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

Abstract

When cooled below ~7°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 clathrindependent endocytotic mechanisms in temporal isolation from those events, such as Ca 2+ 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 Ca 2+ dependence of endocytosis at the snake nerve-muscle synapse. Endocytosis was fast at RT (complete in < 1 min) and markedly faster still at 35°C. Moreover, the rate of endocytosis varied significantly with change in [Ca 2+ ] o ; the rate at 7.2 mM (single exponential time constant, 3 s) was approximately double that at 0 mM (single exponential time constant, ~7 s). Thus, membrane retrieval via clathrin is rapid and, due to its dependence on [Ca 2+ ] o , potentially regulated by changes in the milieu of the synaptic cleft during neural activity.

Another dimension to calcium signaling: a look at extracellular calcium

“…Using a temperature trick to separate temporally endoand exocytosis in garter snake motor neuron boutons, Teng and Wilkinson (Teng and Wilkinson, 2003) found increased rates of endocytosis when extracellular [Ca 2+ ] was elevated incrementally over the range of 0-7.2 mM. These effects were independent of intracellular [Ca 2+ ].…”

Synaptic Vesicle Mobility in Mouse Motor Nerve Terminals with and without Synapsin

“…Perhaps they are immobilized as a result of incomplete rematuration, for example, failure to lose their clathrin coats (cf. Teng and Wilkinson, 2003). To test this, we pulsed the temperature to 37°C (Fig.…”

Clathrin‐mediated endocytosis in snake motor terminals is directly facilitated by intracellular Ca2+

“…The freshly cut end of the muscle nerve was placed into the well, which was covered with petroleum jelly to prevent drying. The preparation was incubated for 4 h (RT), rinsed with saline and refrigerated (5 • C) overnight to permit diffusion and equilibration of the indicator into the nerve terminals (details in Teng & Wilkinson, 2003; see also Wu & Betz, 1996). Ratiometric imaging (340 nm and 380 nm excitation wavelengths) was performed using an inverted microscope equipped with an Intelligent Imaging Innovations (Denver, CO, USA) digital camera system and software.…”

“…These observations are limited, however, because they relate enhanced endocytosis to conditions in which very high (tens of micromolar; Neher, 1998) levels of Ca 2+ exist transiently in microdomains, or hot spots, near active calcium channels at AZs (but see also Cousin & Robinson, 2000). Less is known about the influence of bulk intraterminal Ca 2+ , which rises to ∼1 µm or less during activity (250 nm during a 5 Hz tetanus in snake; Teng & Wilkinson, 2003). Bulk levels of Ca 2+ are relevant to the substantial endocytosis that occurs after stimulation (when bulk [Ca 2+ ] i remains elevated but hot spots have dissipated), plus all endocytosis that occurs away from the AZ.…”