biostudies-literatureBourgeois-Jaarsma QUE, Erasmus Mundus Joint Doctorate program, ENC network cycle 4, project 3ZonMw14408https://www.ebi.ac.uk/biostudies/studies/S-EPMC6783474biostudies-literatureUnknown9(1)Communication between neurons involves presynaptic neurotransmitter release which can be evoked by action potentials or occur spontaneously as a result of stochastic vesicle fusion. The Ca²⁺-binding double C₂ proteins Doc2a and -b were implicated in spontaneous and asynchronous evoked release, but the mechanism remains unclear. Here, we compared wildtype Doc2b with two Ca²⁺ binding site mutants named DN and 6A, previously classified as gain- and loss-of-function mutants. They carry the substitutions D218,220N or D163,218,220,303,357,359A respectively. We found that both mutants bound phospholipids at low Ca²⁺ concentrations and were membrane-associated in resting neurons, thus mimicking a Ca²⁺-activated state. Their overexpression in hScientific reportsDoc2b Ca²⁺ binding site mutants enhance synaptic release at rest at the expense of sustained synaptic strength.PMC678347491113022Bourgeois-Jaarsma QGroffen AJVerhage MfalseDoc2b Ca²⁺ binding site mutants enhance synaptic release at rest at the expense of sustained synaptic strength.Communication between neurons involves presynaptic neurotransmitter release which can be evoked by action potentials or occur spontaneously as a result of stochastic vesicle fusion. The Ca²⁺-binding double C₂ proteins Doc2a and -b were implicated in spontaneous and asynchronous evoked release, but the mechanism remains unclear. Here, we compared wildtype Doc2b with two Ca²⁺ binding site mutants named DN and 6A, previously classified as gain- and loss-of-function mutants. They carry the substitutions D218,220N or D163,218,220,303,357,359A respectively. We found that both mutants bound phospholipids at low Ca²⁺ concentrations and were membrane-associated in resting neurons, thus mimicking a Ca²⁺-activated state. Their overexpression in h2019-01-01T00:00:00Z2019 Oct2025-04-05T13:18:19.236Z2019-10-30T08:18:48ZS-EPMC67834743159498010.1038/s41598-019-50684-1