Intracellular Mechanosensation in Intestinal Smooth Muscle: Piezo1 Complexes Amplify Signaling Beyond the Surface

ABSTRACT Mechanosensation is fundamentally viewed as a plasma membrane phenomenon. We challenge this paradigm by introducing intracellular mechanosensation in intestinal smooth muscle. We hypothesized that a distinct, organelle-based signaling axis exists to amplify mechanotransduction from the inside out. To test this, we investigated whether Piezo1, a canonical plasma membrane mechanosensor, also operates within the cell. Using tissue-level wire myography, high-resolution confocal microscopy, proximity ligation assays, and patch-clamp electrophysiology on freshly dissociated cells, we identified a functional intracellular signaling hub that starts at the sarcoplasmic reticulum (SR). Unlike surface transduction, this intracellular mechanism relies on a nanoscale multiprotein complex (<40 nm) comprising an SR sensor (intra-Piezo1) and an amplifier (Ryanodine Receptor, RyR), coupled with a PM effector (large-conductance, Ca2+-activated K+ channels, i.e., BKCa channels). Activating this intracellular complex generated massive BK-mediated outward currents independent of extracellular Ca²⁺ but strictly dependent on internal SR Ca²⁺ stores, confirming intrinsic organellar mechanotransduction. Within this complex, intra-Piezo1 and RyR are positioned to operate as a coupled SR Ca²⁺ release unit that activates BK channels at SR–PM junctions, driving potent membrane hyperpolarization that reduces smooth muscle contractility, revealing the intra-Piezo1 complex as a molecular brake on excitation. These findings demonstrate that mechanotransduction is not confined to the cell surface. Instead, a specialized Sensor–Amplifier–Effector complex originating at intracellular organelles amplifies cellular sensitivity to physical force, providing a critical gain-control system that restrains smooth muscle excitability and regulates GI motility. Key Points Intracellular organelles contribute to mechanosensory signaling in GI smooth muscle cells, complementing plasma membrane mechanisms. Intra-Piezo1 form a nanoscale signaling complex (<40nm) on the sarcoplasmic reticulum (SR), linking the mechanosensor Piezo1 with RyR and large conductance, Ca2+-activated K+ channels. Unlike surface sensors, this intracellular complex functions via a “Sensor-Amplifier-Effector” mechanism in which intra-Piezo1 detects mechanical stress and triggers SR Ca2+ release, thereby activating a nearby RyR and large-conductance, Ca2+-activated K+ channel. Engaging this intracellular Piezo1-mediated axis significantly dampens smooth muscle contractility, identifying a critical gain-control system essential for regulating GI motility. Competing Interest Statement The authors have declared no competing interest.