BPS2026 – Cold activation of TRPM8 using high-throughput automated patch clamp

Transient receptor potential (TRP) channels are widely distributed throughout the mammalian nervous system. They can be activated by ligands, heat or cold and mechano-stimulation, and are important targets in drug discovery for the treatment of pain, respiratory diseases, cancer and immune disorders. TRPM8 is activated by menthol, icilin and eucalyptol, and by low-temperatures, <25°C. TRPM8 has been implicated to play a role in chronic and neuropathic pain and is a potential target in drug discovery; therefore, it is essential to have reliable methods for recording TRPM8 activated using different stimuli to find TRPM8 modulators. Here, we activated TRPM8 using cold solution on three automated patch clamp (APC) systems. On the SyncroPatch 384 continuous cooling of the recording chamber to temperatures of 10–12°C were used to activate TRPM8 expressed in CHO cells. Either a voltage ramp or a voltage step protocol was used and large currents were recorded when the recording chamber and solutions were cooled down to 10–12°C. This activation was reversed when the recording chamber was heated (30°C). Progressive cooling of the module and solutions led to increasing current density ranging from 20 pA/pF at 25°C to 120 pA/pF at 10°C. In addition, using the Patchliner, solutions were pre-cooled and rapidly applied to the cells for rapid and transient activation of cold-activated TRPM8-mediated currents. Using the Port-a-Patch, TRPM8 was repetitively activated by cold solution at 10°C using the temperature-controlled perfusion system and capsazepine blocked the current. Alternatively, the ligand menthol could be used to activate TRPM8 on the different APC devices. In this way, we could activate TRPM8 using different stimuli, including continuous or transient cooling to temperatures <12°C and this method could be used for high throughput screening of TRPM8 modulators for potential pain therapeutics.