Rab-interacting molecule (RIM)-binding protein 2 (BP2) is a multidomain protein of the presynaptic active zone (AZ). By binding to RIM, bassoon (Bsn), and voltage-gated Ca²⁺ channels (Ca_V), it is considered to be a central organizer of the topography of Ca_V and release sites of synaptic vesicles (SVs) at the AZ. Here, we used RIM-BP2 knock-out (KO) mice and their wild-type (WT) littermates of either sex to investigate the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers (ANFs) with bushy cells (BCs) of the cochlear nucleus, a fast relay of the auditory pathway with high release probability. Disruption of RIM-BP2 lowered release probability altering short-term plasticity and reduced evoked EPSCs. Analysis of SV pool dynamics during high-frequency train stimulation indicated a reduction of SVs with high release probability but an overall normal size of the readily releasable SV pool (RRP). The Ca²⁺-dependent fast component of SV replenishment after RRP depletion was slowed. Ultrastructural analysis by superresolution light and electron microscopy revealed an impaired topography of presynaptic Ca_V and a reduction of docked and membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography of Ca_V, and promotes SV tethering and docking. This way RIM-BP2 is critical for establishing a high initial release probability as required to reliably signal sound onset information that we found to be degraded in BCs of RIM-BP2-deficient mice in vivo.

SIGNIFICANCE STATEMENT Rab-interacting molecule (RIM)-binding proteins (BPs) are key organizers of the active zone (AZ). Using a multidisciplinary approach to the calyceal endbulb of Held synapse that transmits auditory information at rates of up to hundreds of Hertz with submillisecond precision we demonstrate a requirement for RIM-BP2 for normal auditory signaling. Endbulb synapses lacking RIM-BP2 show a reduced release probability despite normal whole-terminal Ca²⁺ influx and abundance of the key priming protein Munc13-1, a reduced rate of SV replenishment, as well as an altered topography of voltage-gated (Ca_V)2.1 Ca²⁺ channels, and fewer docked and membrane proximal synaptic vesicles (SVs). This hampers transmission of sound onset information likely affecting downstream neural computations such as of sound localization.

Rab 相互作用分子 (RIM) 结合蛋白 2 (BP2) 是突触前活性区 (AZ) 的多域蛋白。通过与 RIM、巴松管 (Bsn) 和电压门控 Ca ²⁺通道 (Ca _V ) 结合，它被认为是 Ca _V地形的中心组织者和突触小泡 (SV) 在 AZ 的释放位点。在这里，我们使用 RIM-BP2 敲除 (KO) 小鼠及其任何性别的野生型 (WT) 同窝仔鼠来研究 RIM-BP2 在具有浓密细胞的听觉神经纤维 (ANF) 的固定突触的端球中的作用(BCs) 耳蜗核，一种具有高释放概率的听觉通路的快速中继。RIM-BP2 的破坏降低了释放概率，改变了短期可塑性并减少了诱发的 EPSC。在高频火车刺激期间对 SV 池动力学的分析表明，具有高释放概率的 SV 减少，但易于释放的 SV 池 (RRP) 的总体大小正常。钙²⁺RRP 消耗减慢后 SV 补充的依赖快速成分。通过超分辨率光学和电子显微镜进行的超微结构分析显示突触前 Ca _{V 的}形貌受损，AZ 处的对接和近膜 SV 减少。我们得出结论，RIM-BP2 组织了 Ca _V的地形，并促进了 SV 的束缚和对接。通过这种方式，RIM-BP2 对于建立高初始释放概率至关重要，以可靠地发出声音起始信息的信号，我们发现这些信息在体内RIM-BP2 缺陷小鼠的 BC 中被降解。

重要性声明Rab 相互作用分子 (RIM) 结合蛋白 (BP) 是活性区 (AZ) 的关键组织者。使用多学科方法处理保持突触的肾盏末端球，以高达数百赫兹的速率以亚毫秒精度传输听觉信息，我们证明了 RIM-BP2 对正常听觉信号的要求。尽管全末端 Ca ²⁺正常流入和关键启动蛋白 Munc13-1 丰度、SV 补充速率降低以及电压门控（Ca _V )2.1 Ca ²⁺通道，以及更少的对接和膜近端突触小泡 (SV)。这阻碍了声音起始信息的传输，可能会影响下游神经计算，例如声音定位。