Synaptotagmin I increases the probability of vesicle fusion at low [Ca2+] in pituitary cells

Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Medical Faculty, 1000 Ljubljana; and Celica Biomedical Science Center, 1000 Ljubljana, Slovenia 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 µM [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+ ]. rat melanotrophs; exocytic module; membrane capacitance; calcium sensor