Abstract
The presynaptic active zone protein Munc13 is essential for neurotransmitter release, playing key roles in vesicle docking and priming. Mechanistically, it is thought that the C2A domain of Munc13 inhibits the priming function by homodimerization, and that RIM disrupts the autoinhibitory homodimerization forming monomeric priming-competent Munc13. However, it is unclear whether the C2A domain mediates other Munc13 functions in addition to this inactivation–activation switch. Here, we utilize mutations that modulate the homodimerization and heterodimerization states to define additional roles of the Munc13 C2A domain. Using electron microscopy and electrophysiology in hippocampal cultures, we show that the C2A domain is critical for additional steps of vesicular release, including vesicle docking. Optimal vesicle docking and priming is only possible when Munc13 heterodimerizes with RIM via its C2A domain. Beyond being a switching module, our data suggest that the Munc13-RIM heterodimer is an active component of the vesicle docking, priming and release complex.
Highlights
The presynaptic active zone protein Munc[13] is essential for neurotransmitter release, playing key roles in vesicle docking and priming
The complete loss of docked synaptic vesicles observed in electron tomography and transmission electron microscopy (TEM) studies using cryofixation techniques on Munc13-1/2 double knockout (DKO) neurons[3,4,5] clearly demonstrate that Munc13s are involved in SV docking
The interaction between the Munc[] C2A domain and the RIM zinc-finger domain (ZF) domain[39] and the proposed role of RIM in vesicle docking[2,11,12,13] suggest that the function of Munc[] in vesicle docking could be mediated by the interaction of the C2A domain with RIM
Summary
The presynaptic active zone protein Munc[13] is essential for neurotransmitter release, playing key roles in vesicle docking and priming. Loss-of-function studies in Caenorhabditis elegans, Drosophila melanogaster and mammalian synapses have identified several AZ protein families that play a key role in vesicle docking and priming, including Munc[13] (refs 1–6), CAPS4,7, a-Liprin[8], ELKS9 and RIM2,10–13. Neurons from Munc13-1/2 double knockout (DKO) lack of a measurable ready releasable pool (RRP) and show no spontaneous or Ca2 þ -dependent evoked release[1,6], similar to the observations made in the corresponding unc-13 mutant in C. elegans[15] and in D. melanogaster dunc-13 mutant[16] This absence of neurotransmission was initially attributed to events downstream of SV docking, based on ultrastructural data from conventional chemical fixation[1]. All Munc13s expressed at central synapses are large multidomain proteins and share a well-conserved C-terminal region, including a diacylglycerol a Ca2 þ /phospholipid binding C2B (DAG) domain, binding a MUN
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