AN ENGINEERED POTASSIUM CHANNEL FOR THERMOGENETICS

Pain, a complex sensory and emotional experience, is essential for protection against environmental threats but becomes maladaptive when chronic, as seen in neuropathic pain. This condition involves pathological hyperexcitability in nociceptive circuits, where ion channels play critical roles in action potential (AP) generation and propagation. While targeting endogenous ion channels offers therapeutic potential, their distinct expression patterns and compensatory mechanisms pose significant challenges. To address these limitations, we developed a synthetic potassium (K⁺) channel, TICK (Temperature-Induced Channel K⁺), designed for selective expression and activation via external thermal stimuli, enabling long-term membrane voltage hyperpolarisation to inhibit nociceptive signalling. TICK was engineered by combining the temperature-sensitive domain of the bacterial BacNavAe1 channel with the viral KcvNTS channel pore. Through single-point mutations in the coiled-coil region, the prototype channel was optimised to activate within the physiological temperature range. Additionally, trafficking and export signals were introduced at the C-terminus to enhance plasma membrane expression. Electrophysiological analyses demonstrated a 3.3-fold increase in current density between 25°C and 40°C, and robust thermosensitivity, with a half-activation temperature (T1/2) of 38.95°C, and a Q10 value of 16. Confocal imaging revealed a >6-fold reduction in endoplasmic reticulum retention compared with earlier constructs, consistent with improved trafficking to the plasma membrane. In vivo validation initially focused on the central nervous system, where viral delivery to the anterior cingulate cortex confirmed neuronal expression but indicated that infrared stimulation through the skull achieved only partial activation of the channel (maximum intracerebral ΔT of +1.68 °C). This limitation prompted the transition to the peripheral nervous system, where AAV-mediated delivery of TICK2.1 to dorsal root ganglia and sensory afferents successfully enabled peripheral expression. Upon localized heating of the hind paw, TICK2.1 activation significantly reduced inflammatory mechanical hypersensitivity in the Complete Freund’s Adjuvant (CFA) pain model, decreasing withdrawal responses by 73% in males and 33–44% in females compared with non-heated controls. These results represent the first demonstration that thermogenetic silencing of peripheral nociceptors can modulate pain behaviour in vivo. Collectively, these findings establish TICK2.1 as a functional prototype for temperature-controlled neuronal inhibition and introduce thermogenetics as a viable strategy for gene-based analgesia. The project also integrated a technology transfer and translational development framework, outlining the preclinical, regulatory, and industrial pathways required to advance TICK2.1 towards clinical application. Future work will focus on refining viral vector design, optimising delivery and stimulation methods, and validating efficacy in chronic neuropathic pain models to support first-in-human studies.