An Exclusion Zone for Ca2+ Channels around Docked Vesicles Explains Release Control by Multiple Channels at a CNS Synapse

The spatial arrangement of Ca2+ channels and vesicles remains unknown for most CNS synapses, despite of the crucial importance of this geometrical parameter for the Ca2+ control of transmitter release. At a large model synapse, the calyx of Held, transmitter release is controlled by several Ca2+ channels in a "domain overlap" mode, at least in young animals. To study the geometrical constraints of Ca2+ channel placement in domain overlap control of release, we used stochastic MCell modelling, at active zones for which the position of docked vesicles was derived from electron microscopy (EM). We found that random placement of Ca2+ channels was unable to produce high slope values between release and presynaptic Ca2+ entry, a hallmark of domain overlap, and yielded excessively large release probabilities. The simple assumption that Ca2+ channels can be located anywhere at active zones, except below a critical distance of ~ 30 nm away from docked vesicles ("exclusion zone"), rescued high slope values and low release probabilities. Alternatively, high slope values can also be obtained by placing all Ca2+ channels into a single supercluster, which however results in significantly higher heterogeneity of release probabilities. We also show experimentally that high slope values, and the sensitivity to the slow Ca2+ chelator EGTA-AM, are maintained with developmental maturation of the calyx synapse. Taken together, domain overlap control of release represents a highly organized active zone architecture in which Ca2+ channels must obey a certain distance to docked vesicles. Furthermore, domain overlap can be employed by near-mature, fast-releasing synapses.Author Summary: Ca2+ channels provide the rise in intracellular Ca2+ concentration necessary to initiate the membrane fusion of transmitter—filled vesicles at synapses. Because Ca2+ diffuses away from Ca2+ channels, the distance between Ca2+ channels and vesicles on the range of tens of nanometers is a crucial determinant of the vesicle fusion probability. However, there is still little experimental evidence on how Ca2+ channels and vesicles co-localize in the nanospace of a single synapse. We show by computational modelling that the channels should be located at some distance to vesicles (~ 30 nm), to allow for release control by several channels, a release mechanism found at many synapses. In realistic synapses with a high density of docked vesicles, this translates into a likely localization of Ca2+ channels at membrane sites not occupied by docked vesicles. Thus, we present a computational model of how Ca2+ channels can be localized in an active zone with several docked vesicles, to enable control of release by several Ca2+ channels.