Endophilin is a membrane binding protein with curvature-generating/sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin, and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles but not of clathrin-coated pits at synapses. The absence of endophilin impairs, but does not abolish synaptic transmission and results in perinatal lethality, while partial endophilin absence causes severe neurological defects, including epilepsy, and neurodegeneration. Our data supports a model in which endophilin recruitment to coated pit necks, due to its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.

Neurotransmitter release is triggered by an increase in the cytosolic Ca2+ concentration ([Ca2+]i), but it is unknown whether the Ca2+-sensitivity of vesicle fusion is modulated during synaptic plasticity. We investigated whether the potentiation of neurotransmitter release by phorbol esters, which target presynaptic protein kinase C (PKC)/munc-13 signalling cascades, exerts a direct effect on the Ca2+-sensitivity of vesicle fusion. Using direct presynaptic Ca2+-manipulation and Ca2+ uncaging at a giant presynaptic terminal, the calyx of Held, we show that phorbol esters potentiate transmitter release by increasing the apparent Ca2+-sensitivity of vesicle fusion. Phorbol esters potentiate Ca2+-evoked release as well as the spontaneous release rate. We explain both effects by an increased fusion 'willingness' in a new allosteric model of Ca2+-activation of vesicle fusion. In agreement with an allosteric mechanism, we observe that the classically high Ca2+ cooperativity in triggering vesicle fusion (approximately 4) is gradually reduced below 3 microM [Ca2+]i, reaching a value of <1 at basal [Ca2+]i. Our data indicate that spontaneous transmitter release close to resting [Ca2+]i is a consequence of an intrinsic property of the molecular machinery that mediates synaptic vesicle fusion.

a-RIMs and Munc13s are active zone proteins that control priming of synaptic vesicles to a readily releasable state, and interact with each other via their N-terminal sequences. The a-RIM N-terminal sequence also binds to Rab3s (small synaptic vesicle GTPases), an interaction that regulates presynaptic plasticity. We now demonstrate that a-RIMs contain adjacent but separate Munc13-and Rab3-binding sites, allowing formation of a tripartite Rab3/RIM/ Munc13 complex. Munc13 binding is mediated by the a-RIM zinc-finger domain. Elucidation of the three-dimensional structure of this domain by NMR spectroscopy facilitated the design of a mutation that abolishes a-RIM/ Munc13 binding. Selective disruption of this interaction in the calyx of Held synapse decreased the size of the readily releasable vesicle pool. Our data suggest that the ternary Rab3/RIM/Munc13 interaction approximates synaptic vesicles to the priming machinery, providing a substrate for presynaptic plasticity. The modular architecture of a-RIMs, with nested binding sites for Rab3 and other targets, may be a general feature of Rab effectors that share homology with the a-RIM N-terminal sequence.

The existence of neuron specific endocytic protein isoforms raises questions about their importance for specialized neuronal functions. Dynamin, a GTPase implicated in the fission reaction of endocytosis, is encoded by three genes, two of which, dynamin 1 and 3, are highly expressed in neurons. We show that dynamin 3, thought to play a predominantly postsynaptic role, has a major presynaptic function. While lack of dynamin 3 does not produce an overt phenotype in mice, it worsens the dynamin 1 KO phenotype, leading to perinatal lethality and a more severe defect in activity-dependent synaptic vesicle endocytosis. Thus, dynamin 1 and 3, which together account for the overwhelming majority of brain dynamin, cooperate in supporting optimal rates of synaptic vesicle endocytosis. Persistence of synaptic transmission in their absence indicates that if dynamin plays essential functions in neurons, such functions can be achieved by the very low levels of dynamin 2.