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 (Ca2+) 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 (IC50 = 9.0 μM) and Yoda2 (IC50 = 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 depolarizes membrane potential and reduces store-operated calcium entry and interleukin-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 Ca2+ signaling is anti-inflammatory, while helping to reconcile existing literature discrepancies regarding Piezo1 activation. SIGNIFICANCE STATEMENT: Targeting Ca2+ channels such as Piezo1 is vital for controlling chronic inflammation, but selectivity is challenging. This study reveals that the best-known Piezo1 agonists exhibit a critical off-target effect by blocking the Kv1.3 channel, which is essential for macrophage and microglial immune function. This concurrent inhibition reduces inflammatory Ca2+ signaling and interleukin-1β secretion. This work underscores the necessity for rigorous selectivity screening of Piezo1 drugs and establishes Kv1.3 inactivation modification as a viable strategy for designing novel anti-inflammatory therapeutics.