At most synapses, presynaptic Ca2+ channels are positioned near vesicle release sites, and increasing this distance reduces synaptic strength. We examined the lateral membrane mobility of presynaptic L-type Ca2+ channels at photoreceptor ribbon synapses of the tiger salamander (Ambystoma tigrinum) retina. Movements of individual Ca2+ channels were tracked by coupling quantum dots to an antibody against the extracellular α2δ4 Ca2+ channel subunit. α2δ4 antibodies labeled photoreceptor terminals and colocalized with antibodies to synaptic vesicle glycoprotein 2 and voltage-gated Ca2+ channel 1.4 (CaV1.4) α1 subunits. The results show that Ca2+ channels are dynamic and move within a confined region beneath the synaptic ribbon. The size of this confinement area is regulated by actin and membrane cholesterol. Fusion of nearby synaptic vesicles caused jumps in Ca2+ channel position, propelling them toward the outer edge of the confinement domain. Channels rebounded rapidly toward the center. Thus, although CaV channels are mobile, molecular scaffolds confine them beneath the ribbon to maintain neurotransmission even at high release rates.
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