An unusual C2-domain in the active-zone protein piccolo:: implications for Ca2+ regulation of neurotransmitter release

Ca2+ regulation of neurotransmitter release is thought to require multiple Ca2+ sensors with distinct affinities. However, no low-affinity Ca2+ sensor has been identified at the synapse. We now show that piccolo/ aczonin, a recently described active-zone protein with C-terminal C(2)A- and C2B-domains, constitutes a presynaptic low-affinity Ca2+ sensor. Ca2+ binds to piccolo by virtue of its C(2)A-domain via an unusual mechanism that involves a large conformational change. The distinct Ca2+-binding properties of the piccolo C(2)A-domain are mediated by an evolutionarily conserved sequence at the bottom of the C(2)A-domain, which may fold back towards the Ca2+-binding sites on the top. Point mutations in this bottom sequence inactivate it, transforming low-affinity Ca2+ binding (100-200 muM in the presence of phospholipids) into high-affinity Ca2+ binding (12-14 muM). The unusual Ca2+-binding mode of the piccolo C(2)A-domain reveals that C-2-domains are mechanistically more versatile than previously envisaged. The low Ca2+ affinity of the piccolo C(2)A-domain suggests that piccolo could function in short-term synaptic plasticity when Ca2+ concentrations accumulate during repetitive stimulation.