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A population of membrane vesicles derived from platelet-dense granules was isolated from platelet osmotic lysates by density gradient centrifugation. The preparation is relatively free from contamination by mitochondrial and soluble enzymatic activities and contains only traces of serotonin and adenine nucleotides, but retains significant amounts of Na+-dependent serotonin transport (plasma membrane) activity. In the presence of ATP and in the absence of Na+, these vesicles accumulate serotonin to intravesicular concentrations over 100 times that of the medium. Transport is a saturable process (Km = 1.15 microM) inhibited by reserpine, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, ammonia, and nigericin (in the presence of external potassium), but not by imipramine. When diluted into assay medium at pH 8.5, the interior of these vesicles remains relatively acidic. Addition of ATP further induced generation of a membrane potential (interior positive). The vesicles accumulate maximal concentrations of serotonin only when the vesicle interior is both acidic and positive with respect to the medium. These results are consistent with a transporter which catalyzes countertransport of at least two protons for each serotonin cation accumulated.
Fishkes et al. (Sat,) studied this question.