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

Area of Science:

• Neuroscience
• Molecular Biology
• Cell Biology

Background:

• Double C2-domain protein (Doc2) is a calcium (Ca2+)-binding protein involved in neurotransmitter release.
• Doc2's C2 domains are critical for sensing Ca2+ and regulating synaptic transmission.

Purpose of the Study:

• To investigate the Ca2+-sensing properties of individual and tandem C2 domains in Doc2.
• To analyze the functional consequences of mutating Ca2+ ligands in Doc2β on neurotransmitter release.
• To explore the relationship between Doc2β's Ca2+ binding, membrane localization, and synaptic function.

Main Methods:

• Site-directed mutagenesis to neutralize Ca2+ ligands in Doc2β's C2 domains.
• Overexpression of wild-type and mutant Doc2β in neurons (including synaptotagmin 1-knockout).
• Electrophysiological recordings to assess neurotransmitter release (asynchronous, spontaneous, and evoked).
• Confocal microscopy to examine Doc2β localization at the plasma membrane.

Main Results:

• Both C2 domains of Doc2 sense Ca2+, with distinct properties when acting in tandem.
• Overexpression of a quadruple Ca2+-ligand mutant Doc2β enhanced asynchronous release in synaptotagmin 1-knockout neurons.
• Mutations in the C2A domain disrupted Ca2+ binding but led to constitutive plasma membrane association.
• Constitutively membrane-bound Doc2β mutants enhanced both fast and slow synaptic transmission by increasing the readily releasable pool and spontaneous release frequency.

Conclusions:

• Doc2's tandem C2 domains possess unique Ca2+-sensing capabilities.
• Disrupting Ca2+ binding in Doc2β can paradoxically enhance its membrane association and synaptic function.
• Mutant Doc2β proteins with altered Ca2+ binding exhibit novel roles in regulating synaptic vesicle pools and release.