title: Measurements of the acidification kinetics of single SynaptopHluorin vesicles.
abstract: Uptake of neurotransmitters into synaptic vesicles is driven by the proton gradient established across the vesicle membrane. The acidification of synaptic vesicles, therefore, is a crucial component of vesicle function. Here we present measurements of acidification rate constants from isolated, single synaptic vesicles. Vesicles were purified from mice expressing a fusion protein termed SynaptopHluorin created by the fusion of VAMP/synaptobrevin to the pH-sensitive super-ecliptic green fluorescent protein. We calibrated SynaptopHluorin fluorescence to determine the relationship between fluorescence intensity and internal vesicle pH, and used these values to measure the rate constant of vesicle acidification. We also measured the effects of ATP, glutamate, and chloride on acidification. We report acidification time constants of 500 ms to 1 s. The rate of acidification increased with increasing extravesicular concentrations of ATP and glutamate. These data provide an upper and a lower bound for vesicle acidification and indicate that vesicle readiness can be regulated by changes in energy and transmitter availability.
authors.affiliation: Department of Chemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Kristi L
authors.initials: KL
authors.lastname: Budzinski
authors.forename: Maxwell
authors.initials: M
authors.lastname: Zeigler
authors.forename: Bryant S
authors.initials: BS
authors.lastname: Fujimoto
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Bajjalieh
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Chiu
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology; D002712:Chlorides / Q000378:metabolism; D004705:Endocytosis; D005971:Glutamates / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D018377:Neurotransmitter Agents / Q000378:metabolism; D010539:Permeability; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013572:Synaptic Vesicles / Q000737:chemistry* / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics
chemical_list: D002712:Chlorides; D005971:Glutamates; D018377:Neurotransmitter Agents; D011522:Protons; D011993:Recombinant Fusion Proteins; C514578:synaptopHIuorin; D049452:Green Fluorescent Proteins