pH-dependent fusion of synaptosomal membrane studied by fluorescence quenching method.
We have found that both the synaptic vesicles (SV) and synaptic plasma membrane vesicles (SPM) have an activity to fuse with phosphatidylcoline/phosphatidylserine liposomes in a pH-dependent manner. The activity increases with decreases in extravesicular pH. At a pH lower than 4.0, the activity is almost 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 pH-dependent fusion processes. To find specific markers, we screened various protein modifiers and found that anion channel blockers, stilbene derivatives (DIDS and SITS) and glibenclamide, affected the fusion process. DIDS and SITS decreased the fusion activity with an IC50 of 180 and 300 microM, respectively, whereas glibenclamide, on the contrary, increased it. From the results of an autoradiogram using 3H-tagged DIDS, a 30 kDa DIDS-binding protein was identified in the synaptic plasma membrane, which is possible to be responsible for the pH-dependent fusion.
Kumamaru
E
E
Division of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, 560-8531, Japan. kuma@bpe.es.osaka-u.ac.jp
Sato
M
M
Yoshida
H
H
Ide
T
T
Kasai
M
M
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000818:Animals; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008081:Liposomes; D008561:Membrane Fusion; D051381:Rats; D017207:Rats, Sprague-Dawley; D013570:Synaptic Membranes / Q000502:physiology* / Q000648:ultrastructure; D013572:Synaptic Vesicles / Q000502:physiology / Q000648:ultrastructure*
D008081:Liposomes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid