Intracellular Piezo1 Modulates Intestinal Smooth Muscle Excitability

Background: The mechanosensitive ion channel Piezo1 is typically defined as a plasma membrane (PM) sensor that mediates Ca 2 + influx and excitation. However, recent studies indicate that Piezo1 localizes intracellularly in GI smooth muscle cells (SMCs) with the loss of Piezo1 disrupting small bowel contractility and tissue mechanics, suggesting a more nuanced role than previously established. Therefore, we tested the hypothesis that Piezo1 functionally couples with key intracellular Ca 2 + handling channels in adult GI smooth muscle cells (SMCs) to modulate feedback mechanisms unique to the intestine. Methods: We used high-resolution confocal microscopy, proximity ligation assays (PLA), and patch-clamp electrophysiology on freshly dissociated SMCs from adult murine small intestine and performed functional contractility assays using wire myography in whole and peeled intestinal muscularis. Results: Contrary to the canonical view of Piezo1, we found Piezo1 clusters highly localized to intracellular SR regions (~80%). PLA demonstrated nanoscale proximity (< 40nm) between Piezo1 and the SR calcium-release channel Ryanodine (RyR), as well as large-conductance calcium-activated potassium channels (BKCa). Functionally, pharmacological activation of Piezo1 using Yoda2 generated large outward currents that persisted in the absence of extracellular Ca 2+ (p< 0.001). Furthermore, these currents were abolished after internal SR Ca 2+ depletion by Thapsigargin. Piezo1-activated outward currents were also inhibited by the BKCa channel blocker (Paxilline) (p< 0.05). At the tissue level, Piezo1 activation significantly weakened muscle contractions (p< 0.05) and disrupted rhythmicity, mirroring the hyperpolarizing current observed at the cellular level. Conclusions: These findings identify a novel signaling pathway in the small intestine in which intercellular Piezo1 clusters localize to the sarcoplasmic reticulum (SR) and form a microdomain with large-conductance, calcium-activated potassium (BKCa) channels. Functionally, Piezo1 activation triggers SR Ca 2 + release, which in turn activates nearby BKCa channels, leading to membrane hyperpolarization and smooth muscle relaxation. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.