Synaptotagmin I increases the probability of vesicle fusion at low [Ca2+] in pituitary cells.
Synaptotagmin I (Syt I), a low-affinity Ca(2+)-binding protein, is thought to serve as the Ca(2+) sensor in the release of neurotransmitter. However, functional studies on the calyx of Held synapse revealed that the rapid release of neurotransmitter requires only approximately micromolar [Ca(2+)], suggesting that Syt I may play a more complex role in determining the high-affinity Ca(2+) dependence of exocytosis. Here we tested this hypothesis by studying pituitary cells, which possess high- and low-affinity Ca(2+)-dependent exocytic pathways and express Syt I. Using patch-clamp capacitance measurements to monitor secretion and the acute antisense deletion of Syt I from differentiated cells, we have shown that the rapid and the most Ca(2+)-sensitive pathway of exocytosis in rat melanotrophs requires Syt I. Furthermore, stimulation of the Ca(2+)-dependent exocytosis by cytosol dialysis with solutions containing 1 microM [Ca(2+)] was completely abolished in the absence of Syt I. Similar results were obtained by the preinjection of antibodies against the CAPS (Ca(2+)-dependent activator protein for secretion) protein. These results indicate that synaptotagmin I and CAPS proteins increase the probability of vesicle fusion at low cytosolic [Ca(2+)].
Kreft
M
M
Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Medical Faculty, 1000 Ljubljana, Slovenia.
Kuster
V
V
Grilc
S
S
Rupnik
M
M
Milisav
I
I
Zorec
R
R
D000818:Animals; D002118:Calcium / Q000172:deficiency*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D004705:Endocytosis / Q000187:drug effects / Q000235:genetics; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects / Q000235:genetics*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016376:Oligonucleotides, Antisense; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D050600:SNARE Proteins; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism*; D050863:Synaptotagmin I; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins
C409027:CADPS protein, human; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D016376:Oligonucleotides, Antisense; D020033:Protein Isoforms; D012333:RNA, Messenger; D050600:SNARE Proteins; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D002118:Calcium