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

Abstract

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Biophysics
• Microscopy
• Molecular/Chemical Probes
• Signal Transduction