Voltage-clamp fluorometry to record flow-activated PIEZO1 currents and fluorometric signals

PIEZO channels sense mechanical forces through conformational rearrangements of a mechanosensory domain called blade. To probe these rearrangements in real time, we have inserted conformational-sensitive cyclic-permuted GFP into several positions of PIEZO1’s blade. Here, we describe the step-by-step experimental procedure we developed to simultaneously measure flow-activated ionic currents and fluorometric signals in cells expressing these engineered constructs. We describe steps for performing transfection, seeding cells on coverslips, setting up a perfusion-based fluid shear application system, and performing voltage-clamp fluorometry. For complete details on the use and execution of this protocol, please refer to Ozkan et al. (2023).1 [Display omitted] •Multiplexing electrophysiology, fluorescence imaging, and mechanical stimulations•Detailed procedures from sample preparation to data acquisition•Flow-rate optimization assay to maximize fluorometric signals Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. PIEZO channels sense mechanical forces through conformational rearrangements of a mechanosensory domain called blade. To probe these rearrangements in real time, we have inserted conformational-sensitive cyclic-permuted GFP into several positions of PIEZO1’s blade. Here, we describe the step-by-step experimental procedure we developed to simultaneously measure flow-activated ionic currents and fluorometric signals in cells expressing these engineered constructs. We describe steps for performing transfection, seeding cells on coverslips, setting up a perfusion-based fluid shear application system, and performing voltage-clamp fluorometry.