Characterization of two distinct modes of endophilin in clathrin-mediated endocytosis

► Endophilin E264A and E264R represent endophilin open and closed modes respectively. ► E264R significantly reduces its interaction with dynamin or Ca2+ channels. ► E264A enhances the short-term synaptic depression in cultured hippocampal neurons. ► Modal switching at E264 is crucial for regulating endophilin function in endocytosis.