Noncanonical calcium-independent TRPM4 activation governs intestinal fluid homeostasis

Domain: chemistry
Published: 2026-01-08
Editorial outcome: major_revision

Abstract
Imbalance in intestinal fluid homeostasis leads to nutrient malabsorption, intestinal tissue destruction, and systemic inflammation. Transient receptor potential melastatin 4 (TRPM4) is a calcium-activated, non-selective monovalent cation channel converting chemical signals (Ca2+) into electrical signals (membrane depolarization). Here, we show the TRPM4 channel as a direct target of bisacodyl (BIC), a widely used clinical drug for chronic constipation management, and its active metabolite, deacetyl bisacodyl (DAB). DAB-induced laxative effects are abolished in global and intestinal epithelium-specific TRPM4-knockout mice, establishing the essential role of TRPM4 in intestinal fluid regulation. Furthermore, our structural work reveals DAB bound to an uncharacterized pocket, marking it as a non-Ca2+ TRPM4 agonist and unveiling a noncanonical Ca2+-independent activation mechanism. Additionally, we delineate a signaling axis, TRPM4→VGCC/NCX→ANO1, that governs ion homeostasis in the epithelium. Together, these findings establish TRPM4 as a key regulator of intestinal fluid balance and reveal its noncanonical calcium-independent activation as a therapeutic strategy for constipation.

Introduction
Effective intestinal fluid homeostasis is essential for maintaining digestive health by balancing nutrient absorption with the elimination of waste. Disruptions in this balance manifest as constipation or diarrhea—conditions that pose significant health challenges. Although not lethal, chronic constipation arising from a variety of etiologies, including neurogenic bowel dysfunction, opioid-induced constipation, colorectal malignancy, and constipation-predominant irritable bowel syndrome, affects approximately one-third of the U.S. population, with a higher prevalence among the elderly and women. This condition not only significantly increases the health burden and impacts quality of life1 but can also lead to complications such as fecal impaction and hemorrhoids. Conversely, chronic diarrhea may result in dehydration, electrolyte imbalances, malabsorption, and systemic inflammation, often signaling more complex underlying disorders. Moreover, these conditions are not merely isolated ailments; they frequently serve as clinical indicators of broader molecular dysregulation, arising either from intrinsic gastrointestinal diseases or as adverse effects of pharmacological treatments. Despite the high prevalence and severe clinical implications of these disorders, the molecular mechanisms governing intestinal fluid dynamics remain poorly understood.

Among the therapeutic strategies for managing these conditions, Bisacodyl (BIC)—a stimulant laxative of the diphenylmethane family—has long been employed to treat chronic constipation2. Its active metabolite, deacetyl bisacodyl (DAB), is responsible for its efficacious laxative effect2. Clinical studies have consistently shown that BIC is more effective than alternatives such as prucalopride, lubiprostone, linaclotide, and tegaserod3. However, despite its popular clinical utility since the discovery in the 1950s, the precise molecular targets and intracellular signaling pathways underlying BIC’s action remain unknown. This critical gap in our understanding highlights the urgent need to decipher the molecular basis of intestinal fluid regulation, which is fundamental to developing more targeted and effective treatments for both constipation and diarrhea.

Recent advances in ion channel research have begun to shed light on the contribution of transient receptor potential (TRP) channels to gastrointestinal disorders. Among these, transient receptor potential melastatin 4 (TRPM4)—a calcium-activated, non-selective monovalent cation channel is widely expressed in tissues including the colon4. TRPM4 is responsible for transducing intracellular calcium (Ca2+) signals into membrane depolarization5. In excitable tissues such as the heart and brain, TRPM4 is well known for its roles in modulating cardiac conduction and neuronal excitability6,7. In the immune system, it is expressed in many immune cells, predominantly in T lymphocytes, where it regulates Ca2+ signaling critical for cell activation and migration8. While TRPM4 has been reported to be expressed in the colon tissues4, the pathophysiological role of TRPM4 in the gastrointestinal system remains largely unexplored.

Structurally, TRPM4 is organized as a homotetramer, with each subunit comprising six transmembrane helices and extensive intracellular N- and C-terminal domains that are essential for channel gating9,10. Recent cryo-electron microscopy studies have provided high-resolution insights into its architecture by delineating key binding pockets for agonist and modulators including Ca2+, ATP and decavanadate (DVT)9–11. Notably, emerging evidence highlights temperature-dependent structural dynamics that further modulate TRPM4 function, a feature that may be particularly relevant for understanding its physiological roles12.

Here, our in vitro and in vivo experiments demonstrate that anti-constipation drug BIC and its active metabolite DAB regulate ion transport by directly targeting TRPM4 in the intestinal epithelial cells, establishing a critical role of epithelial-specific TRPM4 as a regulator of intestinal fluid homeostasis and revealing noncanonical Ca2+-independent activation as a therapeutic strategy for chronic constipation. These findings underpin the laxative action of BIC in the treatment of chronic constipation and lay a molecular foundation for the rational design of targeted therapeutics for constipation arising from a variety of etiologies.

Results
BIC and DAB are potent TRPM4 activators
TRPM4 is widely expressed throughout the human body and has recently emerged as an attractive drug target due to its critical role in cancer, immune disorders and heart diseases13. However, there is lack of specific TRPM4 modulators, and Ca2+ remains the only known agonist. To address this gap, we conducted an unbiased high-throughput screen of 2154 structurally diverse compounds using a fluorescence-based Na⁺ mobilization assay in HEK-293 cells stably expressing human TRPM4 (HEK293-TRPM4) (Supplementary Fig. 1A). We discovered that BIC and its active metabolite, DAB activate TRPM4. Treatment with 10 µM BIC resulted in a gradual increase in Na⁺ influx, an effect that was absent in null HEK-293 cells (Supplementary Fig. 1B). DAB elicits a more rapid Na+ influx in HEK-293-TRPM4 cells, but not in null HEK-293 cells, suggesting a more potent effect on TRPM4 activation than its parent compound BIC (Supplementary Fig. 1E). This aligns with previous report that DAB is responsible for the efficacious laxative effect of BIC. Further concentration-response analyses confirmed that both compounds stimulate Na⁺ influx in a concentration-dependent manner, with EC50 values of 2.64 µM for BIC and 0.14 µM for DAB (Supplementary Fig. 1C, D, F, G), demonstrating that DAB is ~20-fold more potent than BIC.

Intestinal epithelial TRPM4 is essential for DAB-induced laxative effects
Given that TRPM4 is expressed in intestinal epithelial cells4 and functions as a monovalent ion channel contributing to sodium influx, we speculate that TRPM4 could be the effective molecular target of BIC and DAB, which alter electrolyte gradients and impact fluid movement in the intestine. To test our hypothesis, we performed in vivo animal experiments. Because DAB is the active metabolite of BIC and exhibits markedly higher potency, we focused on DAB in the following in vivo and in vitro experiments.

Immunofluorescence staining revealed strong TRPM4 expression in the colon tissues. The majority of TRPM4 fluorescence signals co-localized with CK-18, a well-established marker of intestinal epithelial cells, confirming TRPM4’s predominant expression in this cell type (Fig. 1A, upper panel). In contrast, TRPM4 knockout (Trpm4-/-) mice showed no detectable TRPM4 fluorescence in the colon (Fig. 1A, lower panel).

