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L-type calcium channels are involved in fast endocytosis at the mouse neuromuscular junction

Authors

  • Paula P. Perissinotti,

    1. Departmento de Fisiología Biología Molecular y Celular, Universidad de Buenos Aires, Instituto de Fisiología Biología Molecular y Neurociencias, UBA CONICET, Argentina
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  • Bárbara Giugovaz Tropper,

    1. Departmento de Fisiología Biología Molecular y Celular, Universidad de Buenos Aires, Instituto de Fisiología Biología Molecular y Neurociencias, UBA CONICET, Argentina
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  • Osvaldo D. Uchitel

    1. Departmento de Fisiología Biología Molecular y Celular, Universidad de Buenos Aires, Instituto de Fisiología Biología Molecular y Neurociencias, UBA CONICET, Argentina
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Dr O. D. Uchitel, as above.
E-mail: odu@fbmc.fcen.uba.ar

Abstract

We used fluorescence microscopy of FM dyes-labeled synaptic vesicles and electrophysiological recordings to examine the functional characteristics of vesicle recycling and study how different types of voltage-dependent Ca2+ channels (VDCCs) regulate the coupling of exocytosis and endocytosis at mouse neuromuscular junction.

Our results demonstrate the presence of at least two different pools of recycling vesicles: a high-probability release pool (i.e. a fast destaining vesicle pool), which is preferentially loaded during the first 5 s (250 action potentials) at 50 Hz; and a low-probability release pool (i.e. a slow destaining vesicle pool), which is loaded during prolonged stimulation and keeps on refilling after end of stimulation.

Our results suggest that a fast recycling pool mediates neurotransmitter release when vesicle use is minimal (i.e. during brief high-frequency stimulation), while vesicle mobilization from a reserve pool is the prevailing mechanism when the level of synaptic activity increases.

We observed that specific N- and L-type VDCC blockers had no effect on evoked transmitter release upon low-frequency stimulation (5 Hz). However, at high-frequency stimulation (50 Hz), L-type Ca2+ channel blocker increased FM2-10 destaining and at the same time diminished quantal release. Furthermore, when L-type channels were blocked, FM2-10 loading during stimulation was diminished, while the amount of endocytosis after stimulation was increased.

Our experiments suggest that L-type VDCCs promote endocytosis of synaptic vesicles, directing the newly formed vesicles to a high-probability release pool where they compete against unused vesicles.

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