Abstract
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+)].
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Calcium / deficiency*
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Calcium Signaling / drug effects
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Calcium Signaling / physiology*
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Calcium-Binding Proteins / antagonists & inhibitors
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Calcium-Binding Proteins / metabolism
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Cells, Cultured
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DNA, Complementary / genetics
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Endocytosis / drug effects
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Endocytosis / genetics
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Epithelial Cells / drug effects
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Epithelial Cells / metabolism*
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Exocytosis / drug effects
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Exocytosis / genetics*
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Male
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / metabolism*
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Membrane Proteins / drug effects
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Membrane Proteins / metabolism
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Nerve Tissue Proteins / genetics
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Nerve Tissue Proteins / metabolism*
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Oligonucleotides, Antisense
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Pituitary Gland / drug effects
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Pituitary Gland / metabolism*
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Protein Isoforms / genetics
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Protein Isoforms / metabolism
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RNA, Messenger / genetics
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Rats
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SNARE Proteins
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Secretory Vesicles / drug effects
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Secretory Vesicles / metabolism*
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Synaptotagmin I
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Synaptotagmins
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Vesicular Transport Proteins*
Substances
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CADPS protein, human
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Calcium-Binding Proteins
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DNA, Complementary
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Membrane Glycoproteins
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Membrane Proteins
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Nerve Tissue Proteins
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Oligonucleotides, Antisense
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Protein Isoforms
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RNA, Messenger
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SNARE Proteins
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Synaptotagmin I
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Syt1 protein, rat
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Vesicular Transport Proteins
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Synaptotagmins
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Calcium