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Sphingomyelinase Disables Inactivation in Endogenous PIEZO1 Channels.

Cell reports (2020-10-08)
Jian Shi, Adam J Hyman, Dario De Vecchis, Jiehan Chong, Laeticia Lichtenstein, T Simon Futers, Myriam Rouahi, Anne Negre Salvayre, Nathalie Auge, Antreas C Kalli, David J Beech
ABSTRACT

Endogenous PIEZO1 channels of native endothelium lack the hallmark inactivation often seen when these channels are overexpressed in cell lines. Because prior work showed that the force of shear stress activates sphingomyelinase in endothelium, we considered if sphingomyelinase is relevant to endogenous PIEZO1. Patch clamping was used to quantify PIEZO1-mediated signals in freshly isolated murine endothelium exposed to the mechanical forces caused by shear stress and membrane stretch. Neutral sphingomyelinase inhibitors and genetic disruption of sphingomyelin phosphodiesterase 3 (SMPD3) cause PIEZO1 to switch to profoundly inactivating behavior. Ceramide (a key product of SMPD3) rescues non-inactivating channel behavior. Its co-product, phosphoryl choline, has no effect. In contrast to ceramide, sphingomyelin (the SMPD3 substrate) does not affect inactivation but alters channel force sensitivity. The data suggest that sphingomyelinase activity, ceramide, and sphingomyelin are determinants of native PIEZO gating that enable sustained activity.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Collagenase from Clostridium histolyticum, Type IA, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid, For general use
Sigma-Aldrich
Sphingomyelin, from chicken egg yolk, ≥95%
Sigma-Aldrich
2-Methacryloyloxyethyl phosphorylcholine, contains ≤100 ppm MEHQ as inhibitor, 97%
Sigma-Aldrich
Desipramine hydrochloride, ≥98% (TLC), powder
Sigma-Aldrich
Sphingomyelinase from Bacillus cereus, buffered aqueous glycerol solution, ≥100 units/mg protein (Lowry)
Sigma-Aldrich
Altenusin, ≥98% (HPLC)