RIM-BP2 is required for rapid neurotransmitter release through regulation of Ca²⁺ channel clustering at hippocampal mossy fiber terminals

Reviewer #1 (Public Review):

This nice study by Miyano combines slice electrophysiology and superresolution microscopy to address the role of RBP2 in Ca2+ channel clustering and neurotransmitter release at hippocampal mossy fiber terminals. While a number of studies demonstrated a critical role for RBPs in clustering Ca2+ channels at other synapses and some provided evidence for a role of the protein in molecular coupling of Ca2+ channels and release sites, the present study targets another key synapse that is an important model for presynaptic studies and offers access to a microdomain controlled synaptic vesicle (SV) release mechanism with low initial release probability.

Summarizing a large body of high-quality work, the authors demonstrate reduced Ca2+ currents and a reduced release probability. They attribute the latter to the reduced Ca2+ influx and can restore release by increasing Ca2+ influx. Moreover, they propose an altered fusion competence of the SVs, which is not so strongly supported by the data in my view.

The effects are relatively small, but I think the careful analysis of the RBP role at the mossy fiber synapse is an important contribution.

Reviewer #2 (Public Review):

The proper expression and organization of CaV channels at the presynaptic release sites are subject to coordinative and redundant control of many active zone-specific molecules including RIM-BPs. Previous studies have demonstrated that ablation of RIM-BPs in various mammalian synapses causes significant impairment of synaptic transmission, either by reducing CaV expression or decoupling CaV from synaptic vesicles. The mechanisms remain unknown.

In the manuscript, Sakaba and colleagues aimed to examine the specific role of RIM-BP2 at the hippocampal mossy fiber-CA3 pyramidal cell synapse, which is well-characterized by low initial release probability and strong facilitation during repetitive stimulation. By directly recording Ca2+ currents and capacitance jumps from the MF boutons, which is very challenging but feasible, they showed that depolarization-evoked Ca2+ influx was reduced significantly (~39%) by KO of RIM-BP2, but no impacts on Ca-induced exocytosis and RRP (measured by capacitance change). They used STED microscopy to image the spatial distribution of the CaV2.1 cluster but found no change in the cluster number with a slight decrease in cluster intensity (~20%). They concluded that RIM-BP2 functions in tonic synapses by reducing CaV expression and thus differentially from phasic synapses by decoupling CaV-SV.

In general, they provide solid data showing that RIM-BP2 KO reduces Ca influx at MF-CA3 synapse, but the phenotype is not new as Moser and colleagues have also used presynaptic recording and capacitance measurement and shown that RIM-BP2 KO reduces Ca2+ influx at hair cell active zone (Krinner et al., 2017), although at different synapse model expressing CaV1.3 instead of CaV2.1. Further, the concept that RIM-BP2 plays diverse functions in transmitter release at different central synapses has also been proposed with solid evidence (Brockmann et al., 2019).