Human TRPM8 Stable Cell Line - HEK293

Rapamycin (sirolimus) is a macrocyclic lactone compound isolated from Streptomyces hygroscopicus; it is widely used as an oral medication to prevent transplant rejection and treat lymphangioleiomyomatosis. It is also employed in coronary artery stent coatings to prevent restenosis and in topical formulations for treating skin disorders. Here, researchers demonstrate that rapamycin activates TRPM8, a cation channel expressed at sensory nerve endings that serves as the primary cold sensor in mammals. Through a combination of electrophysiology, saturation transfer triple-difference (STTD) NMR spectroscopy, and site-directed mutagenesis guided by molecular docking, it was confirmed that rapamycin binds directly to human TRPM8. The researchers identified a rapamycin binding site located in the groove between the voltage-sensor-like domain and the pore domain; this site is distinct from the interaction sites of cooling agents and known TRPM8 agonists such as menthol and icilin. Related macrocyclic lactone immunosuppressants act as partial agonists of TRPM8 and compete with rapamycin for the same binding site. These findings reveal a novel molecular target for rapamycin and provide new insights into the activation mechanism of TRPM8, facilitating the development of therapies targeting this ion channel. Furthermore, the results suggest that caution is warranted when using rapamycin-induced dimerization-based molecular tools to study ion channel regulation.

In Fura-2-based assays of intracellular Ca2+ concentration ([Ca2+]i) using human TRPM8 stably expressing HEK293 cells (HEK-TRPM8 cells), researchers observed that rapamycin (10 µM) induced a significant increase in [Ca2+]i, with a magnitude comparable to the response elicited by 50 µM menthol, a prototypical TRPM8 agonist (Figure 1A). The responses induced by both rapamycin and menthol were completely inhibited by the specific TRPM8 antagonist AMTB (N-(3-aminopropyl)–2-[(3-methylphenyl) methoxy] -N-(2-thienylmethyl) benzamide hydrochloride; 2 µM) (Figure 1A). The effect of rapamycin was concentration-dependent, saturating at concentrations ≥10 µM, with an EC50 value of 3.8 ± 2.0 µM (Figure 1B). Similar results were obtained in assays using 96-well plates: the EC50 value was 6.0 ± 0.3 µM at room temperature, whereas it was 10.1 ± 0.2 µM at 37 °C. Likewise, whole-cell patch-clamp recordings from HEK-TRPM8 cells at room temperature showed that rapamycin induced significant TRPM8 currents; the currents rapidly returned to baseline levels upon washout of rapamycin and were completely inhibited by AMTB (2 µM) (Figure 1C). The concentration of rapamycin required to induce half-maximal activation of whole-cell currents at +120 mV was 4.5 ± 1.8 µM (Figure 1D). Importantly, neither menthol nor rapamycin induced any detectable calcium signaling or increase in current in untransfected HEK293 cells.

Figure 1. Rapamycin activates TRPM8 in HEK293 cells and sensory neurons. (Tóth B I, et al., 2025)