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

Tharaka Wijerathne; Jerome Lacroix

doi:10.1016/j.xpro.2023.102789

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

STAR Protoc. 2024 Mar 15;5(1):102789. doi: 10.1016/j.xpro.2023.102789. Epub 2023 Dec 15.

Authors

Tharaka Wijerathne¹, Jerome Lacroix²

Affiliations

¹ Department of Basic Medical Sciences, Western University of Health Sciences, 309 East Second St., Pomona, CA 91766, USA.
² Department of Basic Medical Sciences, Western University of Health Sciences, 309 East Second St., Pomona, CA 91766, USA. Electronic address: jlacroix@westernu.edu.

Abstract

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.

Keywords: Biophysics; Microscopy; Molecular/Chemical Probes; Signal Transduction.

MeSH terms

Fluorometry / methods
Patch-Clamp Techniques*
Protein Conformation

Grants and funding

R01 GM130834/GM/NIGMS NIH HHS/United States