Promotion of endocytosis efficiency through an ATP‐independent mechanism at rat calyx of Held terminals

Abstract

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 increases drastically. This paradox raises the interesting question of whether increased activity recruits ATP-independent mechanism(s) to accelerate endocytosis at the same time as preserving ATP availability for other tasks. To address this issue, we studied ATP requirement in three typical forms of endocytosis at 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 days 8-15, suggesting its existence before and after hearing onset. This endocytosis was not affected by a reduction of exocytosis using the light chain of botulinum toxin C, nor by block of clathrin-coat maturation. It was abolished by EGTA, which preferentially blocked endocytosis of retrievable membrane pre-existing at the surface, and was 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. The results of the present study 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.

Synaptic Vesicle Endocytosis in Different Model Systems

“…Moreover, a GTP-independent slow endocytic pathway ( τ = 14 s) is also observed upon repeated RRP-depleting pulses (Xu et al, 2008 ), suggesting the involvement of dynamin-independent bulk endocytosis (Wu et al, 2014 ). On the other hand, blocking clathrin-mediated endocytosis slows down the slow capacitance recovery induced by 20 ms depolarizing pulses (Yue et al, 2017 ). Therefore, clathrin-mediated endocytosis might cooperate with bulk endocytosis in the slow pathway.…”

“…Compounds that inhibit formin and non-muscle myosin II block slow endocytosis at the calyx (Soykan et al, 2017 ), indicating the involvement of actin and myosin dynamics. Pitstop 1, which disrupts amphiphysin-clathrin interactions, impairs but does not block slow endocytosis at the calyx (Yue et al, 2017 ). DNF, a peptide that disrupts amphiphysin-AP2 interactions, has a similar effect (Yue et al, 2017 ).…”

“…Pitstop 1, which disrupts amphiphysin-clathrin interactions, impairs but does not block slow endocytosis at the calyx (Yue et al, 2017 ). DNF, a peptide that disrupts amphiphysin-AP2 interactions, has a similar effect (Yue et al, 2017 ). These results imply that different components of synaptic vesicle endocytosis at the calyx have differential molecular requirements.…”

Lipid signaling to membrane proteins: From second messengers to membrane domains and adapter-free endocytosis

“…Other types of Ca-dependent endocytosis that might be related to delayed MEND are “bulk endocytosis” ( Cheung et al, 2010 ) and “excess endocytosis” ( Engisch and Nowycky, 1998 ), which occurs in secretory cells. Although recent work on bulk endocytosis suggests an involvement of actin and tropomyosin in some secretory cells ( Gormal et al, 2017 ), Ca-dependent endocytosis that occurs in rat calyx of Held terminals appears to be rather similar to Ca-activated MEND ( Yue et al, 2017 ). Ca-activated endocytosis in astrocytes ( Jiang and Chen, 2009 ) has very similar characteristics to the fast forms of Ca-activated MEND ( Lariccia et al, 2011 ).…”

Membrane Tension Inhibits Rapid and Slow Endocytosis in Secretory Cells

“…Dehydration may regulate endocytosis and exocytosis by regulating cell volume and membrane tension. The often-reported endocytosis overshoot (23,(42)(43)(44)(45), which is facilitated by a reduced membrane tension (Figs. 1 and 3), may be a physiological mechanism to control the membrane area of nerve terminals and endocrine cells, which in turn may regulate membrane tension and thus provide a feedback loop for the regulation of membrane tension.…”