Abstract
The Ca2+-sensor synaptotagmin-1 that triggers neuronal exocytosis binds to negatively charged membrane lipids (mainly phosphatidylserine (PtdSer) and phosphoinositides (PtdIns)) but the molecular details of this process are not fully understood. Using quantitative thermodynamic, kinetic and structural methods, we show that synaptotagmin-1 (from Rattus norvegicus and expressed in Escherichia coli) binds to PtdIns(4,5)P2 via a polybasic lysine patch in the C2B domain, which may promote the priming or docking of synaptic vesicles. Ca2+ neutralizes the negative charges of the Ca2+-binding sites, resulting in the penetration of synaptotagmin-1 into the membrane, via binding of PtdSer, and an increase in the affinity of the polybasic lysine patch to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). These Ca2+-induced events decrease the dissociation rate of synaptotagmin-1 membrane binding while the association rate remains unchanged. We conclude that both membrane penetration and the increased residence time of synaptotagmin-1 at the plasma membrane are crucial for triggering exocytotic membrane fusion.
Highlights
Synaptic transmission in the nervous system is mediated by the regulated release of neurotransmitters from presynaptic nerve terminals
We monitored Forster resonance energy transfer (FRET) between the tryptophan residues of the C2AB fragment and dansyl-labeled phospholipids that were incorporated into the liposomes
Our results show that the SNARE complex binds the C2AB fragment with higher affinity than it does the C2B domain, and that K326 and K327 play an important role in this binding (Figure 4h,i), in agreement with recent findings (Brewer et al, 2015; Wang et al, 2016)
Summary
Synaptic transmission in the nervous system is mediated by the regulated release of neurotransmitters from presynaptic nerve terminals. Upon arrival of an action potential, voltage-gated Ca2+-channels open, leading to a rise in cytoplasmic Ca2+ concentration, which triggers the fusion of synaptic vesicles with the presynaptic plasma membrane. The C2 domains of syt-1 are composed of structurally conserved eight-stranded anti-parallel b-sandwiches of ~130 residues. These domains contain Ca2+binding loops at one end of the b-sandwich motif. Coulombic attraction develops towards the head groups of anionic phospholipids, leading to binding of the C2 domains to the membrane associated with completion of the Ca2+ coordination sphere. A conserved polybasic lysine patch located on the C2B domain binds to anionic lipid in the absence of Ca2+, being attracted to multivalent phosphoinositides (PtdIns)
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