To functionally assess the role of TRPM4 in DAB-induced laxative effects, we administered DAB (20 mg kg-1) via oral gavage to both male and female wild-type (WT) and Trpm4-/- mice (Fig. 1B). In WT mice, DAB treatment led to a marked increase in fecal water content and an elevated Bristol stool score (a widely used measurement of stool consistency)14, indicative of an efficacious laxative effect (Fig. 1C, D; Supplementary Fig. 2A). These laxative effects were completely abolished in both male and female Trpm4-/- time-matched littermates, demonstrating that TRPM4 is indispensable for mediating DAB-induced intestinal fluid secretion and stool softening (Fig. 1C, D; Supplementary Fig. 2A).

To further confirm the role of epithelial-specific TRPM4 in this process, we generated intestinal epithelium-specific TRPM4 knockout mice (Trpm4ΔIEC) by cross-mating Trpm4flox/flox mice with Villin-Cre (Vil1-Cre) transgenic mice (Supplementary Fig. 3A). Immunofluorescent staining confirmed successful epithelium-specific deletion of TRPM4, as TRPM4 expression was robustly detected in the colon of Trpm4flox/flox mouse but completely absent in Trpm4ΔIEC mice, while the expression in the skin tissues remained unchanged (Supplementary Fig. 3B). We administrated DAB to both male and female Trpm4flox/flox and Trpm4ΔIEC littermates. In Trpm4flox/flox mice, DAB markedly increased fecal water content and Bristol stool scores, replicating effects observed in WT mice. However, such effect was not observed in DAB-treated Trpm4ΔIEC littermates (Fig. 1E, F; Supplementary Fig. 2B).

Biophysical characterization of DAB-induced activation on TRPM4
Our in vivo findings provide strong evidence that TRPM4 is the key mediator of DAB-induced epithelial fluid secretion and laxative action, we next looked into the biophysical characterization of DAB-induced activation on TRPM4 to find definitive evidence whether DAB directly activates TRPM4, and whether their effects are influenced by temperature or Ca2+ levels, two factors that critically impact TRPM4 activation12.

Using whole-cell patch-clamp recordings in HEK293-TRPM4 cells, we found that, at low concentrations (≤ 0.3 µM), DAB elicited outwardly rectified Na+ currents, whereas at higher concentrations, the I-V curves became linear (Fig. 2A–C), consistent with voltage-dependent gating of TRPM4 channels15. Importantly, introducing 1 µM free Ca2+ into the pipette solution did not alter the potency of DAB, suggesting that DAB-induced TRPM4 activation is independent of intracellular Ca2+ concentration (Fig. 2D–F; Supplementary Table 1). Furthermore, as Ca2+-dependent TRPM4 activation is temperature-dependent with substantial increase in current magnitude at higher temperature12, we explored the thermo-sensitivity of DAB-induced TRPM4 activation. Interestingly, DAB-induced TRPM4 activation showed no obvious difference between 25°C and 37°C (Fig. 2; Supplementary Table 1). Together, these findings suggest that DAB activates TRPM4 independent of both intracellular Ca2+ and temperature, implying an activation mechanism distinct from that of the endogenous agonist, Ca2+. In addition, DAB also activates mouse (m) TRPM4, but has no effect on hTRPM3, hTRPM5 and hTRPM8 (Supplementary Fig. 4A–D), demonstrating that DAB is a selective agonist of TRPM4.

Structure of DAB—TRPM4 complex
Our in vivo and in vitro evidence establishes DAB as a non-Ca2+ agonist selective for TRPM4, highlighting the need to dive into molecular level to identify their binding site and underlying mechanism of activation.

To this end, we determined the cryo-EM structures of human TRPM4 bound to DAB in the presence or absence of Ca²⁺ at physiological temperature, achieving overall resolutions of 3.1 and 3.3 Å, respectively (Fig. 3A–C; Supplementary Fig. 5; Supplementary Table 2). The overall structures resemble the previously published TRPM4 structures of a tetramer assembly with a transmembrane domain (TMD) and a large intracellular domain (ICD). In both conditions, we observed a well-defined density within the S1-S4 domain of each subunit, positioned above the Ca2+ TMD binding site (Fig. 3D, E). This density is tripod-shaped, with one pod smaller than the other two, closely resembling the shape of DAB. Notably, it seems this density has not been observed in any previously reported TRPM4 structures, suggesting a previously unrecognized ligand binding site in TRPM4.

The putative DAB binding site is primarily surrounded by both hydrophobic residues and hydrophilic residues (Fig. 3D). Alanine substitution of these key residues increased the EC50 values of DAB by 1–2 orders of magnitude (Fig. 3H; Supplementary Fig. 6; Supplementary Table 3), confirming this as the functional binding site. We further determined the structures of TRPM4 in the presence of BIC, the precursor of DAB. Despite attempting different concentrations up to 1 mM, we did not observe the density for BIC in the cryo-EM maps. We speculate that the two extra acetyl groups in BIC impede optimal engagement with the binding pocket, leading to reduced binding affinity, which may explain its weaker potency on TRPM4 compared to DAB.

DAB activates TRPM4 via a TMD-focused mechanism
Our structural data reveal that while DAB and Ca2+ bind to adjacent sites within the S1-S4 domain of TRPM4, they interact with entirely different sets of residues and activate TRPM4 independently (Fig. 3D). This distinction underscores the functional and pharmacological significance of the DAB binding site.

Previous studies have established that in non-excitable cells, Ca2+-induced activation of TRPM4 and its close homolog TRPM5 at physiologically relevant negative membrane potentials requires Ca2+ binding at two sites: the TMD agonist site and the ICD modulatory site12,16. While the Ca2+ TMD site functions as the orthosteric agonist site, the Ca2+ ICD site is essential for triggering conformational changes—specifically, an upward movement of the ICD toward the TMD—that prime the channel for opening. This movement reflects an allosteric communication between the ICD and TMD, which is critical for activation at negative potentials. In the absence of Ca2+ binding, or more precisely, without the associated ICD conformational dynamics, TRPM4 requires membrane depolarization to open.

In contrast, our electrophysiological data show that DAB activates TRPM4 at negative membrane potentials independently of Ca2+, indicating a distinct mechanism (Fig. 2A–F). This DAB-induced activation appears confined to the TMD, where DAB binds, and does not rely on ICD movement triggered by Ca2+ binding at Ca2+ ICD. Supporting this, TRPM4 adopts the same TMD conformation whether bound to DAB alone, Ca2+ alone, or both DAB and Ca2+, despite showing different ICD conformations depending on Ca2+ presence (Fig. 3F, G). Further supporting this distinction, DAB-induced activation is temperature independent (Fig. 2A–C, G–I), whereas Ca2+-induced activation requires both elevated temperature and Ca2+ binding to Ca2+ ICD to stabilize the ICD in its active conformation12.

Together, these findings suggest that DAB activates TRPM4 exclusively through its action at the TMD, bypassing the TMD–ICD allosteric communication required for Ca2+-dependent activation. This may provide a valuable strategy for developing TRPM4-targeted therapeutics. Importantly, unlike Ca2+-mediated gating, which is tightly controlled by fluctuations in cytosolic Ca2+ levels and relies on ICD conformational dynamics, TMD-focused agonists like DAB and BIC operate independently of these regulatory mechanisms. As a result, they may offer more consistent pharmacological effects across diverse tissues and physiological conditions, particularly in settings like the intestine, where cytosolic Ca2+ levels can vary despite stable physiological temperature.

