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

Molecular Biology of the Cell
Jon D GaffaneyEdwin R Chapman

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 w...Continue Reading

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Citations

Jul 22, 2015·Proceedings of the National Academy of Sciences of the United States of America·Renhao XueEdwin R Chapman
Jan 27, 2015·Traffic·Devon C Crawford, Ege T Kavalali
Oct 13, 2016·Journal of Neurochemistry·Paulo S PinheiroJakob B Sørensen
Jun 22, 2017·Proceedings of the National Academy of Sciences of the United States of America·Cristina Ferrer-OrtaNúria Verdaguer
Apr 18, 2018·Diabetes·Arianne AslamyDebbie C Thurmond
May 31, 2018·Proceedings of the National Academy of Sciences of the United States of America·Renhao XueEdwin R Chapman
Oct 9, 2019·Scientific Reports·Quentin Bourgeois-JaarsmaAlexander J Groffen

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Methods Mentioned

BETA
isothermal titration calorimetry
confocal microscopy
PCR
transfection

Software Mentioned

pClamp
Prism
GraphPad
Igor
GelEval
Clampfit
FrogDance

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