Kv7.2/7.3 (KCNQ2/3) potassium channels underlie the neuronal M-current, a key regulator of membrane excitability and action potential firing. Pharmacological activation of these channels represents a validated strategy for reducing neuronal hyperexcitability in disorders such as epilepsy and neuropathic pain. Kv7.2/7.3 activators can exhibit distinct mechanisms of action, including modulation of channel gating through interactions with the pore-forming or voltage-sensing domains. This study developed an integrated platform to characterise the pharmacological and physiological effects of Kv7.2/7.3 activators across complementary assay systems.
Using a fluorescent thallium flux assay, automated voltage-clamp and current-clamp electrophysiology, and manual patch-clamp recordings from sensory neurons, we assessed retigabine, flupirtine, ML213, ICA 069673, XEN1101 and opakalim. FLIPR potency measurements enabled high-throughput compound profiling, while Qube 384 recordings provided mechanistic insight into concentration-dependent effects on voltage dependence of activation and maximal conductance. QPatch 48 current-clamp recordings demonstrated concentration-dependent hyperpolarisation of resting membrane potential, with effects approaching the potassium equilibrium potential. Sensory neuron recordings further demonstrated physiological effects at native Kv7 channels. Overall, this integrated workflow linked compound potency and mechanism of action to physiological effects on neuronal excitability, supporting the mechanistic and translational profiling of Kv7.2/7.3 activators.
Optical voltage imaging of human iPSC-derived cardiomyocytes was used to assess electrophysiological effects of compounds beyond hERG inhibition. Action potential waveform analysis revealed compound-specific and concentration-dependent changes, enabling mechanistic differentiation of multichannel activity and demonstrating a human-relevant approach for translational cardiac safety assessment.
Development of a robust hNaV1.9 high-throughput screening assay on the Sophion Qube384 platform. This is complemented by a suite of ion channel selectivity assays and sensory neuron recordings to create a versatile screening cascade to support NaV1.9 drug discovery programmes.