Although DAB activates TRPM4 and produces an near-linear current-voltage relationship at high concentrations (Fig. 2B, E, H, K), the DAB-bound TRPM4 structures exhibit a closed pore, regardless of Ca2+ presence (Fig. 3I–K). A plausible explanation is that additional factors, such as endogenous lipids, may be required to stabilize the open conformation which is missing in our cryo-EM study compared to the in vivo and in vitro study. To gain molecular insight into how DAB facilitates channel activation, we compared the DAB-bound structure with two reference states: a Ca2+-bound closed state and an open state induced by Ca2+ in combination with the positive modulator DVT (Fig. 3L). This comparison revealed that DAB triggers activation through a mechanism that partially overlaps with the Ca2+/DVT-induced opening pathway.

Compared to the Ca2+-bound closed state, DAB binding induces a conformational change in the S4 helix. This change propagates through the S4–S5 linker to the adjacent portion of the S5 helix and the TRP helix (Fig. 3L), causing an upward movement of the ICD toward the TMD. Notably, these DAB-induced conformational changes resemble those observed when DVT converts the Ca2+-bound closed state into an open state12, suggesting that DAB alone partially mimics the combined effect of Ca2+ and DVT. However, unlike in the Ca2+/DVT-bound structure, the structural rearrangement triggered by DAB does not extend to the distal portion of the S5 helix adjacent to S6, and therefore the S6 helices remain in their closed conformation, and the ion-conducting pore does not open (Fig. 3L). This difference likely arises because DAB binds exclusively within the S1–S4 domain and does not directly engage the S5–S6 pore domain, as DVT does12. Since the DAB-bound structure adopts a conformation that is intermediate between the Ca2+-bound closed state and the Ca2+/DVT-induced open state (Fig. 3L), and the channel did not show any desensitization during a prolonged patch-clamp electrophysiological recording, we speculate that it represents a pre-open, partially activated state of TRPM4.

TRPM4→VGCC/NCX→ANO1 coupling in intestinal epithelium mediates the laxative effect of DAB
Given that the strong laxative effect of DAB was abolished in Trpm4ΔIEC mice, we next investigated the mechanism by which DAB disrupts ion homeostasis in intestinal epithelial cells. Na+ imaging revealed that DAB induced a robust increase in intracellular sodium concentration, an effect eliminated by pre-treatment with the TRPM4-selective inhibitor 9-Phe, indicating that TRPM4 is the primary molecular target mediating DAB-induced Na+ influx in HT-29 cells (Fig. 4A). Consistently, whole-cell patch-clamp recordings showed that DAB (10 μM) elicited a robust current in HT-29 cells, which was abolished by 9-Phe (Fig. 4B).

In intestinal epithelial cells, passive water movement into the intestinal lumen is driven by Cl- efflux, primarily mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and/or Ca2+-activated Cl- channels (CaCCs)17. To determine whether TRPM4-mediated Na⁺ influx couples to either or both Cl⁻ efflux systems, we conducted electrophysiological experiments. We found that DAB-induced currents were partially suppressed by Ani9, an inhibitor of Anoctamin 1 (ANO1, a Ca2+-activated Cl- channel)18, but not by CFTRinh-172, a selective CFTR inhibitor19 (Fig. 4C, F; Supplementary Fig. 7). This suggests that DAB-induced current in HT-29 cells arises from Na⁺ influx through TRPM4 and Cl⁻ efflux through ANO1 (rather than CFTR). Consistent with this model, Ani9 no longer suppressed DAB-induced currents when either extracellular Ca2+ was removed (0 mM Ca2+ without EGTA) or intracellular Ca2+ was cheated with EGTA, whereas 9-Phe still completely abolished the response under both conditions (Fig. 4D–F). This suggests that DAB-induced Ca2+ influx is required for ANO1 activation and subsequent Cl- efflux. Together, these findings indicate that Na⁺ influx through TRPM4 functionally couples to Ca2+-dependent Cl⁻ efflux via ANO1.

To validate the TRPM4–ANO1 coupling in vivo, mice were pre-treated with CaCCinh-A01 (A01), a specific ANO1 inhibitor with favorable pharmacokinetic properties20, prior to DAB administration. Pretreatment with A01—but not CFTR inhibitor CFTRinh-172—completely abolished DAB-induced laxative effect, as assessed by both Bristol score and fecal water content (Fig. 4G–I; Supplementary Fig. 8), confirming the functional relevance of the TRPM4–ANO1 axis in vivo.

We next explored how DAB might elevate intracellular Ca2+ to activate ANO1. In HT-29 cells, DAB triggered a rapid increase in cytosolic Ca2+ concentration, which was blocked by the pretreatment of 9-Phe, indicating the involvement of TRPM4 (Fig. 4J). Notably, this Ca2+ rise was completely abolished when extracellular Ca2+ was removed (0 mM Ca²⁺ without EGTA) (Fig. 4K), confirming that Ca2+ influx from the extracellular space is required for ANO1 activation.

To further dissect the route of Ca2+ entry, we found that DAB-induced Ca2+ influx was nearly eliminated by nifedipine, a blocker of voltage-gated Ca2+ channels (VGCCs), and modestly reduced by SEA0400, an inhibitor of the Na+-Ca2+ exchanger (NCX) (Fig. 4L, M). These findings support a model in which TRPM4-mediated Na⁺ promotes Ca²⁺ entry through two synergistic mechanisms: (1) membrane depolarization activates voltage-gated Ca2+ channels (VGCCs); and (2) the combination of elevated intracellular Na⁺ and membrane depolarization favors reverse-mode operation of NCX, leading to additional Ca2+ influx. The resulting rise in cytosolic Ca2+ activates ANO1, driving Cl⁻ efflux and contributing to the laxative effect of DAB.

Methods
All animal experiments were carried out in concordance with the National Institutes of Health Guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978) and approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University (#SYXK 2021-0011, approved for Jan 25th, 2021 to Jan 24th, 2026).

Reagents and antibodies
Bisacodyl (BIS, #603-50-9), 9-Phenanthrol (9-Phe, #484-17-3), triphenylphosphine oxide (TPPO, #791-28-6), CIM0216 (#1031496-06-6), menthol (#2216-51-5), Ani9 (#356102-14-2), SEA0400 (#223104-29-8), nifedipine (#21829-25-4), CaCCinh-A01 (A01, #407587-33-1), CFTRinh-172 (172, #307510-92-5), Blasticidin S (#3513-03-9), Hygromycin B (#31282-04-9) and doxycycline (#564-25-0) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Deacetyl Bisacodyl (DAB, #603-41-8) was purchased from FishReag (Nanjing, Jiangsu, China). Poly-D-lysine (PDL, #27964-99-4), pluronic® F-127 (#9003-11-6), Leupeptin (#103476-89-7), Aprotinin (#9087-70-1), Pepstatin (#26305-03-3), PMSF (#329-98-6) and all the inorganic reagents were purchased from Sigma-Aldrich (St. Louis, MA, USA). Lipofectamine® 2000 reagent (#12566014), CoroNa™ Green/AM (#C36676), Fetal bovine serum (FBS, #A5670701), Penicillin and streptomycin (P/S, #15140122), Geneticin (G418, #108321-42-2), and Dulbecco minimum essential medium (DMEM, #12800017) were from Thermo Fisher Scientific (Waltham, NJ, USA). Fluo-4/AM (#20550) was purchased from AAT Bioquest (Sunnyvale, CA, USA). Primary antibodies against CK-18 (#bsm-33101M, RRID:AB_3678929) and TRPM4 (#ACC-044, RRID:AB_2040250) were from Bioss Antibodies (Beijing, China) and Alomone Labs (Jerusalem, IL, USA), respectively. Alexa Fluor 555 conjugated goat anti-mouse IgG (H + L) (#4409, RRID:AB_1500655) and Alexa Fluor 488-conjugated goat Anti-rabbit IgG (H + L) (#4412, AB_3067986) secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). GDN (#GDN10125GM) was from Anatrace (Maumee, OH, USA). Strep-Tactin superflow high capacity resin (#2-1208-500) was obtained from IBA Lifesciences GmbH (Göttingen, Germany). Superose 6 Increase 10/300 GL (#29091596) was purchased from GE Health Care Technologies Inc. (Chicago, Illinois, USA).

