Non - conserved Ca 2 + / calmodulin binding sites in Munc 13 s differentially 1 control synaptic short - term plasticity 2 3

39 40 Munc13s are presynaptic proteins that mediate synaptic vesicle priming and thereby control 41 the size of the readily releasable pool of vesicles. During high synaptic activity, Munc13-1 42 and its closely related homolog ubMunc13-2 bind Ca 2+ /calmodulin, resulting in enhanced 43 priming activity and in changes of short-term synaptic plasticity characteristics. Here, we 44 studied whether bMunc13-2 and Munc13-3, two remote isoforms of Munc13-1 with a 45 neuronal subtype-specific expression pattern, mediate synaptic vesicle priming and regulate 46 short-term synaptic plasticity in a Ca 2+ /calmodulin-dependent manner. We identified a single 47 functional Ca 2+ /calmodulin binding site in these isoforms, and provide structural evidence 48 that all Munc13s employ a common mode of interaction with calmodulin, despite the lack of 49 sequence homology between their Ca 2+ /calmodulin binding sites. Electrophysiological 50 analysis showed that, during high frequency activity, Ca 2+ /calmodulin binding positively 51 regulates the priming activity of bMunc13-2 and Munc13-3, resulting in an increase in the 52 size of the readily releasable pool of vesicles and subsequently in strong short-term synaptic 53 enhancement of neurotransmission. We conclude that Ca 2+ /calmodulin-dependent regulation 54 of priming activity is structurally and functionally conserved in all Munc13 proteins, and that 55 the composition of Munc13 isoforms in a neuron differentially controls its short-term 56 synaptic plasticity characteristics.