Doc2 Proteins Are Not Required for the Increased Spontaneous Release Rate in Synaptotagmin-1-Deficient Neurons

Abstract

Regulated secretion is controlled by Ca 2ϩ sensors with different affinities and subcellular distributions. Inactivation of Syt1 (synaptotagmin-1), the main Ca 2ϩ sensor for synchronous neurotransmission in many neurons, enhances asynchronous and spontaneous release rates, suggesting that Syt1 inhibits other sensors with higher Ca 2ϩ affinities and/or lower cooperativities. Such sensors could include Doc2a and Doc2b, which have been implicated in spontaneous and asynchronous neurotransmitter release and compete with Syt1 for binding SNARE complexes. Here, we tested this hypothesis using triple-knock-out mice. Inactivation of Doc2a and Doc2b in Syt1-deficient neurons did not reduce the high spontaneous release rate. Overexpression of Doc2b variants in triple-knock-out neurons reduced spontaneous release but did not rescue synchronous release. A chimeric construct in which the C2AB domain of Syt1 was substituted by that of Doc2b did not support synchronous release either. Conversely, the soluble C2AB domain of Syt1 did not affect spontaneous release. We conclude that the high spontaneous release rate in synaptotagmin-deficient neurons does not involve the binding of Doc2 proteins to Syt1 binding sites in the SNARE complex. Instead, our results suggest that the C2AB domains of Syt1 and Doc2b specifically support synchronous and spontaneous release by separate mechanisms. (Both male and female neurons were studied without sex determination.)

Post-tetanic potentiation lowers the energy barrier for synaptic vesicle fusion independently of Synaptotagmin-1

“…Previously, we concluded that changes in spontaneous release rates after genetic or biochemical manipulations did not correspond with the energy barrier shifts measured with HS ( Schotten et al, 2015 ). Here we showed both in Syt1 KO ( Figure 1 ) and Syt1 9Pro expressing synapses ( Figure 2 ) that mEPSC frequencies were increased, in line with previous studies in autapses ( Ruiter et al, 2019 ; Díez-Arazola et al, 2020 ; Huson and Cornelisse, 2019 ) and networks ( Xu et al, 2009 ; Bai et al, 2016 ; Liu et al, 2014 ; Courtney et al, 2018 ). It remains enigmatic why others found a similar increase in spontaneous release in networks but not in autapses after Syt1 deletion ( Liu et al, 2009 ; Wierda and Sørensen, 2014 ), but differences in culture protocol ( Bekkers, 2020 ), or genetic background could play a role.…”

“…1) and Syt1 9Pro expressing synapses (Fig. 2) that mEPSC frequencies were increased, in line with previous studies in autapses [5,20,55] and networks [28,29,36,56]. It remains enigmatic why others found a similar increase in spontaneous release in networks but not in autapses after Syt1 deletion [57,58], but differences in culture protocol [59], or genetic background could play a role.…”

Post-tetanic potentiation lowers the energy barrier for synaptic vesicle fusion independently of Synaptotagmin-1

“…1) and Syt1 9Pro expressing synapses (Fig. 2) that mEPSC frequencies were increased, in line with previous studies in autapses [5,20,55] and networks [28,29,36,56]. It remains enigmatic why others found a similar increase in spontaneous release in networks but not in autapses after Syt1 deletion [57,58], but differences in culture protocol [59], or genetic background could play a role.…”

A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release

“…9 ), indicating that SNT-1 still functions as a fast Ca 2+ sensor even when it is not tethered on SVs. Interestingly, the cytoplasmic region of vertebrate Syt1 retains the ability to synchronize SV release in response to a train stimulus ( Díez-Arazola et al, 2020 ). In contrast, we show that SNT-3 is cytoplasmic but mediates delayed neurotransmitter release.…”