Animals
Both male and female C57BL/6J mice (8 weeks, 20–22 g) were purchased from the Animal Core Facility of Nanjing Medical University (Nanjing, Jiangsu, China). Villin-Cre (Vil1-Cre) mice (Strain #021504) were purchased from The Jackson Laboratory (Bar Harbor, MI, USA). Global TRPM4 knockout (Trpm4-/-, Strain #T014306) and Trpm4flox/flox (Strain #T013555) mice were obtained from GemPharmatech Co., Ltd. (Nanjing, Jiangsu, China). Mice were kept in individual ventilation cage in an SPF animal facility under a 12/12 h light–dark cycle at 23 ± 2 °C with ad libitum access to food and water. Intestinal epithelium-specific TRPM4 knockout (Vil1+/--Trpm4flox/flox or Trpm4ΔIEC) mice were generated through cross-mating Vil1-Cre mice with Trpm4flox/flox mice. The genotypes of Trpm4-/-, Trpm4flox/flox and Trpm4ΔIEC mice were validated by RT-PCR and the expression levels of TRPM4 in colon tissues of these mice were accessed by immunofluorescence staining.

Cells, plasmid and
HEK-293-TRPM8 cells were the same as previously described34. Human colorectal cancer epithelial cells (HT-29 cells) were purchased from the Chinese Academy of Medical Sciences (Beijing, China). The gene encoding full-length hTRPM3 (UniProtKB: Q9HCF6), hTRPM4 (UniProtKB: Q8TD43) and mTRPM4 (UniProtKB: Q7TN37), hTRPM5 (UniProtKB: Q9NZQ8) were synthesized by BioBasic (Amherst, NY, USA).

Animal model
DAB laxative effect was evaluated in C57BL/6J mice at 8 weeks old (both male and female, 20–22 g). After acclimation for 2 days, DAB (20 mg kg−1) was administrated by intragastric gavage (i.g.) at a volume of 20 ml kg−1 every 12 h. To access the role of ANO1 and CFTR on DAB-induced laxative effect, CaCCinh-A01 (3 mg kg−1), a specific ANO1 inhibitor and CFTRinh-172 (1 mg kg−1), a specific CFTR inhibitor, were intraperitoneal (i.p.) injected (10 ml kg−1) into the mouse 30 min prior to the administration of DAB, every 12 h. All the compounds were dissolved in 2% DMSO + 2% Tween-80 and 0.5% sodium carboxymethyl cellulose (CMC-Na) solution. Mice in control group were gavaged with vehicle (0.5% CMC-Na + 2% DMSO + 2% Tween-80). After 24 h, the stool of each mouse was collected, pictured, and the Bristol score was used to assess the stool consistency based on cohesion and surface characteristics categorized on a 7-point scale14. The freshly expelled fecal pellets were weighed and dried at 75 °C for 24 h. Water content of the feces was calculated by the following formula: Water content % = [wet weight of the feces (mg) − dried weight of the feces (mg)] / wet weight of the feces (mg) × 100.

To access the role of TRPM4 on DAB laxative effects, time-matched C57BL/6J wild type (WT) and Trpm4-/- littermates as well as Trpm4flox/flox and Trpm4ΔIEC littermates at 8 weeks were used.

Cell cultures
HEK-293 cells and HT-29 cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin (P/S) and 1% HEPES. HEK-293-TRPM8 cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin (P/S), 1% HEPES, Blasticidin (5 μg ml−1) and Hygromycin B (50 μg ml−1). TRPM8 expression was induced with 1 µg ml−1 of doxycycline for 24 h before recording. HEK-293-hTRPM4 cells were cultured in DMEM supplemented with 10% FBS, 1% P/S and 500 μg ml-1 G418. All cells were cultured in a humidified incubator at 37 °C with 5% CO2.

Transfection and site-directed mutagenesis
HEK-293 cells were transfected with cDNA constructs using Lipofectamine® 2000 reagent according to the manufacturer’s protocol. To facilitate the identification of hTRPM3, hTRPM4, mTRPM4 or hTRPM5 transfected cells, a green fluorescence protein (GFP) was genetically linked to the C terminus of these genes. Patch-clamp recordings were performed after 24–48 h transfection. Mutants of hTRPM4 were generated using KOD plus mutagenesis kit (#KOD-401, Toyobo, Osaka, Japan) as described previously35. All mutants were confirmed by DNA sequencing. Transiently transfected cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin (P/S) and 1% HEPES.

Sodium influx assay
To discover TRPM4 ligands, changes in intracellular Na+ levels were monitored using Na+ sensitive dye, CoroNa™ Green/AM in HEK-293-TRPM4 cells36. Briefly, HEK-293-TRPM4 cultured in black-walled clear bottom 96-well plates (#3603, Corning, New York, USA) were incubated with 5 μM of CoroNa™ Green/AM (dissolved in Lock’s buffer containing 0.2 mg ml-1 Pluronic® F-127) for 60 min at 25 °C. After washing, plates were loaded onto the compound plate position of FLIPR TETRA® (Molecular Devices, Sunnyvale, CA). Emitted fluorescence excited at 488 nm wavelength was recorded at 510–570 nm. Compounds were automatically added to the cell plate by a programmable automatic pipetting system after 60 s baseline fluorescence recording and the fluorescence signals were continuously recorded for an additional 240 s. Data are presented as F/F0 where F is the fluorescence signal at any time point whereas F0 is the fluorescence signal averaged from initial 5 data points. To quantify the concentration-response relationships of BIS and DAB activation of TRPM4, the area under the curve (AUC) calculated from a 240 s duration right after the addition in each well was used.

To investigate the changes of [Na+]i in HT-29 cells, HT-29 cells were loaded with 5 µM CoroNa Green/AM for 1 h, and the fluorescence was digitalized using a Flash 4.0 V2 digital CMOS camera attached to a Leica DMi8 microscope at a sampling rate of 1 Hz. The excitation wavelength was filtered through a 460–495 nm excitation filter, and the emitted fluorescence was collected through a 510–550 nm filter. The arbitrary fluorescence unit of each region of interest (ROI) was quantified. Drugs were applied by bulk perfusion after a baseline fluorescence recording. Fluorescent signal at any time point was denoted as F and the mean of the first 5 data points was denoted as F0. Data are expressed as F/F0. Inhibitors were preincubated for 10 min before fluorescence recording.

