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 C₂A- and C₂B-domains, constitutes a presynaptic low-affinity Ca²⁺sensor. Ca²⁺binds to piccolo by virtue of its C₂A-domain via an unusual mechanism that involves a large conformational change. The distinct Ca²⁺-binding properties of the piccolo C₂A-domain are mediated by an evolutionarily conserved sequence at the bottom of the C₂A-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 μM in the presence of phospholipids) into high-affinity Ca²⁺binding (12-14 μM). The unusual Ca²⁺-binding mode of the piccolo C₂A-domain reveals that C₂-domains are mechanistically more versatile than previously envisaged. The low Ca²⁺affinity of the piccolo C₂A-domain suggests that piccolo could function in short-term synaptic plasticity when Ca²⁺concentrations accumulate during repetitive stimulation.