Intracellular mechanosensation in intestinal smooth muscle: Piezo1 complexes amplify signalling beyond the surface.
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 signalling axis exists to amplify mechanotransduction from the inside out. To test this we investigated whether Piezo1, a canonical plasma membrane (PM) 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 previously uncharacterized Piezo1-RyR-BKCa signalling axis in small intestinal smooth muscle cells (SMC). This intracellular mechanism relies on a nanoscale signalling complex (<40 nm) comprising an intracellular 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 paxilline-sensitive outward currents independent of extracellular Ca2+ and dependent on internal SR Ca2+ stores, consistent with 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 support a model in which 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 gastrointestinal (GI) motility. KEY POINTS: This study advances a framework in which intracellular organelles contribute to mechanosensory signalling in gastrointestinal (GI) smooth muscle cells, complementing plasma membrane mechanisms. Piezo1 is predominantly intracellular in intestinal smooth muscle, where this pool, intra-Piezo1, forms a nanoscale signalling complex on the sarcoplasmic reticulum (SR) that positions it within <40 nm of RyR and of sarcolemmal large-conductance, Ca2+-activated K+ (BKCa) channels. Electrophysiological recordings show that this 'sensor-amplifier-effector' mechanism generates potent paxilline-sensitive hyperpolarizing currents that depend on SR Ca2+ release and persist without extracellular Ca2+, amplifying the cell's response to mechanical stress when intra-Piezo1 is activated from the inside out. Activation of this intra-Piezo1-mediated axis significantly dampens smooth muscle contractility, acting as a molecular 'brake' that supports the stretch-induced-relaxation feedback mechanism essential for intestinal function.