Electrophysiology
Electrophysiological recordings were performed using an EPC-10 amplifier controlled by the PatchMaster software at 25 °C unless mentioned. Recording pipettes were pulled from borosilicate glass using P-1000 Micropipette Puller to 4-6 MΩ. For recording of hTRPM4, mTRPM4 and hTRPM5 currents, the pipette and extracellular solution contains (in mM) 130 NaCl, 3 HEPES, 5 EGTA. To investigate the influence of Ca2+ on DAB response on TRPM4. The pipette solution contains a free Ca2+ concentration of 1 µM obtained by a combination of CaCl2 (3.11 mM) and EGTA (5 mM) according to the following website: https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaEGTA-TS.htm. To recording the hTRPM8 currents in HEK-293 cells, the pipette solution contains (in mM) 150 NaCl, 5 MgCl2, 5 EGTA and 10 HEPES, pH 7.4 with NaOH, while the extracellular solution is (in mM) 150 NaCl, 1 MgCl2 and 10 HEPES, pH 7.4 with NaOH. The pipette solution for recording of hTRPM3 contains (mM) 110 CsCl, 20 tetraethylammonium chloride, 3.5 MgCl2, 2.5 Na2ATP, 2.41 CaCl2, 5 HEPES, 5 EGTA, pH 7.3 (with CsOH) while the extracellular solution is (mM) 144 NaCl, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 0.7 Na2HPO4, 10 HEPES and 5.6 D-glucose, pH 7.4 (with NaOH). When recording the DAB induced currents in HT-29 cells, the extracellular solution contains (in mM) 150 NaCl, 2 CaCl2 (or 0, without EGTA), 10 HEPES, PH 7.3, and the pipette solution comprises (mM) 140 KCl, 10 NaCl, 2 MgCl2, 10 HEPES, 0 Ca2+ (without EGTA or with 5 EGTA), PH 7.3. Cells were held at 0 mV and the currents, elicited by a voltage ramp from -100 mV to +100 mV in 400 ms, were recorded at 5 kHz. The ramp was applied every 1 s. Compounds were applied through a press-driven multichannel system (ALA Scientific Instruments, Farmingdale, NY, USA) with the outlet placed approximately 50 µm away from the cell being recorded.

Immunofluorescence
Immunofluorescence staining was conducted as described previously37. Briefly, after deparaffinization and antigen recovery, sections were blocked with 10% goat serum for 30 min and incubated with primary antibodies overnight at 4 °C. The primary antibodies were rabbit anti-TRPM4 (1:100) and mouse anti-CK-18 (1:200). After washing, sections were incubated with Alexa Fluor 555 goat anti-mouse (1:1,000) or Alexa Fluor 488 goat anti-rabbit (1:1,000) secondary antibodies for 1 h. Hoechst 33342 (10 μg ml−1) was added and incubated for 10 min to stain the nuclei of cells. Fluorescent images were taken using a confocal laser scanning microscope.

Intracellular Ca2+ levels determination
To investigate the DAB effects on intracellular Ca2+ levels, HT-29 cells were loaded with 5 µM Fluo-4/AM (dissolved in Lock’s buffer containing 5 mg ml−1 BSA) for 2 h, and the fluorescence was digitalized using a Flash 4.0 V2 digital CMOS camera attached to a Leica DMi8 microscope at a sampling rate of 0.5 or 1 Hz. The excitation wavelength was set at 460–495 nm, and the emitted fluorescence was recorded at 510–550 nm. The arbitrary fluorescence unit of each region of interest (ROI) was quantified. DAB was applied by bulk perfusion after a baseline fluorescence recording. Fluorescent signal at any time point was denoted as F and the mean of the first 5 data points was denoted as F0. Data are expressed as F/F0. Inhibitors were preincubated for 10 min before fluorescence recording.

Human TRPM4 protein expression and purification
The gene encoding human full-length TRPM4 (UniProtKB: Q8TD43) was subcloned into pEGBacMam vector with a 2× Strep tag, GFP and a thrombin-cleavage site at the N terminus38. Bacmid and baculovirus of TRPM4 in a BacMam vector were generated, and P2 viruses were used to infect a suspension of tsA cells. Cells were incubated at 37 °C for 8 h and 10 mM sodium butyrate was added to the culture and the temperature was changed to 30 °C. The cells were collected 72 h after infection and resuspended in a buffer containing 100 mM Tris pH 8.0 and 150 mM NaCl (TBS buffer) in the presence of 1 mM phenylmethylsulphonyl fluoride, 0.8 μM aprotinin, 2 μg ml−1 leupeptin and 2 mM pepstatin A. The cells were lysed by sonication and the membrane fraction was collected by centrifugation at 186,000 g using a 45 Ti rotor (Beckman Coulter) for 1 h at 4 °C. The membrane then homogenized with a Dounce homogenizer in TBS buffer supplemented with protease inhibitors. The protein was extracted from the membrane with TBS buffer supplemented with 1% GDN and protease inhibitors for 3 h at 4 °C. The solubilized proteins were loaded to Strep-Tactin resin. After washing with TBS buffer supplemented with 0.02% GDN, TRPM4 was eluted with the same buffer, supplemented with 10 mM desthiobiotin. The GFP tag was cleaved, and proteins were concentrated and further purified by size-exclusion chromatography (Superose 6). The peak fractions containing the TRPM4 were pooled and concentrated to 8 mg ml−1.

EM sample preparation and data acquisition
Purified TRPM4 was incubated with 5 mM calcium chloride or 5 mM EGTA, as required for each experiment, for 30 s at 37 °C. Then 0.1 mM DAB was added and further incubation for 2 min. 2.5 μl sample was applied to a glow-discharged Quantifoil holey carbon grid (gold, 2/1 μm hole space, 300 mesh). The grids were blotted for 1.5 s in the Vitrobot Mark III set to 100% humidity and 37 °C with a 15 s wait time before being plunge-frozen into liquid ethane cooled by liquid nitrogen.

For the Ca2+ and DAB sample, images were recorded using the FEI Titan Krios electron microscope at 300 kV and a nominal magnification of 105,000×. Data were collected on the Gatan K3 Summit direct electron detector in super-resolution mode, resulting in a binned pixel size of 0.828 Å. SerialEM39 was used for automated acquisition. Nominal defocus ranged from −1.2 to −1.9 μm.

For the DAB and EGTA sample, images were recorded on an FEI Glacios electron microscope at 200 kV and a nominal magnification of ×130,000. A Falcon 4i direct electron detector with Selectris energy filter was used resulting a pixel size of 0.91 Å. EPU was used for automated acquisition. Nominal defocus ranged from −0.5 to −1.4 μm.

Cryo-electron microscopy data analysis procedure
The detailed workflow for the data-processing procedure is summarized in Supplementary Fig. 5. In general, the raw movies for each dataset were motion-corrected and 2× binned (for K3 data) using MotionCor2 (v. 1.1.0)40. The per-micrograph defocus values were estimated using ctffind (v. 4.1.10)41. Particle picking was performed using relion Laplacian-of-Gaussian blob42 and topaz (v. 0.2.4)43. Junk particles were removed by rounds of 3D heterogeneous refinement using CryoSPARC44. Good particles were selected for non-uniformed refinement with C4 symmetry in CryoSPARC to generate a 3D map. Multiple rounds of CTF refinement and Bayesian polishing were performed in RELION to further improve the map resolution.

For Ca2+ and DAB dataset, symmetry expansion at the single subunit level was done from the map refined with C4 symmetry, followed by monomer subtraction. The subtracted images of the monomer underwent a round of local refinement with C1 symmetry followed by 3D classification without image alignment in RELION to improve the local resolution of MHR1/4 domain. Similar procedures were performed at the TMD to further increase the local resolution. Map resolution estimates were based on the gold standard Fourier shell correlation 0.143 criterion for all datasets.

Model building
Atomic models were generated by rigid-body fitting of the TMD, MHR1/2 and MHR3/4 and C-terminal domains from a published human TRPM4 model (PDB: 5WP6) into the final cryo-EM maps. Ligands were fitted into the density through real-space refinement using COOT45. The CIF file of DAB was generated using grade web server (Global Phasing Ltd.). The models were then manually adjusted in COOT and subjected to phenix.real_space_refine46 to improve the model metrics. The final models were validated using phenix.molprobity47. Figures were generated using UCSF ChimeraX48.

