Sniffing calcium from the outside: an extracellular calcium sensor for synaptic vesicle recycling.

In order for neurons to maintain communication with one another, following exocytosis, synaptic vesicles within the nerve terminal must be internalized from the plasma membrane and be refilled with neurotransmitter molecules for subsequent bouts of transmitter release. Calcium influx into the nerve terminal, which triggers exocytosis, may be an important temporal regulator of vesicle recycling. Several studies designed to address the calcium dependence of synaptic vesicle endocytosis have led to conflicting conclusions that intracellular calcium speeds up (Klingauf et al. 1998; Sankaranarayanan & Ryan, 2001), slows down (von Gersdorff & Matthews, 1994) or has little effect (Wu & Betz, 1996) on the rate of vesicle recycling. However, it is difficult to study the calcium dependence of endocytosis in isolation from exocytosis because these processes proceed concurrently and because exocytosis itself requires calcium influx. Thus, the role of calcium during synaptic vesicle recycling is still unclear. In this issue of The Journal of Physiology, a study of snake motor boutons measures uptake of the membrane dye sulphorhodamine 101 (SR101) to show that calcium regulates vesicle recycling via an extracellular sensor (Teng & Wilkinson, 2003). The calcium dependence of vesicle recycling was revisited by temporally separating this process from the immediate effects of calcium influx and exocytosis. To do so, the preparation was cooled to 7 °C following stimulation in order to delay membrane retrieval well beyond the time when calcium had returned to resting levels and exocytosis was complete. The ‘endocytotic debt’ incurred by delaying endocytosis lasted for several hours and was recovered at any time by warming the preparation to room temperature. When the magnitude and time course of dye uptake were measured in the presence of increasing extracellular calcium concentrations, delayed endocytosis proceeded more rapidly. Large changes in extracellular calcium had no observable effect on resting intraterminal calcium levels, as measured by a calcium indicator that reports nanomolar calcium concentrations. Therefore, calcium is a positive regulator of the rate of membrane retrieval, and these experiments implicate an extracellular calcium sensor in synaptic vesicle recycling. These same authors reported previously that delayed endocytosis is mediated by a temperature blockade of clathrin-coated vesicle formation and uncoating (Teng & Wilkinson, 2000). Thus, the current results suggest that extracellular calcium might regulate the rate of coated vesicle formation. This hypothesis is supported by the results of an electron microscope study of the lamprey giant synapse, in which synaptic vesicle recycling from the plasma membrane was temporarily blocked by the removal of extracellular calcium following stimulation (Gad et al. 1998). Subsequent recovery of synaptic vesicles was triggered via a rapid burst in coated vesicle formation by simply adding back 11 μM extracellular calcium. Although the interpretation was that minimal intracellular calcium is needed for synaptic vesicle recycling, these experiments may have inadvertently measured the affinity of the putative extracellular calcium sensor. The mechanism by which the endocytotic calcium sensor translocates a signal across the plasma membrane to trigger coated vesicle formation remains unclear. In the endocrine system, a G-protein-coupled extracellular calcium receptor regulates parathyroid hormone release by activating a cascade of pathways including protein kinase C and mitogen-activated protein kinase (Tfelt-Hansen et al. 2003), and the mechanisms for detecting extracellular calcium at synapses might be similar. During synaptic activity, a nerve terminal is exposed to changes in both extracellular and intracellular calcium, and thus the extracellular calcium sensor may coordinate with intracellular calcium effectors to regulate the early and late stages of coated vesicle formation. Calcium influx into the presynaptic terminal is likely to decrease calcium transiently in the synaptic cleft. As a result, vesicle recycling would be slowed until exocytosis is complete and extracellular calcium is returned to basal levels. Meanwhile, the entering calcium could trigger a cascade of events leading to the dephosphorylation of clathrin-associated proteins and their consequent assembly into multimolecular endocytotic complexes (Slepnev et al. 1998). Restoration of basal extracellular calcium levels could then trigger the extracellular sensor and speed up the early stages of endocytosis at a time when the intracellular endocytotic complexes are prepared to promote a burst of coated vesicle formation. In this way, changes in both intracellular and extracellular calcium could regulate synaptic vesicle recycling via coated vesicle formation. The current study by Teng & Wilkinson (2003) also highlights several other features of synaptic vesicle endocytosis in snake motor boutons. First, only half of the membrane retrieval was mediated by delayed endocytosis, indicating that a second mode of endocytosis exists, as is seen in other nerve terminals (e.g. Aravanis et al. 2003; Gandhi & Stevens, 2003). In snake nerve terminals, the second mode of membrane retrieval is likely to be mediated by macropinocytosis, which resembles bulk endocytosis and from which coated vesicles emanate (Teng & Wilkinson, 2000). Whether calcium affects the rate of macropinocytosis is unknown. Second, the ‘endocytotic debt’ incurred by delaying endocytosis was completely restored within 1 min, indicating that coated vesicle formation can proceed rapidly. Finally, because removal of extracellular calcium never completely blocked delayed endocytosis, calcium must be a regulator rather than an essential component of membrane retrieval. Identification of the putative extracellular calcium sensor and the mechanism by which it regulates the speed of synaptic vesicle recycling awaits further investigation.