Piezo1 Agonists are Potent Off-Target Inhibitors of the Kv1.3 Channel via Gating Modification

The mechanosensitive Piezo1 channel is crucial for the regulation of calcium (Ca 2+ ) signaling within myeloid tissue macrophages. However, the current lack of comprehensive pharmacological characterization for existing Piezo1 agonists complicates the assessment of Piezo1 agonism as a viable therapeutic strategy. We report inhibition of the voltage-gated Kv1.3 potassium (K + ) channel as a critical off-target effect of the most widely used Piezo1 agonists, Yoda1 and Yoda2. Kv1.3 is essential for the activation and innate immune functions of tissue macrophages. Patch-clamp electrophysiological experiments demonstrate that Yoda1 (IC 50 = 9.0 μM) and Yoda2 (IC 50 = 5.9 μM) state-independently inhibit Kv1.3 by accelerating channel inactivation. Functionally, blockade of Kv1.3 by Yoda2 in Piezo1-deficient bone marrow derived macrophages (BMDM) depolarizes membrane potential, reduces store-operated calcium entry (SOCE) and IL-1β release. Using RosettaLigand docking and molecular dynamics simulations, we identified a network of key residues near the Kv1.3 outer pore as the potential binding site. Our results suggest that Kv1.3 blockade likely contributes substantially to the reported anti-inflammatory effects of Yoda1 and Yoda2. This provides a plausible mechanism that aligns with the established principle that reducing Ca 2+ signaling is anti-inflammatory, while helping to reconcile existing literature discrepancies regarding Piezo1 activation.