Synucleinopathy such as Parkinsons disease are associated with missense or multiplication mutations of α-synuclein,which is expressed in nerve terminals.α-Synuclein mutations can impair synaptic transmission at the presymptomatic stage,but the underlying mechanisms remain unclear.Taking advantage of electrophysiological analysis at a giant nerve terminal in mouse/rat,the calyx of Held,we found that overexpression of the disease-related human α-synucleinA53T and acute supply of α-synucleinA53T or α-synuclein impaired vesicle exocytosis by inhibiting both voltage-dependent calcium channels and endocytosis.Thus calcium channels and endocytosis are the presymptomatic targets of α-synucleinmutations.This work not only sheds light on the physiological functions of α-synuclein but also revealsmechanisms underlying the early synaptic dysfunctions in synucleinopathy,which has been difficult to study at small conventional synapses.