Voltage-clamp fluorometry to record flow-activated PIEZO1 currents and fluorometric signals.
Biophysics
Microscopy
Molecular/Chemical Probes
Signal Transduction
Journal
STAR protocols
ISSN: 2666-1667
Titre abrégé: STAR Protoc
Pays: United States
ID NLM: 101769501
Informations de publication
Date de publication:
15 Mar 2024
15 Mar 2024
Historique:
received:
14
09
2023
revised:
13
11
2023
accepted:
01
12
2023
pubmed:
17
12
2023
medline:
17
12
2023
entrez:
16
12
2023
Statut:
ppublish
Résumé
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).
Identifiants
pubmed: 38103195
pii: S2666-1667(23)00756-6
doi: 10.1016/j.xpro.2023.102789
pmc: PMC10770629
pii:
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
102789Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM130834
Pays : United States
Informations de copyright
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of interests The authors declare no competing interests.