The anchor domain is critical for Piezo1 channel mechanosensitivity

Abstract

The mechanosensitive channel Piezo1 is a crucial membrane mechanosensor ubiquitously expressed in mammalian cell types. Critical to its function in mechanosensory transduction is its ability to change conformation in response to applied mechanical force. Here, we interrogate the role of the anchor domain in the mechanically induced gating of human Piezo1 channels. Using the insertion of glycine residues at each corner of the triangular-shaped anchor domain to decouple this domain we provide evidence that the anchor is important in Piezo1 mechanogating. Insertion of two extra glycine residues between the anchor and the outer helix of human Piezo1 causes abrogated inactivation and reduced mechanosensitivity. Whereas inserting two glycine residues at the apex of the anchor domain at the conserved amino acid P2113 causes the channel to be more sensitive to membrane forces. Correlation of stretch sensitivity with the volume of the neighboring amino acid, natively a phenylalanine (F2114), suggests this is caused by removal of steric hindrance on the inner pore-lining helix. Smaller volume amino acids at this residue increase sensitivity whereas larger volume reduces mechanosensitivity. The combined data show that the anchor domain is a critical region for Piezo1-mediated force transduction.

Loss-of-Function Piezo1 Mutations Display Altered Stability Driven by Ubiquitination and Proteasomal Degradation

“…Previous work points towards defective trafficking of G2029 R (Lukacs et al, 2015) but that S217L had normal membrane localization (Faucherre et al, 2020). We showed that both S217L and G2029R displayed reduced N-glycosylation (Li et al, 2020) [in addition to other trafficking defective mutants generated during structure-function studies (Vero Li et al, 2021)] similar to trafficking defective disease causing variants in ion channels such as K v11.1 , which causes long QT syndrome type 2 (Vandenberg et al, 2012). Herein we provided further insight into the molecular mechanisms by which these mutations influence Piezo1 function.…”

“…The cycloheximide chase procedure has permitted visualization of the degradation kinetics of the steady state population of a variety of cellular proteins (Kao et al, 2015). To understand the turnover rate of Piezo1 channels, we applied cycloheximide (CHX) to the culture media of Piezo1 −/− HEK293T cells expressing wild-type (WT) or mutant Piezo1 (S217L and G2029R) fused to GFP (Cox et al, 2016;Li et al, 2020;Vero Li et al, 2021;Ridone et al, 2020;Buyan et al, 2020).…”

“…These N-glycans may act as molecular tethers and participate in force sensing (particularly shear stress sensing) as shown for the epithelial sodium channel ENaC (Knoepp et al, 2020). Previously, we showed that trafficking defective Piezo1 mutations (disease linked or from structure function studies (Vero Li et al, 2021)) display modified N-linked glycosylation (Li et al, 2021). Specifically, we noted that loss-of-function variants of Piezo1 such as S217L and G2029R show reduced N-linked glycosylation.…”

Modified N-linked glycosylation status predicts trafficking defective human Piezo1 channel mutations

“…Probing N-glycosylation status in this manner provides a rapid and reliable method to determine if human Piezo1 variants, that are generated for structure-function studies 33 or in studies of disease-linked variants, exhibit aberrant trafficking exemplified by our data on G2029R, a known trafficking defective Piezo1 variant 28 . We also used glycosylation status to attempt to rescue G2029R using two widely used strategies; low temperature treatment and a pharmacological chaperone 22 .…”

“…Also, the presence of the larger N-glycosylated species of Piezo1 was seen to be a surrogate for the mature membrane protein as it was absent in trafficking-defective mutants. These findings will be useful when assessing the effects of mutations generated during structure-function studies 33 , or when exploring disease-linked PIEZO1 variants. As a proof of concept, we showed that loss-of-function Piezo1 variants such as G2029R that are known to be trafficking-defective 28 lack a larger N-glycosylated species when expressed in vitro.…”