Statistics and reproducibility
Data were analyzed and plotted using GraphPad Prism software (ver 8.04, GraphPad Software, Inc., San Diego, CA, USA). Concentration-response curves were fitted by a non-linear regression analysis. All values are expressed as Mean ± SEM. One-way or two-way ANOVA followed by post hoc Bonferroni tests were used to examine statistical significance. A value of P < 0.05 was considered statistically significant. Each experiment was repeated at least three times. No statistical method was used to predetermine sample size, and no data points were excluded. The experimental procedures were randomized, and the investigators were blinded during experimentation or outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Here, Liu, Hu, Xue and Huang et al. studied the mechanisms underlying bisacodyl treatment of constipation. Remarkably,\nthey identified TRPM4 as the physiological target and further defined that the active metabolite, DAB, acts through a\npreviously unknown binding site in the membrane domain of the receptor. Animal studies, electrophysiology, and cryo-EM\nare integrated with a very clear presentation to connect the mechanistic pieces. I found the study easy to follow and my\nimpression is that it will be quite impactful in revealing both the physiological mechanism for a widely used drug and a new\nbinding side in a TRP channel. I have no major concerns, just a few questions, mainly centered on the structure and\nbiophysics.\n1. Do the EC50’s for BIC and DAB match reasonably with the concentrations achieved in vivo when humans take BIC?\n2. Line 150 and Fig. 2: currents look larger at positive holding potentials than negative holding potentials, indicating outward,\nrather than inward rectification, correct? Maybe I am missing something. Regardless, can the authors speculate on the\nmechanistic basis of the rectification?\n3. Does TRPM4 desensitize in the sustained presence of DAB? Might the DAB-bound structure represent a desensitized\nstate, rather than an intermediate or pre-activated state? Or, do the top panels in Fig. 2 indeed report that DAB does not\ncause TRPM4 to desensitize (and ED Fig. 7A)?\n4. Please elaborate on how asymmetric the Ca+DAB structure was, related to why symmetry expansion was needed and\nhelpful- did this improve ligand density?", "reviewer_2": "This study identifies TRPM4 as the key intestinal epithelial target of the laxative metabolite deacetyl bisacodyl (DAB). DAB\nactivates TRPM4 through a novel calcium-independent mechanism, triggering Na⁺ influx that drives Cl⁻ secretion via the\nTRPM4→VGCC/NCX→ANO1 pathway. Structural analysis reveals a new ligand-binding site in TRPM4, highlighting a\npotential therapeutic target for chronic constipation and other gastrointestinal disorders.\nThis is a well-designed and compelling study, with mechanistic consistency and high-quality structural and functional data.\nThe genetic validation and signaling pathway analyses are particularly robust. However, the broad impact on the field and\ntherapeutic relevance beyond chronic constipation are not fully substantiated. The authors should better contextualize which\ngastrointestinal disorders are most likely to benefit from these findings and moderate claims regarding the immediacy of\ntherapeutic application.\nDetailed comments:\n1. The identification of DAB as the first non-Ca²⁺ TRPM4 agonist represents a clear advance in TRPM4 biology. The\ndiscovery of a novel binding pocket is also noteworthy. However, the manuscript broadly claims a “novel therapeutic\nstrategy” for gastrointestinal disorders, but the specific diseases beyond chronic constipation are not well delineated. The\nclinical relevance to diarrhea, inflammatory bowel disease, or other GI disorders remains speculative. The authors should\nclarify which conditions are most likely to benefit from TRPM4-targeted therapies, supported by preclinical and more\nextensive epidemiological rationale.\n2. All in vivo studies have been focused on acute effects (24–36 h). How did the authors choose the time points? Chronic\neffects, potential desensitization, or broader impacts on intestinal absorption/electrolyte homeostasis are not addressed.\nThis point limits the translational significance of the findings.\n3. In the paragraph “DAB activates TRPM4 via a TMD-focused mechanism” the authors conclude that the DAB-bound\nchannel pore remains closed, possibly representing a pre-open or partially activated state. However, this appears\ninconsistent with the full channel activation observed in functional assays (in vitro and in vivo). Please clarify how this\nstructural state correlates with full activation, and whether it reflects a true intermediate in the activation pathway.\n4. While the mechanistic findings presented are compelling, the broader physiological significance of TRPM4 activation—\neither under normal homeostatic conditions or in disease contexts—is not sufficiently explored.\n5. The use of HT-29 cells (colon cancer cell line) does not fully recapitulate the human intestinal physiology.\n6. The rationale for the in vivo dosing should be justified, specifically in relation to clinically relevant concentrations.\n7. Most figures report N = number of cells or mice, but it is sometimes unclear whether statistical tests account for technical\nor biological replicates.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "We thank the reviewers for their thoughtful evaluation of our manuscript and for their\nconstructive comments, which have helped us to improve both the clarity and rigor of\nthe work. Below, we provide detailed responses to each point and describe the\ncorresponding revisions made in the manuscript.\nReviewers' comments:\nReviewer #1 (Remarks to the Author)\nHere, Liu, Hu, Xue and Huang et al. studied the mechanisms underlying bisacodyl\ntreatment of constipation. Remarkably, they identified TRPM4 as the physiological\ntarget and further defined that the active metabolite, DAB, acts through a previously\nunknown binding site in the membrane domain of the receptor. Animal studies,\nelectrophysiology, and cryo-EM are integrated with a very clear presentation to\nconnect the mechanistic pieces. I found the study easy to follow and my impression is\nthat it will be quite impactful in revealing both the physiological mechanism for a\nwidely used drug and a new binding side in a TRP channel. I have no major concerns,\njust a few questions, mainly centered on the structure and biophysics.\nResponse: Thank you for your positive and insightful feedback on our manuscript. In\nresponse, we have carefully revised the manuscript to address your concerns.\n1. Do the EC ’s for BIC and DAB match reasonably with the concentrations achieved\n50\nin vivo when humans take BIC?\nResponse: Thank you for the question. It is well established that bisacodyl (BIC) is\nconverted by intestinal deacetylase enzymes into its active metabolite, bis-(p-\nhydroxyphenyl)-pyridyl-2-methane (DAB) 1. Following the administration of BIC\nsuppositories, the parent drug was undetectable in serum, while its metabolite DAB\nreached concentrations of 10-55 μg/L (approx. 36-198 nM). However, only 3.36±0.52%\nof the dosed drug was excreted in urine, indicating its low systemic absorption 2. Thus,\nthe absorption of the BIC or DAB is likely not required and the laxative effect is\nproposed to be occurred locally 3. There is no data available regarding the BIC and\nDAB levels in the intestinal system. Nevertheless, ex vivo studies have established that\na high concentration of BIC (10 μg/mL, ~27 μM) is required to enhance contractility in\nhuman large intestinal muscle strips 4. In contrast, its active metabolite DAB exerts its\neffects—including enhanced colon mucosal secretion and increased muscle tone in both\nthe small bowel and colon—at much lower concentrations (0.5-5 µM) 5. In the\nmanuscript, we demonstrate that DAB and BIC activate TRPM4 with EC values of\n50\n0.14 and 2.64 μM, consistent with the observation that DAB is much more potent than\nBIC to stimulate muscle contraction and mucosal secretion 4,5. The discrepancy\nbetween the low EC values for TRPM4 activation (BIC: 2.64 µM; DAB: 0.14 µM)\n50\nand the higher concentrations required in ex vivo muscle contraction/mucosal secretion\nis likely due to reduced bioavailability and system complexity. In the ex vivo setting,\npenetration through dense tissue barriers lowers the effective concentration of both\ncompounds at their target site. Furthermore, to trigger a measurable functional\ncontraction, a threshold numbers of TRPM4 channels must be activated, a requirement\nthat is absent in simplified in vitro systems. We have incorporated this information into\nthe first paragraph of Discussion section (Lines 350-362).