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

Abstract

Ca2+-dependent neurotransmitter release requires synaptotagmins as Ca2+ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains—C2A and C2B—to Ca2+. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca2+ sensor in Caenorhabditis elegans. Here, we report a new Ca2+ sensor, SNT-3, which triggers delayed Ca2+-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca2+ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca2+ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca2+ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini–mediated membrane tethering. Our findings therefore reveal a novel dual Ca2+ sensor system in C. elegans and provide significant insights into Ca2+-regulated exocytosis.

The Role of Calmodulin vs. Synaptotagmin in Exocytosis

“…Later studies have found that it is the C2 domain protein Syt, but not the EF-hand protein, acts as the primary Ca 2+ sensor for membrane fusion. This notion was confirmed by studies in many species, including mouse (Geppert et al, 1994;Nishiki and Augustine, 2004), Drosophila (Lee et al, 2013), zebra fish (Wen et al, 2010), as well as the recent studies in the nematode Caenorhabditis elegans (Li et al, 2018(Li et al, , 2021. Meanwhile, EF-hand protein CaM, has been found to play an active role in exocytotic steps prior to fusion via interaction with multiple functional proteins in a Ca 2+ -dependent manner (Junge et al, 2004;Zikich et al, 2008).…”

Sexually Dimorphic Neurotransmitter Release at the Neuromuscular Junction in Adult Caenorhabditis elegans

“…Sex difference in extracellular Ca 2+ sensitivity of the neurotransmission at the NMJs indicates different plasticity properties of the NMJs between adult males and hermaphrodites. Previous research demonstrated that synaptic plasticity at the cholinergic NMJs of adult hermaphrodites was characterized with synaptic depression in response to a train stimulus in motor neurons (Liu et al, 2009;Li et al, 2021). We therefore sought to investigate whether biological sex affects synaptic plasticity at the cholinergic NMJs.…”

“…Weaker depression in male worms may arise from a faster replenishment rate of synaptic vesicles in the presynaptic terminals. We thus calculated the replenishment rate by fitting the slope of the cumulative amplitude of evoked EPSCs as previously described Li et al, 2021) and found that adult male worms displayed faster replenishment rates in response to both 1− and 5-Hz stimulus compared to adult hermaphrodites (Figures 5G-J). Taken together, our results demonstrated the synaptic depression at the cholinergic NMJs of both adult males and hermaphrodites, and revealed weaker depression and faster replenishment rates of vesicle release at the cholinergic NMJ in adult males.…”

Protocols for electrophysiological recordings and electron microscopy at C. elegans neuromuscular junction

“…The quality of the electrophysiology data is directly determined by the quality of the preparation. A good dissection allows to acquire high quality of mEPSC, mIPSC, and evoked EPSC ( Figure 3 ) ( Li et al., 2019 ), or sucrose- or drug-activated currents ( Figure 3 ) ( Li et al., 2021 ). For wild-type worms, the mEPSC and mIPSC frequencies vary from 20 Hz–80 Hz, with an average of around 45 Hz.…”

“…For complete details on the use and execution of this protocol, please refer to Liu et al. (2021) and Li et al. (2021) .…”