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Mutations that disrupt Ca²?-binding activity endow Doc2? 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 Doc2?, 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) Doc2?, 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, Doc2? affects only asynchronous release; in contrast, Doc2? 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 Doc2? that were intended to disrupt Ca(2+) binding result in an anomalous enhancement of constitutive membrane-binding activity and endow Doc2? with novel functional properties.

SUBMITTER: Gaffaney JD

PROVIDER: S-EPMC3923640 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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

Gaffaney Jon D JD Xue Renhao R Chapman Edwin R ER

Molecular biology of the cell 20131219 4

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 ...[more]

PMID: 24356452

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Project description:Using postsynaptically tethered calcium sensor GCaMP, we investigated spontaneous synaptic transmission at individual active zones (AZs) at the Drosophila (both sexes) neuromuscular junction. Optical monitoring of GCaMP events coupled with focal electrical recordings of synaptic currents revealed "hot spots" of spontaneous transmission, which corresponded to transient states of elevated activity at selected AZs. The elevated spontaneous activity had two temporal components, one at a timescale of minutes and the other at a subsecond timescale. We developed a three-state model of AZ preparedness for spontaneous transmission and performed Monte Carlo simulations of the release process, which produced an accurate quantitative description of the variability and time course of spontaneous transmission at individual AZs. To investigate the mechanisms of elevated activity, we first focused on the protein complexin, which binds the SNARE protein complex and serves to clamp spontaneous fusion. Overexpression of Drosophila complexin largely abolished the high-activity states of AZs, while complexin deletion drastically promoted it. A mutation in the SNARE protein Syntaxin-1A had an effect similar to complexin deficiency, promoting the high-activity state. We next tested how presynaptic Ca2+ transients affect the states of elevated activity at individual AZs. We either blocked or promoted Ca2+ influx pharmacologically, and also promoted Ca2+ release from internal stores. These experiments coupled with computations revealed that Ca2+ transients can trigger bursts of spontaneous events from individual AZs or AZ clusters at a subsecond timescale. Together, our results demonstrated that spontaneous transmission is highly heterogeneous, with transient hot spots being regulated by the SNARE machinery and Ca2+ SIGNIFICANCE STATEMENT Spontaneous synaptic transmission is a vital component of neuronal communication, since it regulates the neuronal development and plasticity. Our study demonstrated that spontaneous transmission is highly heterogeneous and that nerve terminals create transient "hot spots" of spontaneous release of neuronal transmitters. We show that these hot spots are regulated by the protein machinery mediating the release process and by calcium ions. These results contribute to our understanding of spontaneous synaptic transmission as a dynamic, plastic, and tightly regulated signaling mechanism and unravel fundamental biophysical properties of neuronal communication.

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Mutations that disrupt Ca²?-binding activity endow Doc2? with novel functional properties during synaptic transmission.

Publications

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

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