An unusual C 2 ‐domain in the active‐zone protein piccolo: implications for Ca 2+ regulation of neurotransmitter release

Ca 2+ regulation of neurotransmitter release is thought to require multiple Ca 2+ sensors with distinct affinities. However, no low‐affinity Ca 2+ 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 C 2 B‐domains, constitutes a presynaptic low‐affinity Ca 2+ sensor. Ca 2+ binds to piccolo by virtue of its C 2 A‐domain via an unusual mechanism that involves a large conformational change. The distinct Ca 2+ ‐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 Ca 2+ ‐binding sites on the top. Point mutations in this bottom sequence inactivate it, transforming low‐affinity Ca 2+ binding (100–200 μM in the presence of phospholipids) into high‐affinity Ca 2+ binding (12–14 μM). The unusual Ca 2+ ‐binding mode of the piccolo C 2 A‐domain reveals that C 2 ‐domains are mechanistically more versatile than previously envisaged. The low Ca 2+ affinity of the piccolo C 2 A‐domain suggests that piccolo could function in short‐term synaptic plasticity when Ca 2+ concentrations accumulate during repetitive stimulation.