"Delayed" endocytosis is regulated by extracellular Ca2+ in snake motor boutons

When cooled below approximately 7 degrees C, recently endocytosed vesicles in the motor terminals of the garter snake fail to shed their clathrin coats. Perhaps as a result, the terminals complete only about one-half of the compensatory endocytosis expected after a given period of stimulation. Upon return to room temperature (RT), endocytosis resumes immediately and is complete within minutes. This "delayed" endocytosis following release from cold block provides an opportunity to study clathrin-dependent endocytotic mechanisms in temporal isolation from those events, such as Ca2+ entry and consequent exocytosis, that are normally associated with the activation of nerve terminals. We have taken advantage of clathrin decoating blockade to examine the rate, temperature dependence and extracellular Ca2+ dependence of endocytosis at the snake nerve-muscle synapse. Endocytosis was fast at RT (complete in < 1 min) and markedly faster still at 35 degrees C. Moreover, the rate of endocytosis varied significantly with change in [Ca2+]o; the rate at 7.2 mM (single exponential time constant, approximately 3 s) was approximately double that at 0 mM (single exponential time constant, approximately 7 s). Thus, membrane retrieval via clathrin is rapid and, due to its dependence on [Ca2+]o, potentially regulated by changes in the milieu of the synaptic cleft during neural activity.