Synaptotagmin Modulation of Fusion Pore Kinetics in Regulated Exocytosis of Dense-Core Vesicles

Abstract

In the exocytosis of neurotransmitter, fusion pore opening represents the first instant of fluid contact between the vesicle lumen and extracellular space. The existence of the fusion pore has been established by electrical measurements, but its molecular composition is unknown. The possibility that synaptotagmin regulates fusion pores was investigated with amperometry to monitor exocytosis of single dense-core vesicles. Overexpression of synaptotagmin I prolonged the time from fusion pore opening to dilation, whereas synaptotagmin IV shortened this time. Both synaptotagmin isoforms reduced norepinephrine flux through open fusion pores. Thus, synaptotagmin interacts with fusion pores, possibly by associating with a core complex of membrane proteins and/or lipid.

Visualization of synaptotagmin I oligomers assembled onto lipid monolayers

“…We favor a model in which copies of the cytoplasmic domain of syt subunits form lower-ordered oligomers (e.g., dimers or tetramers) on the inner leaflet of the plasma membrane. Assembly of these oligomers might represent structural rearrangements within the fusion complex to regulate the time to opening or dilation kinetics of fusion pores (14). We speculate that syt that has bound to an effector, e.g., membranes and͞or SNARE proteins (30), subsequently assembles into multimers, thereby rearranging components of the fusion complex to regulate fusion (14,57).…”

“…The ability of syt I to bind Ca 2ϩ , coupled to functional studies demonstrating that syt I plays a critical postdocking function in exocytosis, suggests that syt I is a major Ca 2ϩ sensor that regulates release (12,13). Recent studies established that changes in the ratio of syt isoforms can alter fusion pore kinetics, placing syt action in the final events of the fusion reaction (14).…”

In search of the fusion pore of exocytosis

“…In our initial studies of fusion pores, we transfected PC12 cells with two different isoforms of synaptotagmin [10]. Synaptotagmin is a synaptic vesicle protein [11,12], that harbors two C2 domains with the capacity to bind Ca 2+ and engage in interactions with a number of targets [13,14].…”

“…Thus, the time interval from the appearance of the foot until its termination with the onset of the spike increases. Transfecting with another isoform, synaptotagmin IV, has the opposite effect, shortening the fusion pore lifetime [10]. Synaptotagmin IV is much less abundant and has no known Ca 2+ binding activity.…”

Mécanisme de la fusion membranaire

“…Ces données suggèrent que la synaptotagmine est responsable de la dépendance au calcium de la libération de neurotransmetteurs. Il est possible que la synaptotagmine participe à la genèse du pore de fusion [41]: avant l'entrée du calcium, elle bloquerait la fusion, le complexe SNARE étant partiellement assemblé; après l'entrée de calcium, elle se lierait au complexe SNARE et permettrait ainsi la fusion (Figure 2). …”