All‐optical monitoring of excitation–secretion coupling demonstrates that SV2A functions downstream of evoked Ca 2+ entry

Autor: Mazdak M. Bradberry, Edwin R. Chapman
Rok vydání: 2022
Předmět:
Zdroj: J Physiol
ISSN: 1469-7793
0022-3751
Popis: SV2A, an essential transporter-like synaptic vesicle protein, is a major target for antiepileptic drugs and a receptor for clostridial neurotoxins including Botox. While SV2A is required for normal levels of evoked neurotransmitter release, the mechanism underlying this role remains unclear. Here, we introduce a new chemogenetic approach for all-optical monitoring of excitation-secretion coupling, and we demonstrate its use in characterizing the SV2A KO phenotype in cultured hippocampal neurons. This method employs the HaloTag system to target a robust small-molecule Ca(2+) indicator, JF(646)-BAPTA, to the presynaptic compartment. The far-red fluorescence of this indicator enables multiplexing with the fluorescent glutamate sensor iGluSnFR for detection of presynaptic Ca(2+) influx and glutamate release at the same axonal boutons. Evoked glutamate release probability was reduced in SV2A KO neurons without a change in presynaptic Ca(2+) entry, suggesting that SV2A supports vesicle fusion by increasing the functional availability, or efficiency, of the Ca(2+)-regulated membrane fusion machinery.
Databáze: OpenAIRE
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