2022 - Mechanical and Pharmacological Activation of Piezo1 Channels Characterized by High Throughput Electrophysiology
SyncroPatch 384 poster, BPS 2022 (0.66 MB)
PIEZO channels are mechanically-activated cation channels that play important roles in biological functions including touch, proprioception, shear stress and stretch sensation as well as blood pressure regulation. Mutations in Homo sapiens PIEZO1 channels are associated with anemia, malarial resistance, lymphatic dysplasia and varicose vein disease, suggesting important red blood cells and vascular roles in humans. The pharmacology of the PIEZO1 channels is in its infancy. Here we sought high throughout methodology for investigating small-molecule modulation in combination with mechanical stimulation. A bottleneck in PIEZO drug development has been the lack of mechanical stimulation in automated patch clamp. Here we show how the optimization of pipetting parameters and the modification of the NPC-384 chip of the SyncroPatch 384 lead to the possibility to mechanically stimulate PIEZO1 channels using high throughput electrophysiology. Data of mouse and human PIEZO1 channels expressed in HEK T-REX 293 cells activated by either mechanical or chemical stimuli will be shown as well as the combination of both methods. Under voltage-clamp we were able to show reliable quantification of PIEZO1 activation by fluid flow, Yoda1 (a small-molecule PIEZO1 agonist) and a Yoda1 analogue. To our knowledge, this is the first time that mechanical stimulation of PIEZO channels in a high throughput planar patch clamp system has been shown. The possibility of comparing and combining mechanical and chemical stimulation in a high throughput electrophysiological assay facilitates the biophysical and pharmacological studies of PIEZO channels. This work was supported by research grants from the Wellcome Trust, British Heart Foundation and has received funding from the European Union’s Horizon 2020 research and innovation program.
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