Chemical transfection reagents induce intracellular Ca2+ signals in TRPA1-expressing cells
- 1KU Leuven
Abstract
Transient receptor potential ankyrin 1 (TRPA1) is a polymodal sensory ion channel whose activity is influenced not only by chemical ligands but also by the physical properties of the plasma membrane. This raises the possibility that membrane-active compounds used routinely in cell biology may alter TRPA1 function. We investigated the acute effects of two widely used chemical transfection reagents, Lipofectamine 3000 and Mirus TransIT-293, on intracellular Ca2+; signaling and TRPA1 activity. For this, we monitored intracellular Ca2+; dynamics using ratiometric Fura-2 imaging in CHO cells stably expressing mouse TRPA1 (CHO-mTRPA1), parental CHO-WT cells, and primary mouse dorsal root ganglion (DRG) neurons. Transfection reagent preparations were applied at different concentrations under controlled temperature and low-flow conditions. The contribution of TRPA1 and Ca2+; influx was assessed using the selective TRPA1 inhibitor HC-030031 and the broad-spectrum Ca2+; channel blocker ruthenium red. We found that Lipofectamine 3000 induced concentration-dependent, irregular Ca2+; transients in CHO-mTRPA1 cells, while simultaneously inhibiting the constitutive TRPA1-dependent Ca2+; activity observed under basal conditions. Its inhibitory effect was evident at concentrations below those producing substantial cellular activation and was rapidly reversible after washout. At higher concentrations, Lipofectamine-induced Ca2+; responses were only partially suppressed by TRPA1 inhibition, indicating the involvement of additional mechanisms. Consistent with this, Lipofectamine also induced Ca2+; transients in CHO-WT cells and primary DRG neurons, where both extracellular Ca2+; influx and intracellular Ca2+; mobilization contributed to the responses. Analysis of the components of the Lipofectamine 3000 formulation further revealed distinct effects of Lipofectamine and the P3000 enhancer. Mirus TransIT-293 similarly induced Ca2+; transients in CHO-mTRPA1 cells and DRG neurons. In CHO-mTRPA1 cells, its response was concentration-dependent and strongly reduced by HC-030031, whereas the response in DRG neurons showed little sensitivity to TRPA1 inhibition. We conclude that chemical transfection reagents can acutely alter intracellular Ca2+; homeostasis and modulate TRPA1 activity. Their effects involve both TRPA1-dependent and TRPA1-independent mechanisms and differ substantially between formulations and cell types. These findings identify membrane-active transfection reagents as previously underappreciated modulators of sensory ion-channel function and highlight their potential to influence the interpretation of experiments performed in transfected cells.
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BibTeX
@misc{milici2026chemical,
title = {{Chemical transfection reagents induce intracellular Ca2+ signals in TRPA1-expressing cells}},
author = {A. Milici and A. Segal and J. B. Startek and K. Talavera},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.16.751488},
url = {https://www.biorxiv.org/content/10.64898/2026.09.16.751488v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Chemical transfection reagents induce intracellular Ca2+ signals in TRPA1-expressing cells
AU - A. Milici
AU - A. Segal
AU - J. B. Startek
AU - K. Talavera
PY - 2026
DA - 2026/09/16
PB - bioRxiv
DO - 10.64898/2026.09.16.751488
UR - https://www.biorxiv.org/content/10.64898/2026.09.16.751488v1
AB - Transient receptor potential ankyrin 1 (TRPA1) is a polymodal sensory ion channel whose activity is influenced not only by chemical ligands but also by the physical properties of the plasma membrane. This raises the possibility that membrane-active compounds used routinely in cell biology may alter TRPA1 function. We investigated the acute effects of two widely used chemical transfection reagents, Lipofectamine 3000 and Mirus TransIT-293, on intracellular Ca2+; signaling and TRPA1 activity. For this, we monitored intracellular Ca2+; dynamics using ratiometric Fura-2 imaging in CHO cells stably expressing mouse TRPA1 (CHO-mTRPA1), parental CHO-WT cells, and primary mouse dorsal root ganglion (DRG) neurons. Transfection reagent preparations were applied at different concentrations under controlled temperature and low-flow conditions. The contribution of TRPA1 and Ca2+; influx was assessed using the selective TRPA1 inhibitor HC-030031 and the broad-spectrum Ca2+; channel blocker ruthenium red. We found that Lipofectamine 3000 induced concentration-dependent, irregular Ca2+; transients in CHO-mTRPA1 cells, while simultaneously inhibiting the constitutive TRPA1-dependent Ca2+; activity observed under basal conditions. Its inhibitory effect was evident at concentrations below those producing substantial cellular activation and was rapidly reversible after washout. At higher concentrations, Lipofectamine-induced Ca2+; responses were only partially suppressed by TRPA1 inhibition, indicating the involvement of additional mechanisms. Consistent with this, Lipofectamine also induced Ca2+; transients in CHO-WT cells and primary DRG neurons, where both extracellular Ca2+; influx and intracellular Ca2+; mobilization contributed to the responses. Analysis of the components of the Lipofectamine 3000 formulation further revealed distinct effects of Lipofectamine and the P3000 enhancer. Mirus TransIT-293 similarly induced Ca2+; transients in CHO-mTRPA1 cells and DRG neurons. In CHO-mTRPA1 cells, its response was concentration-dependent and strongly reduced by HC-030031, whereas the response in DRG neurons showed little sensitivity to TRPA1 inhibition. We conclude that chemical transfection reagents can acutely alter intracellular Ca2+; homeostasis and modulate TRPA1 activity. Their effects involve both TRPA1-dependent and TRPA1-independent mechanisms and differ substantially between formulations and cell types. These findings identify membrane-active transfection reagents as previously underappreciated modulators of sensory ion-channel function and highlight their potential to influence the interpretation of experiments performed in transfected cells.
ER -