title: Mutations that disrupt Ca²⁺-binding activity endow Doc2β with novel functional properties during synaptic transmission.
abstract: 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.
authors.affiliation: Howard Hughes Medical Institute and Department of Neuroscience, University of Wisconsin, Madison, WI 53706.
authors.forename: Jon D
authors.initials: JD
authors.lastname: Gaffaney
authors.forename: Renhao
authors.initials: R
authors.lastname: Xue
authors.forename: Edwin R
authors.initials: ER
authors.lastname: Chapman
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D016716:PC12 Cells; D061251:Primary Cell Culture; D011485:Protein Binding; D021381:Protein Transport; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009435:Synaptic Transmission; D050863:Synaptotagmin I / Q000235:genetics / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; C490418:Doc2a protein, mouse; C490421:Doc2b protein, mouse; D009419:Nerve Tissue Proteins; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; D050863:Synaptotagmin I; C020485:proteoliposomes; D002118:Calcium