RIM Proteins Activate Vesicle Priming by Reversing Autoinhibitory Homodimerization of Munc13

Abstract

At a synapse, the presynaptic active zone mediates synaptic vesicle exocytosis. RIM proteins are active-zone scaffolding molecules that – among others – mediate vesicle priming, and directly or indirectly interact with most other essential presynaptic proteins. In particular, the Zn2+-finger domain of RIMs binds to the C2A-domain of the priming factor Munc13, which forms a homodimer in the absence of RIM, but a heterodimer with it. Here we show that RIMs mediate vesicle priming not by coupling Munc13 to other active zone proteins as thought, but by directly activating Munc13. Specifically, we found that the isolated Zn2+-finger domain of RIMs autonomously promotes vesicle priming by binding to Munc13, thereby relieving Munc13 homodimerization. Strikingly, constitutively monomeric mutants of Munc13 rescued priming in RIM-deficient synapses, whereas wild-type Munc13 did not. Both mutant and wild-type Munc13, however, rescued priming in Munc13-deficient synapses. Thus, homodimerization of Munc13 inhibits its priming function, and RIMs activate priming by disrupting Munc13 homodimerization.

RIM genes differentially contribute to organizing presynaptic release sites

“…In RIM1/2 double-deficient neurons, the RRP is massively decreased because RIM proteins mediate synaptic vesicle priming by disinhibiting inactive Munc13 homodimers (14,19,29). We here found that similar to evoked IPSCs and spontaneous release, deletion of individual RIM genes decreased the RRP size (Fig.…”

“…We here found that similar to evoked IPSCs and spontaneous release, deletion of individual RIM genes decreased the RRP size (Fig. S4), although the size of the decrease is less than that observed with the double deletion (14,19,29). This experiment suggests that RIM1 and RIM2 promote priming in an additive fashion and cannot compensate in priming for each other's loss.…”

“…To address this question, we examined neurotransmitter release in cultured neurons in which the Rims1 gene encoding RIM1α and RIM1β and the Rims2 gene encoding RIM2α, RIM2β, and RIM2γ were acutely deleted either alone or together. In these experiments, we restricted our analyses to inhibitory synapses for two reasons: (i) we showed that excitatory and inhibitory synaptic transmission are similarly affected in RIM double-deficient neurons (14,19), suggesting that the phenotype observed in inhibitory synapses likely applies to all synapses; and (ii) inhibitory synaptic responses are more precisely analyzed in cultured neurons than excitatory responses because they are not contaminated by recurrent network activity (20).…”

“…Glial growth was controlled by continuous presence of cytosine arabinoside starting at 2-3 d in vitro (DIV). Lentiviruses expressing GFP-tagged cre-recombinase, a recombination deficient deletion mutant thereof, or cre-recombinase and RIM2γ by use of bicistronic expression (14,19,21) were generated in transfected HEK293T cells as described (21,22), and the HEK293T cell supernatant containing the lentiviruses was harvested 48 h after transfection. Cell debris was removed by centrifugation (5 min at 750 × g), and neuronal cultures were infected with freshly prepared lentiviruses contained in the HEK293T cell supernatant 3-5 d after plating.…”

Superpriming of synaptic vesicles after their recruitment to the readily releasable pool

“…The present study and previous reports by Wadel et al (3) and Müller et al (7) show that primed SVs just recruited from SRP after a predepolarization are somewhat less Ca 2+ -sensitive than FRP SVs at steady state. Recently, it has been shown that activation of Munc13 requires its interaction with RIM, which renders the MUN domain of Munc13 to be exposed (23,24). Rab3-interacting molecule (RIM) interacts with Ca 2+ channels, and thus may be closely associated with them in the active zone.…”