Allosteric stabilization of calcium and phosphoinositide dual binding engages several synaptotagmins in fast exocytosis

Abstract

Synaptic communication relies on the fusion of synaptic vesicles with the plasma membrane, which leads to neurotransmitter release. This exocytosis is triggered by brief and local elevations of intracellular Ca2+ with remarkably high sensitivity. How this is molecularly achieved is unknown. While synaptotagmins confer the Ca2+ sensitivity of neurotransmitter exocytosis, biochemical measurements reported Ca2+ affinities too low to account for synaptic function. However, synaptotagmin's Ca2+ affinity increases upon binding the plasma membrane phospholipid PI(4,5)P2 and, vice versa, Ca2+-binding increases synaptotagmin's PI(4,5)P2 affinity, indicating a stabilization of the Ca2+/PI(4,5)P2 dual-bound syt. Here we devise a molecular exocytosis model based on this positive allosteric stabilization and the assumptions that (1.) synaptotagmin Ca2+/PI(4,5)P2 dual binding lowers the energy barrier for vesicle fusion and that (2.) the effect of multiple synaptotagmins on the energy barrier is additive. The model, which relies on biochemically measured Ca2+/PI(4,5)P2 affinities and protein copy numbers, reproduced the steep Ca2+ dependency of neurotransmitter release. Our results indicate that each synaptotagmin dual binding Ca2+/PI(4,5)P2 lowers the energy barrier for vesicle fusion by ~5 kBT and that allosteric stabilization of this state enables the synchronized engagement of several (typically three) synaptotagmins for fast exocytosis. Furthermore, we show that mutations altering synaptotagmin’s allosteric properties may show dominant-negative effects, even though synaptotagmins act independently on the energy barrier, and that dynamic changes of local PI(4,5)P2 (e.g. upon vesicle movement) dramatically impact synaptic responses. We conclude that allosterically stabilized Ca2+/PI(4,5)P2 dual binding enables synaptotagmins to exert their coordinated function in neurotransmission.

Electrostatic regulation of the cis- and trans-membrane interactions of synaptotagmin-1

“…4 ). Our data support that local PIP 2 concentration might control Ca 2+ cooperativity by allosterically-stabilized dual binding of synaptotagmin-1 to Ca 2+ and PIP 2 38 .…”

“…4). Our data support that local PIP 2 concentration might control Ca 2+ cooperativity by allosterically-stabilized dual binding of synaptotagmin-1 to Ca 2+ and PIP 2 38 . In this study, we investigate the electrostatic regulation of C2AB binding to vesicle membrane and the PMliposomes.…”

“…We propose that Ca 2+ chelators compete with vesicle membranes that contain anionic phospholipids in binding to Ca 2+ and disrupt the cis -interaction of synaptotagmin-1 by charge screening 10 . However, PIP 2 overcomes this inhibitory effect of ATP, because PIP 2 dramatically enhances the Ca 2+ -binding affinity of synaptotagmin-1 21 , 38 ; this high Ca 2+ affinity of the C2AB domain to PIP 2 -containing membranes is not affected by ATP 10 .…”

Electrostatic regulation of thecis- andtrans-membrane interactions of synaptotagmin-1

“…We propose that Ca 2+ chelators compete with vesicle membranes that contain anionic phospholipids in binding to Ca 2+ and disrupt the cis -interaction of synaptotagmin-1 by charge screening 10 . However, PIP 2 overcomes this inhibitory effect of ATP, because PIP 2 enhances the Ca 2+ -binding affinity of synaptotagmin-1 21, 38 ; this high Ca 2+ affinity of the C2AB domain to PIP 2 -containing membranes is not affected by ATP 10 .…”

Rate-limiting recovery processes in neurotransmission under sustained stimulation

“…Transmitter release is typically induced by presynaptic action potentials, i.e., brief de-and re-polarisations of the cellular membrane potential that lead to the opening of voltage gated Ca 2+ ion channels [5][6][7]. The resulting elevation of the presynaptic Ca 2+ concentration following Ca 2+ influx through these channels triggers synaptic vesicle fusion by activating the vesicular Ca 2+ sensing protein Synaptotagmin [5,8,9].…”