Mechanosensing is an integral part of many physiological processes including stem cell differentiation, fibrosis, and cancer progression. Two major mechanosensing systems—focal adhesions and mechanosensitive ion channels—can convert mechanical features of the microenvironment into biochemical signals. We report here unexpectedly that the mechanosensitive calcium-permeable channel Piezo1, previously perceived to be diffusive on plasma membranes, binds to matrix adhesions in a force-dependent manner, promoting cell spreading, adhesion dynamics, and calcium entry in normal but not in most cancer cells tested except some glioblastoma lines. A linker domain in Piezo1 is needed for binding to adhesions, and overexpression of the domain blocks Piezo1 binding to adhesions, decreasing adhesion size and cell spread area. Thus, we suggest that Piezo1 is a previously unidentified component of focal adhesions in nontransformed cells that catalyzes adhesion maturation and growth through force-dependent calcium signaling, but this function is absent in most cancer cells.

Pressure overload-induced cardiac hypertrophy is a maladaptive response with poor outcomes and limited treatment options. The transient receptor potential melastatin 4 (TRPM4) ion channel is key to activation of a Ca2+/calmodulin-dependent kinase II (CaMKII)-reliant hypertrophic signaling pathway after pressure overload, but TRPM4 is neither stretch-activated nor Ca2+-permeable. Here we show that Piezo1, which is both stretch-activated and Ca2+-permeable, is the mechanosensor that transduces increased myocardial forces into the chemical signal that initiates hypertrophic signaling via a close physical interaction with TRPM4. Cardiomyocyte-specific deletion of Piezo1 in adult mice prevented activation of CaMKII and inhibited the hypertrophic response: residual hypertrophy was associated with calcineurin activation in the absence of its usual inhibition by activated CaMKII. Piezo1 deletion prevented upregulation of the sodium–calcium exchanger and changes in other Ca2+ handling proteins after pressure overload. These findings establish Piezo1 as the cardiomyocyte mechanosensor that instigates the maladaptive hypertrophic response to pressure overload, and as a potential therapeutic target.

Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like most plasma membrane ion channel proteins they must pass through biosynthetic quality control in the endoplasmic reticulum that results in them reaching their destination at the plasma membrane. Here we show that N-linked glycosylation of two highly conserved asparagine residues in the ‘cap’ region of mechanosensitive Piezo1 channels are necessary for the mature protein to reach the plasma membrane. Both mutation of these asparagines (N2294Q/N2331Q) and treatment with an enzyme that hydrolyses N-linked oligosaccharides (PNGaseF) eliminates the fully glycosylated mature Piezo1 protein. The N-glycans in the cap are a pre-requisite for N-glycosylation in the ‘propeller’ regions, which are present in loops that are essential for mechanotransduction. Importantly, trafficking-defective Piezo1 variants linked to generalized lymphatic dysplasia and bicuspid aortic valve display reduced fully N-glycosylated Piezo1 protein. Thus the N-linked glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance.

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.

Missense mutations in the gene that encodes for the mechanically-gated ion channel Piezo1 have been linked to a number of diseases. Gain-of-function variants are linked to a hereditary anaemia and loss-of-function variants have been linked to generalized lymphatic dysplasia and bicuspid aortic valve. Two previously characterized mutations, S217L and G2029R, both exhibit reduced plasma membrane trafficking. Here we show that both mutations also display reduced stability and higher turnover rates than wild-type Piezo1 channels. This occurs through increased ubiquitination and subsequent proteasomal degradation. Congruent with this, proteasome inhibition using N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) reduced the degradation of both mutant proteins. While ALLN treatment could not rescue the function of S217L we show via multiple complementary methodologies that proteasome inhibition via ALLN treatment can not only prevent G2029R turnover but increase the membrane localized pool of this variant and the functional Piezo1 mechanosensitive currents. This data in combination with a precision medicine approach provides a new potential therapeutic avenue for the treatment of Piezo1 mediated channelopathies.