Doc2b Ca2+-binding site mutants act as a gain of function at rest and loss of function during neuronal activity

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 Ca 2+ -binding double C 2 proteins Doc2a and –b regulate both spontaneous and asynchronous evoked release, but the mechanism remains unclear. Here, we compared wildtype Doc2b with two Ca 2+ binding site mutants named DN and 6A, respectively considered gain- and loss-of function mutants and carrying the substitutions D218,220N or D163,218,220,303,357,359A. We found that both mutants bound phospholipids at low free Ca 2+ concentrations and were membrane-associated in neurons at rest, mimicking a Ca 2+ activated state. Their overexpression in hippocampal primary neurons culture had similar effects on spontaneous and evoked release, inducing higher 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 but as loss-of-function during neuronal activity.