Doc2b 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 hippocampal primary cultured neurons had similar effects on spontaneous and evoked release, inducing high mEPSC frequencies and increased short-term depression. Together, these data suggest that the DN and 6A mutants both act as gain-of-function mutants at resting conditions.