Spatial Distribution of Calcium Entry Evoked by Single Action Potentials within the Presynaptic Active Zone

Abstract

The nature of presynaptic calcium (Ca 2ϩ ) signals that initiate neurotransmitter release makes these signals difficult to study, in part because of the small size of specialized active zones within most nerve terminals. Using the frog motor nerve terminal, which contains especially large active zones, we show that increases in intracellular Ca 2ϩ concentration within 1 msec of action potential invasion are attributable to Ca 2ϩ entry through N-type Ca 2ϩ channels and are not uniformly distributed throughout active zone regions. Furthermore, changes in the location and magnitude of Ca 2ϩ signals recorded before and after experimental manipulations (-conotoxin GVIA, diaminopyridine, and lowered extracellular Ca 2ϩ ) support the hypothesis that there is a remarkably low probability of a single Ca 2ϩ channel opening within an active zone after an action potential. The trial-to-trial variability observed in the spatial distribution of presynaptic Ca 2ϩ entry also supports this conclusion, which differs from the conclusions of previous work in other synapses.

Ca2+from One or Two Channels Controls Fusion of a Single Vesicle at the Frog Neuromuscular Junction

“…If we fix the geometry and the number of open channels in our model, then the total number of vesicles released depends on two parameters: the average single Ca 2ϩ channel current during an AP and the affinity of the Ca 2ϩ binding sites. Rapid highresolution Ca 2ϩ imaging of AP-mediated presynaptic Ca 2ϩ transients at the frog NMJ indicates trial-to-trial variability in the spatial distribution of Ca 2ϩ entry within single AZs (Wachman et al, 2004). This work suggests that an AP triggers the opening of a small number of Ca 2ϩ channels per AZ, which is consistent with our experimental findings and the modeling.…”

“…Several putative Ca 2ϩ channels are located within 100 nm of each vesicle (Heuser et al, 1974;Pumplin et al, 1981), so a high degree of channel cooperativity is possible. However, indirect electrophysiological analysis (Yoshikami et al, 1989;Augustine et al, 1991) and a recent Ca 2ϩ imaging study (Wachman et al, 2004) are consistent with only a few channels opening with each action potential (AP), suggesting low cooperativity. We measured cooperativity by reducing the number of open channels with -conotoxin GVIA ( -CTX) and monitoring Ca 2ϩ influx fluorometrically while measuring postsynaptic potentials (PSPs).…”

Deletion of the Presynaptic Scaffold CAST Reduces Active Zone Size in Rod Photoreceptors and Impairs Visual Processing

“…Some synapses, such as the neuromuscular junction or ribbon synapses of photoreceptors, and hair cells use large active zones to accommodate high rates of synaptic transmission. There, tens of Ca 2ϩ channels and docked synaptic vesicles are organized in well-ordered arrays, thought to adhere to a specific topographic principle that favors tight control of vesicle fusion by nearby Ca 2ϩ channels (Harlow et al, 2001;Wachman et al, 2004;Brandt et al, 2005;Frank et al, 2010;Jarsky et al, 2010). The CAZ proteins Bassoon, RIM, Piccolo, CAST/ERC2 (termed CAST in the following), and ELKS/ ERC1 are thought to mediate these organizational principles during and after synapse formation (Fejtova and Gundelfinger, 2006;Hida and Ohtsuka, 2010).…”

Synaptotagmins 1 and 2 as mediators of rapid exocytosis at nerve terminals: The dyad hypothesis

“…Again, although there is evidence that the Ca 2+ channels at nerve endings are located in close proximity to the sites of regulated exocytosis (Pumplin et al, 1981;Robitaille et al, 1990;Cohen et al, 1991;Wachman et al, 2004), the issue of Ca 2+ channel localization is 7 secondary to the development of the current model. Of central importance is the presumption that one or more proteins must initiate membrane fusion by triggering a process that can occur even in the absence of protein.…”