Neurotransmission relies on membrane endocytosis to maintain vesicle supply and membrane stability. Endocytosis has been generally recognized as a major ATP-dependent function, which efficiently retrieves more membrane at elevated neuronal activity when ATP consumption within nerve terminals drastically increases. This paradox raises an interesting question whether increased activity recruits ATP-independent mechanism(s) to accelerate endocytosis while preserving ATP availability for other tasks. To address this issue, we studied ATP requirement in three typical forms of endocytosis at the rat calyx of Held terminals by whole-cell membrane capacitance measurements. At room temperature, blocking ATP hydrolysis effectively abolished slow endocytosis and rapid endocytosis, but only partially inhibited excess endocytosis following intense stimulation. The ATP-independent endocytosis occurred at calyces from postnatal 8–15 days, suggesting its existence before and after hearing onset. This endocytosis was not affected by reduction of exocytosis using the light chain of botulinum toxin C, or by block of clathrin-coat maturation. It was abolished by EGTA, which preferentially blocked endocytosis of retrievable membrane pre-existing at surface, and impaired by oxidation of cholesterol and inhibition of neutral sphingomyelinase. ATP-independent endocytosis became more significant at 34–35°C, and recovered membrane by an amount that on average was close to exocytosis. Our results suggest that activity and temperature recruit ATP-independent endocytosis of pre-existing membrane, in addition to ATP-dependent endocytosis, to efficiently retrieve membrane at nerve terminals. This less understood endocytosis represents a non-canonical mechanism regulated by lipids such as cholesterol and sphingomyelinase.
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