\n2. Line 150 and Fig. 2: currents look larger at positive holding potentials than negative\nholding potentials, indicating outward, rather than inward rectification, correct?\nMaybe I am missing something. Regardless, can the authors speculate on the\nmechanistic basis of the rectification?\nResponse: Thanks reviewer points this out. We are sorry for the typo in the original\nmanuscript (Inwardly rectified Na+ current, Line 153). At the lower concentration (≤\n0.3 μM), DAB elicits outwardly rectified Na+ currents, whereas at higher concentrations,\nthe I-V curves became linear. Such effect is consistent with voltage-dependent gating\nof TRPM4 channels 6. We have changed the description in the manuscript accordingly\n(Lines 154-155).\n3. Does TRPM4 desensitize in the sustained presence of DAB? Might the DAB-bound\nstructure represent a desensitized state, rather than an intermediate or pre-activated\nstate? Or, do the top panels in Fig. 2 indeed report that DAB does not cause TRPM4 to\ndesensitize (and ED Fig. 7A)?\nResponse: Thank you very much for raising your concern, and we fully agree that it\ncould represent a desensitized state. However, we prefer to interpret this structure as\nrepresenting a pre-open pore conformation, for the following reasons:\nFunctionally, DAB at low concentrations gradually increased the TRPM4 currents [both\ninward (-100 mV) and outward (+100 mV)] with the higher concentrations reach the\nplateau much faster. During the recording (10 min), we didn’t observe channel\ndesensitization. Moreover, the laxative effect of DAB persists for at least 48 h indicating\nits sustained activation in vivo. Calcium, the canonical activator, rapidly stimulates the\nTRPM4 currents with rapid desensitization to a plateau level that can be reversed by\naddition of PIP 7. However, DAB activation of TRPM4 is not dependent on the Ca2+\n2\nions.\nStructurally, although it could represent a desensitized state, we think it is more likely\nto be a pre-open state. First, how TRPM4 becomes desensitized structurally is not clear,\nso we do not have evidence to support that this is a desensitized state. Second, we\nspeculate that opening of the channel requires other factors besides DAB in our cryo-\nEM experiment. The absence of those factors, together with this DAB-bound\nconformation being in between the closed and open conformations, makes us think this\nis more likely to be a pre-open or intermediate state rather than a desensitized state.\nTogether, we interpret the TRPM4 structure as a pre-open state, while\nDAB\nacknowledging that a desensitized conformation cannot be entirely ruled out.\n4. Please elaborate on how asymmetric the Ca2+ DAB structure was, related to why\nsymmetry expansion was needed and helpful- did this improve ligand density?\nResponse: We thank the reviewer for this insightful question. The Ca2+-DAB structure\nis overall C4 symmetric, with all four subunits clear and consistent density for both the\nCa and DAB in the consensus C4 reconstruction. Symmetry expansion was not\nTMD\napplied to enhance the ligand or transmembrane domain density, which were already\nwell resolved in the initial consensus map. Instead, it was specifically used to improve\nthe local resolution of the MHR1/4 region, which exhibited conformational variability\nand reduced local resolution in the consensus reconstruction. By performing symmetry\nexpansion followed by focused classification, we were able to sort out this\nheterogeneity and obtain improved density for the MHR1/4 region. We have clarified\nthis point in the Methods (Lines 595-596).\nReviewer #2 (Remarks to the Author):\nThis study identifies TRPM4 as the key intestinal epithelial target of the laxative\nmetabolite deacetyl bisacodyl (DAB). DAB activates TRPM4 through a novel calcium-\nindependent mechanism, triggering Na⁺ influx that drives Cl⁻ secretion via the\nTRPM4→VGCC/NCX→ANO1 pathway. Structural analysis reveals a new ligand-\nbinding site in TRPM4, highlighting a potential therapeutic target for chronic\nconstipation and other gastrointestinal disorders.\nThis is a well-designed and compelling study, with mechanistic consistency and high-\nquality structural and functional data. The genetic validation and signaling pathway\nanalyses are particularly robust. However, the broad impact on the field and\ntherapeutic relevance beyond chronic constipation are not fully substantiated. The\nauthors should better contextualize which gastrointestinal disorders are most likely to\nbenefit from these findings and moderate claims regarding the immediacy of\ntherapeutic application.\nResponse: Thank you very much for reviewing our manuscript and providing\nconstructive comments to improve our manuscript. We fully agree with your point that\nthe broad impact on the field and the therapeutic relevance beyond chronic constipation\ncould be better substantiated.\n1. The identification of DAB as the first non-Ca²⁺ TRPM4 agonist represents a clear\nadvance in TRPM4 biology. The discovery of a novel binding pocket is also noteworthy.\nHowever, the manuscript broadly claims a “novel therapeutic strategy” for\ngastrointestinal disorders, but the specific diseases beyond chronic constipation are not\nwell delineated. The clinical relevance to diarrhea, inflammatory bowel disease, or\nother GI disorders remains speculative. The authors should clarify which conditions\nare most likely to benefit from TRPM4-targeted therapies, supported by preclinical and\nmore extensive epidemiological rationale.\nResponse: Thank you for these constructive comments. We agree that the statement\n“novel therapeutic strategy for gastrointestinal disorders” remains speculative. Thus, in\nthe revised manuscript, we have revised the statement “novel therapeutic strategy for\ngastrointestinal disorders” to be “a novel therapeutic strategy for constipation arising\nfrom a variety of etiologies, including neurogenic bowel dysfunction, opioid-induced\nconstipation, colorectal malignancy, and constipation-predominant irritable bowel\nsyndrome (IBS-C)” in the Introduction (Lines 38-43; 84) and Discussion section\n(Lines 405-407). This is because bisacodyl is widely used to treat acute and chronic\nconstipation arising from a variety of etiologies, including neurogenic bowel\ndysfunction, opioid-induced constipation, colorectal malignancy, and constipation-\npredominant irritable bowel syndrome (IBS-C) 3,8. It is also used to prepare the colon\nfor colonoscopy.\nWe have previously explored the role of TRPM4 on the onset and development of\nulcerative colitis. However, deletion of Trpm4 had no significant effect on the onset and\ndevelopment in mouse ulcerative colitis model (data not shown) suggesting that\nTRPM4 is not critical in the ulcerative colitis progression. We haven’t tested the role of\nTRPM4 in other GI disorders that we will continue to explore. In this manuscript, we\nindeed aimed to discover a TRPM4 probe that is not readily available in literature, and\nwe identified BIC and its active metabolite DAB are potent TRPM4 activators. Given\nBIC is a widely used laxative in clinical practice, we explored its effect on the intestinal\nfluid homeostasis and demonstrated that DAB triggers its laxative effect through\nTRPM4, extending the role of TRPM4 in the GI system. DAB thus may represent a\nmolecular probe to investigate the role of TRPM4 pathophysiology.\n2. All in vivo studies have been focused on acute effects (24–36 h). How did the authors\nchoose the time points? Chronic effects, potential desensitization, or broader impacts\non intestinal absorption/electrolyte homeostasis are not addressed. This point limits the\ntranslational significance of the findings.