Mutations that disrupt Ca²⁺-binding activity endow Doc2β with novel functional properties during synaptic transmission.

Double C2-domain protein (Doc2) is a Ca(2+)-binding protein implicated in asynchronous and spontaneous neurotransmitter release. Here we demonstrate that each of its C2 domains senses Ca(2+); moreover, the tethered tandem C2 domains display properties distinct from the isolated domains. We confirm that overexpression of a mutant form of Doc2β, in which two acidic Ca(2+) ligands in the C2A domain and two in the C2B domain have been neutralized, results in markedly enhanced asynchronous release in synaptotagmin 1-knockout neurons. Unlike wild-type (wt) Doc2β, which translocates to the plasma membrane in response to increases in [Ca(2+)](i), the quadruple Ca(2+)-ligand mutant does not bind Ca(2+) but is constitutively associated with the plasma membrane; this effect is due to substitution of Ca(2+) ligands in the C2A domain. When overexpressed in wt neurons, Doc2β affects only asynchronous release; in contrast, Doc2β Ca(2+)-ligand mutants that constitutively localize to the plasma membrane enhance both the fast and slow components of synaptic transmission by increasing the readily releasable vesicle pool size; these mutants also increase the frequency of spontaneous release events. Thus, mutations in the C2A domain of Doc2β that were intended to disrupt Ca(2+) binding result in an anomalous enhancement of constitutive membrane-binding activity and endow Doc2β with novel functional properties.