Synaptotagmin Increases the Dynamic Range of Synapses by Driving Ca2+-Evoked Release and by Clamping a Near-Linear Remaining Ca2+ Sensor

Abstract

Ca²+-evoked transmitter release shows a high dynamic range over spontaneous release. We investigated the role of the Ca²+ sensor protein, Synaptotagmin2 (Syt2), in both spontaneous and Ca²+-evoked release under direct control of presynaptic [Ca²+](i), using an in vivo rescue approach at the calyx of Held. Re-expression of Syt2 rescued the highly Ca²+ cooperative release and suppressed the elevated spontaneous release seen in Syt2 KO synapses. This latter release clamping function was partially mediated by the poly-lysine motif of the C₂B domain. Using an aspartate mutation in the C₂B domain (D364N) in which Ca²+ triggering was abolished but release clamping remained intact, we show that Syt2 strongly suppresses the action of another, near-linear Ca²+ sensor that mediates release over a wide range of [Ca²+](i). Thus, Syt2 increases the dynamic range of synapses by driving release with a high Ca²+ cooperativity, as well as by suppressing a remaining, near-linear Ca²+ sensor.

Molecular mechanisms governing Ca2+ regulation of evoked and spontaneous release

“…The two synaptic proteins Synaptotagmin and Complexin enable synchronous and efficient Ca 2+ -triggered release 34,103 , but also regulate spontaneous release. Deletion of Syt1 or Syt2 leads to an enhanced spontaneous release in nearly all preparations and species studied 52,68,[104][105][106][107] , and this effect has been interpreted as a 'clamping' function of Syt1 and Syt2 (refs. 52,104).…”

“…52), indicating that loss of Syt2 unmasks the action of a secondary Ca 2+ sensor. Genetic deletion of Syt2 at the calyx also leads to a strongly increased mini frequency (~20-40 fold 52,107 ); however, the spontaneous release in Syt2 knockout mice is sensitive to BAPTA (unlike spontaneous release at the wild-type calyx 52 ; see above) and thus likely depends on the coupling between the slow sensor and an unknown intracellular Ca 2+ release pathway 52 . Thus, part of the unclamped spontaneous release might represent an additional gain of function effect observed in the Syt2 knockout mice (see also ref.…”

Modulation of Presynaptic Release Probability by the Vertebrate-Specific Protein Mover

“…The calyx offers the possibility to combine detailed functional analyses of synaptic transmission with perturbations of protein function in vivo (e.g., Wimmer et al, 2004;Young and Neher, 2009;Schwenger and Kuner, 2010;Kochubey and Schneggenburger, 2011a). We generated an in vivo knockdown of Mover using adeno-associated virus (AAV)-mediated small hairpin RNA (shRNA) expression in globular bushy cells of the ventral cochlear nucleus (VCN), the projection neurons forming the calyces of Held.…”

Linker mutations reveal the complexity of synaptotagmin 1 action during synaptic transmission

“…Syt and Doc2 have also both been proposed to function as Ca 2+ sensors for spontaneous release (minis) 20,21 , but the question of whether glutamatergic minis are regulated by Ca 2+ is currently the subject of debate 22,23 . Interestingly, loss of syt results in an increase in mini frequency (see Ref.…”