Piezo1 is a mechanosensitive ion channel that plays a central role in diverse physiological processes, including vascular development, red blood cell homeostasis, and tumor progression. Pharmacological modulation of Piezo1 is of increasing interest for both therapeutic development and mechanistic studies; however functional assays capable of screening modulators with sufficient throughput remain limited. Recently, we developed an automated patch clamp (APC) “M-Stim” approach, which applies high-flow pipetting in 384-well APC systems to reproducibly activate Piezo1 currents. Here, we adapted the M-Stim assay to investigate Piezo1 pharmacology across both recombinant and endogenous expression systems. Using the small-molecule agonist Yoda2 and the non-selective mechanosensitive channel blocker gadolinium chloride as a reference compounds, we validated the assay in recombinant Piezo1-expressing cells. We then transferred the methodology to the fibrosarcoma cell line HT1080, which expresses Piezo1 endogenously, and report for the first time the application of M-Stim in an endogenous context to assess the efficacy of a subset of Piezo1 inhibitors. The ability to probe inhibitors in physiologically relevant, native cellular environments is critical for understanding the pharmacology of Piezo1 and identifying compounds with translational potential. Importantly, the APC-based M-Stim platform enables parallel testing in 384 wells, offering a substantial increase in throughput compared to traditional patch clamp methods. This scalability not only accelerates discovery of Piezo1 modulators but also provides a robust and standardized framework for cross-system pharmacological comparisons. Our findings establish M-Stim as a versatile high-throughput tool for assessing Piezo1 pharmacology in both recombinant and endogenous systems, advancing efforts to identify and characterize novel Piezo1 modulators.
Strassmaier et al. (2026) studied this question.