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J. Biol. Chem., Vol. 277, Issue 7, 5506-5513, February 15, 2002
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From the Departments of Medicine and Physiology, Cardiovascular
Research Institute, University of California,
San Francisco, California 94143-0521
A novel long wavelength fluorescent
Cl
Chloride Concentration in Endosomes Measured Using a Ratioable
Fluorescent Cl
Indicator
EVIDENCE FOR CHLORIDE ACCUMULATION DURING
ACIDIFICATION*
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 M1.
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.
*
This study was supported by National Institutes of Health
Grants HL59198, DK35124, HL60288, and DK43840 and by Grant R613 from
the Cystic Fibrosis Foundation.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.
To whom correspondence should be addressed: Cardiovascular
Research Institute, 1246 Health Sciences East Tower, Box 0521, University of California, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; E-mail: verkman@itsa.ucsf.edu.
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