Transmitter release face Ca²⁺ channel clusters persist at isolated presynaptic terminals

Ca²⁺ influx through N-type Ca²⁺ channels (CaV2.2) is known to be critical for transmitter release at many synapses. These channels are known to be localized to transmitter release sites, but their anchoring mechanism remains unknown. Recent studies have demonstrated that presynaptic organization is subject to interactions with the postsynaptic cell or the intervening extracellular matrix. We used a previously described high-affinity antibody against the N-type Ca²⁺ channels, Ab571, to localize Ca ²⁺ channel clusters at the release face of an isolated giant calyx-type synapse to test whether the maintenance of these clusters requires an intact extracellular matrix or contact with the postsynaptic cell. Because the number of Ca²⁺ channel clusters was unchanged after extracellular matrix dispersal or nerve terminal isolation, we conclude that presynaptic transmitter release face Ca²⁺ clusters can be maintained independently of extracellular influences. Our results suggest that a presynaptic molecular scaffold is responsible for the maintenance of release site Ca²⁺ channel clusters.