RIM-binding protein 2 regulates release probability by fine-tuning calcium channel localizytion at murine hippocampal synapses

The tight spatial coupling of synaptic vesicles and voltage-gated Ca channels (CaVs) ensures efficient action potential-triggered neurotransmitter release from presynaptic active zones (AZs). Rab-interacting molecule-binding proteins (RIM-BPs) interact with Ca channels and via RIM with other components of the release machinery. Although human RIM-BPs have been implicated in autism spectrum disorders, little is known about the role of mammalian RIM-BPs in synaptic transmission. We investigated RIM-BP2 deficient murine hippocampal neurons in cultures and slices. Short-term facilitation is significantly enhanced in both model systems. Detailed analysis in culture revealed a reduction in initial release probability, which presumably underlies the increased short-term facilitation. Superresolution microscopy revealed an impairment in CaV2.1 clustering at AZs, which likely alters Ca nanodomains at release sites and thereby affects release probability. Additional deletion of RIM-BP1 does not exacerbate the phenotype, indicating that RIM-BP2 is the dominating RIM-BP isoform at these synapses. Significance Highly regulated and precise positioning of Ca channels at the active zone (AZ) controls Ca nanodomains at release sites. Their exact localization affects vesicular release probability (PVR) and is important for proper synaptic transmission during repetitive stimulation. We provide a detailed analysis of synaptic transmission combined with superresolution imaging of the AZ organization in mouse hippocampal synapses lacking Rab-interacting molecule-binding protein 2 (RIMBP2). By dual and triple channel time-gated stimulated emission depletion (gSTED) microscopy we directly show that RIM-BP2 fine-tunes voltage-gated Ca channel 2.1 (CaV2.1) localization at the AZ. We reveal that RIM-BP2 likely regulates the Ca nanodomain by positioning CaV2.1 channels close to synaptic vesicle release sites. Loss of RIM-BP2 reduces PVR and alters short term plasticity. Introduction At the presynapse, coupling between action potentials (APs) and synaptic vesicle fusion is exquisitely precise, ensuring high temporal fidelity of neuron-to-neuron signaling in the nervous system. Two properties are thought to be responsible for this remarkable precision: a highly efficient release apparatus that transduces Ca signals into vesicle fusion and a tightly organized active zone (AZ), where the release apparatus and voltage-gated Ca channels (CaVs) are spatially coupled. Rab-interacting molecules (RIM) are thought to contribute to both properties, because loss of RIM impairs vesicle priming (1) and CaV localization at the AZ (2). RIM-binding proteins (RIM-BPs) directly interact with RIM (3), the pore-forming subunits of CaV1 and CaV2 channels (2, 4, 5), and Bassoon (5), and have therefore been suggested to play a role in presynaptic CaV localization. The Drosophila homolog of RIM-binding proteins (DRBP) is indeed crucial for neurotransmitter release at the AZ of neuromuscular junctions (NMJs) because loss of DRBP reduces CaV abundance and impairs the integrity of the AZ scaffold (6). DRBP-deficient flies show severe impairment of neurotransmitter release along with increased short-term facilitation (6, 7). Recently, Acuna et al. (8) published a report on the combined loss of RIM-BP1 and RIM-BP2 in mouse synapses. The authors report that although RIM-BPs are not essential for synaptic transmission, AP-triggered neurotransmitter release is more variable and the sensitivity to the Ca chelator EGTA is increased at the Calyx of Held, suggesting a larger coupling distance of CaV and the release machinery. In the present study, we further investigated the consequences of constitutive deletion of RIM-BP2 on the structure and function of mouse hippocampal synapses. We show that loss of RIM-BP2 leads to a moderate reduction in initial release probability, which translates into profound changes in short-term plasticity (STP). This deficit can be overcome by increasing extracellular Ca. We established triple channel time-gated stimulated emission depletion (gSTED) microscopy for RIM-BP2, Munc13-1 and Bassoon, as well as for CaV2.1, RIM and the postsynaptic marker protein Homer1. Using this technique, we demonstrate that although synapse number and molecular architecture appear essentially intact, RIM-BP2 is necessary for proper coclustering of the P/Q-type CaV subunit CaV2.1 with the AZ protein Bassoon at hippocampal CA3-CA1 synapses. We hypothesize that the observed change in CaV localization causes a discrete alteration in the coupling of Ca influx and exocytosis, and thereby modifies release probability and, consequently, STP. Additional deletion of RIM-BP1 did not strengthen the changes in short-term facilitation, supporting our hypothesis that RIM-BP2 is the major RIM-BP paralog at glutamatergic hippocampal synapses.