Intersectin‐1 enhances calcium‐dependent replenishment of the readily releasable pool of synaptic vesicles during development

Intersectin-1 (Itsn1) is a scaffold protein that plays a key role in coupling exocytosis and endocytosis of synaptic vesicles (SVs). However, it is unclear whether and how Itsn1 regulates these processes to support efficient neurotransmission during development. To address this, we examined the calyx of Held synapse in the auditory brainstem of wild-type and Itsn1 mutant mice before (immature) and after (mature) the onset of hearing. Itsn1 was present in the pre- and postsynaptic compartments at both developmental stages. Loss of function of Itsn1 did not alter presynaptic action potentials, Ca²⁺ entry via voltage-gated Ca²⁺ channels (VGCCs), transmitter release or short-term depression (STD) induced by depletion of SVs in the readily releasable pool (RRP) in either age group. Yet, fast Ca²⁺-dependent recovery from STD was attenuated in mature mutant synapses, while it was unchanged in immature mutant synapses. This deficit at mature synapses was rescued by introducing the DH-PH domains of Itsn1 into the presynaptic terminals. Inhibition of dynamin, which interacts with Itsn1 during endocytosis, had no effect on STD recovery. Interestingly, we found a developmental enrichment of Itsn1 near VGCCs, which may underlie the Itsn1-mediated fast replenishment of the RRP. Consequently, the absence of Itsn1 in mature synapses led to a higher failure rate of postsynaptic spiking during high-frequency synaptic transmission. Taken together, our findings suggest that Itsn1 translocation to the vicinity of VGCCs during development is crucial for accelerating Ca²⁺-dependent RRP replenishment and sustaining high-fidelity neurotransmission. KEY POINTS: Itsn1 is expressed in the pre- and postsynaptic compartments of the calyx of Held synapse. Developmental upregulation of vesicular glutamate transporter-1 is Itsn1 dependent. Itsn1 does not affect basal synaptic transmission at different developmental stages. Itsn1 is required for Ca²⁺-dependent recovery from short-term depression in mature synapses. Itsn1 mediates the recovery through its DH-PH domains, independent of its interactive partner dynamin. Itsn1 translocates to the vicinity of presynaptic Ca²⁺ channels during development. Itsn1 supports high-fidelity neurotransmission by enabling rapid recovery from vesicular depletion during repetitive activity.