TY  - JOUR
A1  - Pathe-Neuschäfer-Rube, Andrea
A1  - Neuschäfer-Rube, Frank
A1  - Püschel, Gerhard Paul
T1  - Cell-based reporter release assay to determine the activity of calcium-dependent neurotoxins and neuroactive pharmaceuticals
JF  - Toxins / Molecular Diversity Preservation International (MDPI)
N2  - The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca2+-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K+-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K+-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release.
KW  - cell-based assay
KW  - neurotoxins
KW  - muscarinic acetylcholine receptor
KW  - voltage-dependent calcium channels
KW  - VGCC
Y1  - 2021
U6  - https://doi.org/10.3390/toxins13040247
SN  - 2072-6651
VL  - 13
IS  - 4
PB  - MDPI
CY  - Basel
ER  -