pH-dependent fusion of synaptosomal membrane studied by fluorescence quenching method

We have found that both the synaptic vesicles (SV) and synaptic plasma memb rane vesicles (SPM) have an activity to fuse with phosphatidylcoline/phosph atidylserine liposomes in a pH-dependent manner. The activity increases wit h decreases in extravesicular pH. At a pH lower than 4.0, the activity is a lmost steady at its maximum value, and there was a rapid drop around pH 5.5 . The pH-dependent fusion was inhibited by proteolysis with trypsin; hence, at least in part, some membrane proteins play an important role in these p H-dependent fusion processes. To find specific markers, we screened various protein modifiers and found that anion channel blockers, stilbene derivati ves (DIDS and SITS) and glibenclamide, affected the fusion process. DIDS an d SITS decreased the fusion activity with an IC50 Of 180 and 300 mu M, resp ectively, whereas glibenclamide, on the contrary, increased it. From the re sults of an autoradiogram using H-3-tagged DIDS, a 30kDa DIDS-binding prote in was identified in the synaptic plasma membrane, which is possible to be responsible for the pH-dependent fusion.