PROJECT SUMMARY/ABSTRACT Coordinated gastrointestinal (GI) tract motility is fundamental for normal GI tract function. Several cell types combine to regulate GI motility, with the smooth muscle cell (SMC) as the workhorse required to provide the physical power for contractions. Disruptions in SMC function contribute to common GI disorders, may occur after infections and inflammation, and associate with rare but devastating GI motility disorders like visceral myopathies and pseudo-obstruction. The gut wall is a highly complex multilayered structure under mechanical stress at baseline and constantly moving. Therefore, cells in the GI tract experience a range of types and amounts of mechanical stimuli. The normal coordinated motility requires an ability to sense and adjust to forces. In multiple cycles of this grant, we have dissected mechanisms of smooth muscle mechanotransduction, have made discoveries that advanced GI physiology and pathophysiology, and provided novel drug targets. However, our current understanding of SMC mechanosensing remains incomplete. It is established that SMCs, even as single cells, adjust their contractions in response to force in a process called the myogenic reflex. In vascular SMCs, the myogenic reflex depends on mechanogated ion channels, but in the GI tract, cellular and molecular mechanisms remain poorly understood. Therefore, the overall objective of our research is to determine the primary mechanogated ion channels involved in GI SMC mechanosensitivity. For this proposal, we created novel animal models and used cutting-edge techniques to generate compelling preliminary data. Our preliminary studies show that a recently discovered mechanogated ion channel Tmem63a is expressed in a subpopulation of SMCs, which are optimized for force sensing and distributed across the tissue to detect force. Indeed mechanosensitive ionic currents in a population of primary mouse GI SMCs have unique biophysical properties consistent with Tmem63a, the activation of which by force leads to a Ca2+ increase, modulating small and large bowel contractions and whole gut transit time. Interestingly, our data also show that patients with slow transit constipation have a decrease in Tmem63a. Thus, the central hypothesis that a mechanogated ion channel Tmem63a significantly contributes to the myogenic reflex will be tested in two Aims. In Aim 1, we determine Tmem63a function, its response to force, and its role in GI SMCs using conventional and cutting-edge techniques electrophysiology and Ca2+ imaging approaches. In Aim 2, we propose experiments to define the Tmem63a+ SMC population and to determine the role of Tmem63a SMCs in regulating GI smooth muscle function. Since Tmem63a is found in a subpopulation of SMCs, we use single-cell and spatial transcriptomics, novel Ca2+ imaging, smooth muscle contractility assays and in vivo whole gut transit. Successful completion of the proposed innovative experiments has both basic significance and clinical impact, evaluating and establishing a novel SMC mechanogated ion channel which contributes to SMC function and the myogenic reflex and, in the long term, may provide a novel target for functional and motility GI disorders.

PROJECT NARRATIVE Gastrointestinal functional and motility disorders affect ~15% of the US population, resulting in chronic morbidity, disruptions in quality of life, loss of productivity and high healthcare costs. Gastrointestinal motility is a complex process that is ultimately executed by the smooth muscle cells. This proposal targets the molecular mechanisms involved in the function and dysfunction in smooth muscle cells, and has the potential to significantly advance basic understanding and potential to provide novel diagnostic approaches and drug targets.