The lipid transporter ORP2 regulates synaptic neurotransmitter release via two distinct mechanisms.

Cholesterol is crucial for neuronal synaptic transmission, assisting in the molecular and structural organization of lipid rafts, ion channels, and exocytic proteins. Although cholesterol absence was shown to result in impaired neurotransmission, how cholesterol locally traffics and its route of action are still under debate. Here, we characterized the lipid transfer protein ORP2 in murine hippocampal neurons. We show that ORP2 preferentially localizes to the presynapse. Loss of ORP2 reduces presynaptic cholesterol levels by 50%, coinciding with a profoundly reduced release probability, enhanced facilitation, and impaired presynaptic calcium influx. In addition, ORP2 plays a cholesterol-transport-independent role in regulating vesicle priming and spontaneous release, likely by competing with Munc18-1 in syntaxin1A binding. To conclude, we identified a dual function of ORP2 as a physiological modulator of the synaptic cholesterol content and a regulator of neuronal exocytosis.