Calcium accelerates endocytosis of vSNAREs at hippocampal synapses

Abstract

A pH-sensitive form of green-fluorescent protein (GFP) fused to the lumenal domain of VAMP (synapto-pHluorin) provides a sensitive optical probe to track the net balance between exocytosis and endocytosis of this protein at small synaptic terminals of the central nervous system. Here we used a reversible proton-pump blocker that prevents vesicle re-acidification upon endocytosis to trap vesicles in the alkaline state during recycling. In combination with optical measurements of synapto-pHluorin, we used alkaline trapping to examine the kinetic components of exocytosis and endocytosis separately at synaptic terminals. Using this approach, we show that, in addition to controlling exocytosis, intracellular calcium levels tightly regulate the speed of endocytosis, increasing it to a maximal speed of approximately one vesicle per second.

Enlargeosome, an Exocytic Vesicle Resistant to Nonionic Detergents, Undergoes Endocytosis via a Nonacidic Route

“…Among the PC12-27 cells investigated by patch clamping, those that responded to stimulation with [Ca 2ϩ ] i rises Ͼ4.5 M exhibited capacitance decrease rates severalfold faster than those that had reached values Ͻ4 M. The enlargeosome endocytosis seems therefore to be regulated, a property that, in other cells and secretory systems, has already attracted great interest (Sankaranarayanan and Ryan, 2001;Sorkin and Von Zastrow, 2002;Maxfield and McGraw, 2004). Moreover, the intracellular vesicles positive for d/A maintained a neutral pH in their lumen.…”

Protein scaffolds in the coupling of synaptic exocytosis and endocytosis

“…Endocytic rate constants in addition to potential regulation by Ca 2+ (REF. 38) are also partially sensitive to exocytic load 39 . under conditions of excessive non-physiological stimulation, endocytic capacity is overrun by exocytic load, resulting in the transient accumulation of synaptic vesicle proteins on the neuronal surface -a condition that may trigger bulk uptake of the presynaptic plasma membrane into vacuolar invaginations, as seen at retinal bipolar cell terminals 40 and in cultured neurons 41 .…”

“…Alternatively, fusion constructs of pH-sensitive green fluorescent protein (GFP) variants with synaptic proteins (so-called pHluorins) have been successfully used as exocytic-endocytic reporters in hippocampal neurons in culture or in slice preparations from transgenic mice that stably express pHluorin reporters 38,117 . The fluorescence of these proteins is quenched while the GFP moiety resides in the low pH of the vesicle lumen, but is activated after exocytosis, when the proteins are exposed to the neutral extracellular milieu.…”

“…The fluorescence of these proteins is quenched while the GFP moiety resides in the low pH of the vesicle lumen, but is activated after exocytosis, when the proteins are exposed to the neutral extracellular milieu. Fusion proteins between pHluorin-GFP and various synaptic vesicle proteins have been reported 38,117 . They differ in the signal-to-noise ratios following stimulation-induced exocytosis-endocytosis, with vesicular glutamate transporter 1 (VGLUT1) pHluorin or synaptophysin pHluorin being considered superior over the more traditionally used synaptobrevin 2 pHluorin.…”

Mécanisme de la fusion membranaire

“…Dans la synapse, la synaptobrévine est recyclée par endocytose d'une manière dépendante du calcium, dans de nouvelles vésicules synaptiques qui participeront aux prochaines vagues de libération de neurotransmetteurs [44] (Figure 2). L'homéostasie cellulaire est ainsi maintenue à chaque instant par un perpétuel recyclage des protéines SNARE laissant apparaître, à l'état d'équilibre, la spécificité de localisation précédemment décrite.…”