Acidification of Small Synaptic Vesicles in SLO-Permeabilized Synaptosomes

2 Acidification of Small Synaptic Vesicles in SLO-Permeabilized Synaptosomes 

The uptake of aminoacid neurotransmitter has been characterized by bioenergetic studies (Hell et ai, 1992 Hartinger and John, 1993). In contrast to catecholamine and GABA uptake, glutamate uptake does not depend on a pH gradient over the vesicle membrane. 

Application of Alpha-Toxin and Streptolysin O as Tools in Cell Biological Research 267 

PC 12 cells were permeabilized with purified SLO as given in section 18.3.1 of this chapter. Protein content was measured using the BCA-method. Values are the mean of three samples SD. Similar results were obtained using either wild-type SLO fusion protein or its alanine mutant. The assay can also be performed using alpha-toxin for permeabilization. 

Uptake of glutamate results in an acidification of the intravesicular lumen. This acidification can be indicated by the fluorescent dye acridine orange, which accumulates in acid compartments. Synapto-somes were prepared (McMahon etal., 1992) and stored as pellets of about 1 mg overlaid with 200 p.1 Krebs-Ringer-HEPES buffer on ice. Acidification was performed with either glutamate or chloride (Hell et al., 1992 Hartinger and Jahn, 1993). To get rid of the non-vesicular glutamate extensive washing of the permeabilized synaptosomes is required. 

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