ABSTRACT

Tentonin 3/TMEM150C (TTN3) is a mechanically activated (MA) channel involved in various mechanotransduction processes. It is rapidly activated by mechanical stimuli and followed by slow inactivation. While it is well-established that many MA channels are modulated by forces derived from membrane lipids and tethering proteins associated with the cytoskeleton, the mechanisms underlying the modulation of TTN3's mechanosensitivity remain largely unexplored. Here, we report that the pharmacological disruption of the F-actin cytoskeleton or downregulation of focal adhesion genes significantly reduced TTN3 MA currents. Strengthening the F-actin cytoskeleton produced robust TTN3 MA currents in Piezo1-deficient cells. Moreover, we identify stomatin-like protein 3 (StomL3) as a key modulator of TTN3, which augments membrane stiffness through cholesterol recruitment and physically associates with TTN3 to enhance its mechanosensitivity. Our findings highlight that TTN3 mechanosensitivity highly depends on cytoskeletal integrity and StomL3-dependent cell stiffness.