Chemical transfection reagents induce intracellular Ca ²⁺ signals in TRPA1-expressing cells

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 Trans IT-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 Trans IT-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.