\nResponse: Thank you for raising this constructive question. In the manuscript, we only\nshowed the DAB laxative effect at 24 h. In the preliminary study, we explored the DAB\nlaxative effects every 12 h as shown in Fig. R1. After 12 h of DAB oral gavage, DAB\ncaused laxative effects that reached plateau at 24 h and persisted to 48 h. We are not\nable to do longer time points since the IUCAU is not permitted.\nFigure R1: Laxative effects of different dose of DAB in mice. (A) Fecal water content\nand (B) Bristol score of the stool collected at different time points. N = 6 mice. *, P <\n0.05; **, P < 0.01, vs. Veh at respective time point. Two-Way ANOVA\n3. In the paragraph “DAB activates TRPM4 via a TMD-focused mechanism” the\nauthors conclude that the DAB-bound channel pore remains closed, possibly\nrepresenting a pre-open or partially activated state. However, this appears inconsistent\nwith the full channel activation observed in functional assays (in vitro and in vivo).\nPlease clarify how this structural state correlates with full activation, and whether it\nreflects a true intermediate in the activation pathway.\nResponse: We thank the reviewer for raising this insightful point. We conclude that the\nstructures determined in the cryo-EM experiments represent a pre-open or intermediate\nstate, as the pore remains closed. Under our cryo-EM conditions, DAB binding alone\nis likely insufficient to trigger full channel opening, since many key cellular factors are\nabsent compared to in vitro and in vivo environment, including membrane potential and\nendogenous lipids. These differences likely account for the discrepancy between the\nstructural and functional results, an observation commonly encountered in ion channel\nstudies. Since all in vitro and in vivo data were obtained under physiological conditions,\nwe believe that the functional assays best reflect the true effect of DAB.\n4. While the mechanistic findings presented are compelling, the broader physiological\nsignificance of TRPM4 activation—either under normal homeostatic conditions or in\ndisease contexts—is not sufficiently explored.\nResponse: Thank you for this thoughtful comment. The primary goal of the current\nstudy was to identify a TRPM4 probe, such tools have been lacking. We identified BIC\nand its active metabolite, DAB, are potent TRPM4 activators. Given BIC is a widely\nused laxative in clinical practice, we further explored its effect on the intestinal fluid\nhomeostasis and demonstrated that DAB triggers its laxative effect through TRPM4\nactivation, thereby extending the role of TRPM4 in the GI system. We fully agree that\nthe broader implications of TRPM4 activation in health and disease are of great interest.\nOngoing studies in our lab are addressing these questions, but they are beyond the scope\nof the present work.\n5. The use of HT-29 cells (colon cancer cell line) does not fully recapitulate the human\nintestinal physiology.\nResponse: Thank you very much for your comments. We agree it is better to use human\nenterocytes to perform the experiment. Given the ethical restriction associated with\nusing primary human enterocytes, we turned to the HT-29 cell line, a standard model\nfor intestinal epithelium, to investigate the laxative mechanism of DAB. In this model,\nwe delineated a pathway where DAB activates TRPM4, leading to Ca2+ influx\npredominantly through L-type Ca2+ channels, which in turn stimulates Ca2+-activated\nCl- channels (CaCCs). This role of CaCCs is confirmed by our in vivo experiments\nusing the inhibitor CaCC -A01, as presented in the manuscript (Fig. 4H&I). Moreover,\ninh\nour conclusion that L-type Ca2+ channels primary mediate Ca2+ influx is consistent with\nthe established finding that DAB-induced secretion in the human intestine is\nindependent of voltage-gated sodium channels but dependent on L-type Ca2+ channels\n5. A sentence “This VGCC-mediated Ca2+ influx is consistent with the established\nfinding that DAB-induced secretion in the human intestine is dependent on L-type Ca2+\nchannels 5.” has been added into the revised manuscript Discussion section (Lines 388-\n390).\n6. The rationale for the in vivo dosing should be justified, specifically in relation to\nclinically relevant concentrations.\nResponse: We thank the reviewer for this comment. In clinical practice, the dose of\nBIC is 5-15 mg/day, that equivalent to doses in mice 1.3 mg/kg- 5 mg/kg. We initially\nperformed the dose-response relationships of DAB laxative effect (Fig. R1). We chose\n20 mg/kg for further study because it produced robust and sustained response. This dose\nis relatively higher than BIC used in clinical practice. However, it should be noted that,\nthe BIC used in clinical practice is formulated to be enteric-coated tablet that enables\nthe drugs to be released in the intestine while in mice, because of the technique difficulty,\nno enteric-coated tablet for mice is available. Thus, in our mouse study, DAB may be\ndistributed in entire gastrointestinal system while in human, BIC is only distributed in\nthe intestinal system and converted to be DAB. Such different formulations may explain\nwhy higher DAB dose should be used in mice.\n7. Most figures report N = number of cells or mice, but it is sometimes unclear whether\nstatistical tests account for technical or biological replicates.\nResponse: We thank the reviewer for the helpful suggestions. In this revised manuscript,\nwe have incorporated the information of the N numbers.\nReference\nWith Chronic Constipation. Clin Gastroenterol Hepatol 9, 577-583,\ndoi:10.1016/j.cgh.2011.03.026 (2011).\nsuppositories in man? Int J Pharm 196, 11-20, doi:10.1016/S0378-\n5173(99)00385-3 (2000).\nclinical evidence to guide use in clinical practice in patients with constipation.\nNeurogastroenterol Motil 33, 14123, doi:10.1111/nmo.14123 (2021).\nhuman colon (author's transl). Klin Wochenschr 53, 493-495,\ndoi:10.1007/BF01468637 (1975).\nhydroxyphenyl)-pyridyl-2-methane (BHPM)—the active metabolite of the\nlaxatives bisacodyl and sodium picosulfate—enhances contractility and\nsecretion in human intestine in vitro. Neurogastroenterol Motil 30, e13311,\ndoi:10.1111/nmo.13311 (2018).\nTRPM4. J Biol Chem 278, 30813-30820, doi:10.1074/jbc.M305127200 (2003).\nphosphatidylinositol 4,5-biphosphate. EMBO J 25, 467-478,\ndoi:10.1038/sj.emboj.7600963 (2006).\nFunction and Quality of Life in Patients with Chronic Constipation—Analysis\nof Pooled Data from Two Randomized Controlled Trials. Open J. Gastroenterol\n07, 32-43, doi:10.4236/ojgas.2017.71005 (2017)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have satisfyingly addressed my comments and questions.", "reviewer_2": "The authors have made some revisions; however, our primary concerns remain largely unresolved, and the manuscript\noverall impact continues to be limited by the insufficient demonstration of disease relevance, translational context, and a fully\nsupported structure–function relationship. Although some textual clarifications were added, these do not adequately address\nthe substantive scientific gaps identified in the first revision.\nThe principal issues are as follows: the requested preclinical data and broader epidemiological justification for point 1\nremain absent, thereby leaving the claims of disease relevance insufficiently supported; the response to point 3 persists as\nlargely speculative in the absence of additional data; point 4 was not addressed in the revised manuscript; and the reply to\npoint 5 still fails to provide a concrete experimental or methodological strategy to overcome the known limitations of the\ncurrent model.\nOverall, the revisions rely primarily on textual adjustments rather than the additional experimental or analytical work\nnecessary to substantiate the central claims, and further data together with deeper mechanistic support would be needed for\nthe manuscript to meet the level of rigor and impact expected for publication in Nature Communications.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": ""}]

Decision letter