pH from 6.91 to 6.05.The low [Cl-] just after internalization (compared with 137 mM solution [Cl-]) was prevented by reducing the interior-negative Donnan potential. [Cl-] in alpha2-macroglobulin-labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1-45 min after internalization, whereas pH decreased from 6.85 to 5.20.Cl- accumulation was prevented by bafilomycin but restored by valinomycin.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
Chloride concentration ([Cl-]) was measured in defined organellar compartments using fluorescently labeled transferrin, alpha2-macroglobulin, and cholera toxin B-subunit conjugated with Cl--sensitive and -insensitive dyes. In pulse-chase experiments, [Cl-] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over approximately 10 min in parallel to a drop in pH from 6.91 to 6.05. The low [Cl-] just after internalization (compared with 137 mM solution [Cl-]) was prevented by reducing the interior-negative Donnan potential. [Cl-] in alpha2-macroglobulin-labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1-45 min after internalization, whereas pH decreased from 6.85 to 5.20. Cl- accumulation was prevented by bafilomycin but restored by valinomycin. A Cl- channel inhibitor slowed endosomal acidification and Cl- accumulation by approximately 2.5-fold. [Cl-] was 49 mM and pH was 6.42 in cholera toxin B subunit-labeled Golgi complex in Vero cells; Golgi compartment Cl- accumulation and acidification were reversed by bafilomycin. Our experiments provide evidence that Cl- is the principal counter ion accompanying endosomal and Golgi compartment acidification, and that an interior-negative Donnan potential is responsible for low endosomal [Cl-] early after internalization. We propose that reduced [Cl-] and volume in early endosomes permits endosomal acidification and [Cl-] accumulation without lysis.

Show MeSH
Related in: MedlinePlus