Introduction: Piezo1 channels are mechanosensitive ion channels that play a pivotal role in sensing mechanical forces in various cell types. Their dysfunction has been associated with numerous pathophysiological states including generalised lymphatic dysplasia, varicose vein disease, dehydrated hereditary stomatocytosis, and malarial resistance. Given their high physiological relevance, investigating Piezo1 is crucial for the pharmaceutical industry that requires scalable techniques to allow for drug discovery. In this regard, several studies have shown the use of high-throughput automated patch clamp (APC) to explore the function and properties of Piezo1 channels in heterologous expression system as well as primary cells (Rotordam et al. 2018, Parsonage et al., 2022; Karamatic Crew et al., 2023) mainly based on usage of Yoda1, a specific gating modifier of Piezo1 channels (Syeda et al., 2015). However, to our knowledge, a combination of solely mechanical stimulation and high-throughput APC has not yet been available for the study of Piezo1 channels.
Methods: Here we show that optimization of pipetting parameters coupled with the possibility to increase the number of cells per well in the NPC-384 chip of the SyncroPatch 384 lead to Piezo1-mediated currents activated by solely mechanical stimulation (M-Stim). Mouse and human Piezo1 channels expressed in HEK293 cells and untransfected HEK293 cells were tested by M-Stim in the absence and presence of Yoda1. Cells showing stable seal resistance and eliciting inward currents higher than −100 pA that could be inhibited by GdCl3 (non-specific blocker of mechanosensitive channels) were considered Piezo1 responding cells.
Results: Our results strongly suggest that applying solutions on top of the cells at a fast pipetting speed is crucial for activating Piezo1 channels by M-Stim on the SP384. Moreover, stimulating 4 cells simultaneously in one well of a NPC-384 chip increased the current amplitudes at peak by 2-fold in mPiezo1, from −387.8 pA, 95% CI [297, 511.4] (n=148) to −894.3 pA, 95% CI [757.9, 1043] (n=579) and by 1.5-fold in hPiezo1, from −398,8 pA, 95% CI [332,5, 518] (n=174) to −612.8 pA, 95% CI [544.8, 685.5] (n=346) for hPiezo1. No currents were detected from the untrasfected cells. In line with this, the number of responding cells increased significantly from approximately 10% (both mPiezo1 and hPiezo1) to 64.17% ± 3.25% for mPiezo1 and to 52.75% ± 1.60% for hPiezo1 when using 4-hole chips. The number of responding cells was close to 100% when using 4-hole chips in combination with Yoda1.
Conclusions: In this study, M-Stim of Piezo1 channels using a high-throughput planar patch clamp system could be demonstrated. The possibility of comparing and combining mechanical and chemical stimulation in a high-throughput patch clamp assay facilitates the biophysical and pharmacological characterization of Piezo1 channels and thereby provides an important experimental tool for drug discovery.