Chloride concentration in endosomes measured using a ratioable fluorescent Cl- indicator: evidence for chloride accumulation during acidification

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.