Double C2-like domain-containing proteins alpha and beta (Doc2 alpha and Doc2 beta) are tandem C2-domain proteins proposed to function as Ca2+ sensors for asynchronous neurotransmitter release. Here, we systematically analyze each of the negatively charged residues that mediate binding of Ca2+ to the beta isoform. The Ca2+ ligands in the C2A domain were dispensable for Ca2+-dependent translocation to the plasma membrane, with one exception: neutralization of D220 resulted in constitutive translocation. In contrast, three of the five Ca2+ ligands in the C2B domain are required for translocation. Importantly, translocation was correlated with the ability of the mutants to enhance asynchronous release when overexpressed in neurons. Finally, replacement of specific Ca2+/lipid-binding loops of synaptotagmin 1, a Ca2+ sensor for synchronous release, with corresponding loops from Doc2 beta, resulted in chimeras that yielded slower kinetics in vitro and slower excitatory postsynaptic current decays in neurons. Together, these data reveal the key determinants of Doc2 beta that underlie its function during the slow phase of synaptic transmission.
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