The regulation of neuronal excitability is essential for processes such as learning, memory, cognition, and the control of muscle contraction. Voltage-gated potassium ion channels, such as the Kv7 family, are key proteins involved in this regulation. Mutations in Kv7.5, one of the five isotypes within the family, are linked to epilepsy, neuropathic pain, and other neurological disorders. While pharmacological modulators of Kv7 channels hold therapeutic promise, the structural basis of ligand activation of Kv7.5 remains poorly understood. This question is of particular importance since Kv7 subunits are highly conserved, making selective targeting challenging and raising the risk of off-target effects.
To address this, we expressed Kv7.5 homotetramers in Expi293F cells using the BacMam expression system and purified them via a GFP-nanobody resin and SEC. Three known modulatory ligands were selected for structural analysis, with the aim of comparing their binding interactions and conformational effects on channel gating. Kv7.5 was then structurally characterised in both apo and ligand-bound conditions using cryo-electron microscopy (cryo-EM). While data processing is ongoing, the apo structure of Kv7.5 with endogenous bound calmodulin was determined at a resolution of 2.6 Å. Patch-clamp electrophysiology will soon be utilised to link observed conformational changes and binding modes to functional modulation.
Through these studies, we hope to gain structural insight into the mechanisms of ligand-dependent activation of Kv7.5. Establishing this framework will be an important step towards understanding subtype-specific modulation within the Kv7 family and could ultimately help with the design of selective therapeutics for neurological disorders.