However, a possible involvement of Ca2+-CaM-Munc13-1 signaling in release site clearance will have to be tested in future experiments. Munc13-1W464R KI calyces from P14–P17 mice exhibit

low PPRs at all inter-stimulus intervals tested (10–500 ms; Figure 7D), indicating higher release probability pr. Homeostatic processes leading to high pr were suggested to occur in the calyx of Held upon perturbation of synaptic transmission at the level of inner hair cells (Erazo-Fischer et al., 2007). It is thus possible that the high pr seen in Munc13-1W464R calyces may reflect a homeostatic compensatory mechanism that occurs in response to the physiological consequences of the Munc13-1W464R mutation in the calyx synapse or upstream of it. Alternatively, the high pr in Munc13-1W464R mutant calyces may indicate a modulatory LY294002 nmr effect of Munc13-1 activity on the release machinery. One such role was proposed based on the phenotype of neurons from KI mutant mice that carry a Munc13-1H567K mutation, which renders Munc13-1

insensitive to diacylglyerol and phorbol esters (Basu et al., 2007; Rhee et al., 2002). Cultured Munc13-1H567K neurons exhibit an increase in pr, which has been interpreted to reflect a gain-of-function effect of the H567K mutation, reducing the energy barrier for SV fusion buy AZD2281 downstream of SV priming (Basu et al., 2007). A similar scenario might arise

in the context of the Munc13-1W464R mutant calyces, which would be supported by our observation that at P14–P17, the fast time constant of release was slightly, albeit not significantly, faster in KI (τ1 = 0.8 ± 0.2 ms, 55%) compared to WT synapses (τ1 = 1.3 ± 0.5 ms, 60%; see Figures 4A and 4B), which is consistent with the slightly higher pr in the former. However, the H567K mutation likely destroys the zinc-finger structure of the C1 domain, thereby promoting an open conformation of Munc13-1 that mediates the gain-of-function effect. In contrast, the W464R mutation does not affect the α-helical structure of the Ca2+-CaM binding motif. Further studies are necessary to determine the reason Metalloexopeptidase for the increased pr in mature Munc13-1W464R calyces and how this might be linked to Munc13-1 regulation and synaptic function. In the present study, we used a combination of mouse genetics and electrophysiological recordings in the calyx of Held synapse to study the role of Ca2+-CaM-Munc13-1 signaling in presynaptic function and plasticity. With the Munc13-1W464R mutation, we were able to specifically pinpoint the role of Ca2+-CaM binding to Munc13-1 and to separate this process from the numerous other signaling pathways that are mediated by Ca2+-CaM and that may be affected upon pharmacological interference with Ca2+-CaM signaling.