This is a preprint review of Doc2b Ca2+-binding site mutants act as a gain of function at rest and loss of function during neuronal activity by Quentin Bourgeois-Jaarsma, Matthijs Verhage and Alexander J Groffen. The preprint was originally posted on bioRxiv on January 31, 2019 (DOI: https://doi.org/10.1101/536581). In this manuscript, Bourgeois-Jaarsma and colleagues work on identifying the role of the Ca2+-binding double C2 protein, Doc2b, in neurotransmission. They focus on two Doc2b mutants (Doc2bDN and Doc2b6A) and observe that the two mutant proteins 1) localize at plasma membrane of neurons less activity-independently than wildtype Doc2b, and 2) show different Ca2+-dependent pattern from wildtype Doc2b when binding with phospholipid in vitro. They also show that in cultured neurons (either wild type or Doc-KO), overexpression of the two mutants 3) both increases the frequency of spontaneous neurotransmitter release at rest, 4) introduces different patterns of response overtime when repetitively stimulated, but 5) doesn’t affect the neurons’ morphology. From these, they conclude that the two mutants share similar behaviors both at rest and during neuronal activity: increased spontaneous activity at rest and decreased activity during neuronal activity, and thus showing Doc2b plays an important role in Ca2+-dependent phospholipid association.In general, the logic of this study is clear, and the authors present their work in a way easy to follow. After visualizing subcellular localization of the mutants, the authors then test their Ca2+-dependency in phospholipid binding, effects of overexpression of them in neurons at rest, during activity and on morphology. Also, different models, strategies and experiments are used to address questions and hypothesis at each step. The combination of in vitro Ca2+ binding experiment, in vivo subcellular localization and physiology recording really makes the story coherent and intriguing to think deeply about. Moreover, the authors consider various possibilities and carefully design their controls.However, one of the weaknesses would be the conclusion that overexpression of the mutant Doc2b proteins would lead to an increased short-term depression. The authors somehow tend to over-normalize the data in some cases and make the interpretations not convincing enough.