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

Abstract

Progressive acidification of vesicles in the endosomal pathway is important for receptor and ligand sorting and vesicular fusion (1, 2). Endosomal acidification is driven by a vacuolartype H ϩ pump that is present in the endosomal-limiting membrane. To maintain electroneutrality, H ϩ entry into the endosomal aqueous lumen must be accompanied by anion entry and/or cation exit. The principal transportable intracellular anion is Cl Ϫ and cation is K ϩ . Studies of organellar pH in living cells and isolated vesicles have provided evidence that Cl Ϫ entry may be the rate-limiting passive conductance in permitting active H ϩ entry in endosomes (3-9). In isolated endocytic vesicles from kidney proximal tubule (10) and liposomes reconstituted with proteins from clathrin-coated vesicles (11), a protein kinase A-activated Cl Ϫ conductance was characterized, and it was proposed that activation of Cl Ϫ channels might regulate endosomal acidification by providing a shunt to dissipate the interior-positive potential produce by the H ϩ pump. There is also evidence that Na ϩ /K ϩ pump activity in early endosomes may alter the driving force for H ϩ entry and thus regulate acidification (12-14). In Golgi, there is evidence that both Cl Ϫ and K ϩ conductances may contribute to acidification (15-17), whereas acidification of secretory granules in synaptic vesicles appears to require the expression of a specific Cl Ϫ channel (18). Although measurements of endosomal pH have been reported utilizing ratioable pH-sensitive fluorescent indicators (19 -22), there have been no measurements of ion concentrations in the endosomal lumen.Physico-chemical considerations indicate that endosomal [Cl Ϫ ] and pH should depend on the activity of the vacuolar H ϩ pump, the magnitude of endosomal cation (K ϩ , Na ϩ , and H ϩ ) and anion (Cl Ϫ and HCO 3 Ϫ ) 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 (23, 24), 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 Ϫ ], endosomal [Cl Ϫ ] is a particularly important parameter because of its implications for relative endosomal ion conductances and membrane potential. If Cl Ϫ conductance is the rate-limiting ion conductance in endosomal acidification, then the interior-positive endosomal electrical potential should produce marked Cl Ϫ accumulation in the endosomal aqueous lumen during acidification.The purpose of this study is to develop and apply methodology to measure endosomal [Cl Ϫ ] quantitatively in living cells. For these measurements, we synthesized a ratioable long wavelength fluorescent Cl Ϫ indicator that is brightly fluorescent, pH-insensitive, sensitive to [Cl Ϫ ] from 0 to Ͼ100 mM, biochemically stable, and membrane-impermeant. The endosomal aqueous lumen in cultured cells was stained with Cl Ϫ an...

Anthrax toxin-induced rupture of artificial lipid bilayer membranes

“…97 However, other ions and their transporters lower the potential to (27 ± 16) mV (positive inside) 98 which is consistent with the expected range estimated through the Goldman-Hodgkin-Katz equation 96,97 or from a kinetic analysis of the known channels and transporters in the membranes. 99 If we assume the distribution of the ψ values is Gaussian, then ≈2% of the endosomes will have transmembrane potentials that are sufficiently great (|V| ≥ 70 mV) that would support the putative LF N or EF N translocation model or the membrane rupture model suggested herein (Fig.…”

In situ measurement of the electrical potential across the phagosomal membrane using FRET and its contribution to the proton-motive force

“…3D). Similar (10-20 mV) inside-positive potentials were proposed to exist in endosomes, based on measurements of chloride concentration and on the assumption that the conductance to this anion is predominant (11).…”

Glycosylphosphatidylinositol-anchored Proteins and Actin Cytoskeleton Modulate Chloride Transport by Channels Formed by the Helicobacter pylori Vacuolating Cytotoxin VacA in HeLa Cells

“…This is likely because (i) the anion channel blocker NPPB inhibits VacA-induced vacuolation (11,12, this study); (ii) it is well established that VacA-induced vacuolation requires the V-ATPase activity (2), and it has been recently documented that the rate-limiting step in late endosome acidification by the V-ATPase is the endosomal influx of chloride ions by specific channels that could be inhibited by NPPB (36,37); and (iii) VacA mutant proteins, with an impaired capacity to form active channels, are unable (or poorly able) to vacuolate cells (38,39). It is not clear, however, where the toxin channel is formed in the cell.…”