Hyperactivity of the mechanosensitive ion channel PIEZO1 promotes pathologic Ca2+ overload in red blood cells (RBCs), driving dehydration, TMEM16F-dependent phosphatidylserine (PS) exposure, microparticle shedding, and increased thrombotic and vaso-occlusive risks in hereditary xerocytosis (HX) and sickle cell disease (SCD). However, clinically deployable PIEZO inhibitors to treat these blood disorders are lacking. Here we report that cannabidiol (CBD), a non-psychoactive cannabinoid commonly used in SCD patients for pain management, inhibits PIEZO1 activity and restores aberrant mechanotransduction in HX and SCD RBCs. Micromolar concentrations of CBD blocks PIEZO1 currents and suppresses PIEZO1-mediated Ca2+ entry. In HX and SCD RBCs, CBD attenuates PIEZO1-TMEM16F coupling, thereby reducing PS exposure, microparticle release, thrombin generation, RBC-endothelium adhesion, and sickling. Beyond RBCs, CBD also blocks PIEZO2 currents and PIEZO2-dependent mechanical sensation in mice, suggesting broader effects of CBD-mediated PIEZO inhibition on nociceptive functions. Together, our findings identify CBD as a potent PIEZO inhibitor that restores calcium and membrane homeostasis, supporting the repurposing of CBD or the development of CBD-derived, PIEZO-selective analogs as a promising disease-modifying strategy for SCD, HX, and other PIEZO-mediated mechanosensing disorders. ### Competing Interest Statement The authors have declared no competing interest. Duke University, https://ror.org/00py81415 American Society of Hematology, https://ror.org/02nw48b86

Temporomandibular disorders (TMD) pain is the most common orofacial pain with limited effective treatments. Here, we observed elevated lysophosphatidic acid (LPA), a bioactive lipid, in blood, trigeminal ganglion (TG), and peri-temporomandibular joint (TMJ) tissues in mouse models of TMD-like pain induced by TMJ inflammation or masseter muscle injury. Notably, LPA levels were also elevated in TMD patients' blood and positively correlated with their pain intensity. LPA receptors (LPAR) 1 and 3 were expressed in mouse and human TG neurons and upregulated in TMD-like pain models. Inhibition or knockout of LPAR1 or LPAR3 attenuated TMD-like pain, while LPA injection into the TMJ or masseter muscle evoked pain. Furthermore, we demonstrated that LPA/LPAR signaling upregulates and sensitizes PIEZO2, a mechanosensitive ion channel, in TG neurons via extracellular signal-regulated kinase (ERK). Specific deletion or inhibition of PIEZO2 and suppression of ERK activation in TG neurons mitigated TMD-like pain. These findings suggest that LPA/LPAR signaling drives TMD-like pain via PIEZO2, offering potential therapeutic targets.

BACKGROUND:Transfer RNA-derived small RNAs (tsRNAs) represent a novel class of noncoding RNAs increasingly implicated in cardiovascular regulation. However, their roles in sepsis-induced cardiomyopathy (SICM) remain largely undefined. This study aimed to investigate the function and underlying mechanism of 5'tiRNA-32-LysCTT-11-a highly upregulated tsRNA in sepsis-induced cardiomyopathy-in modulating myocardial injury. METHODS:A murine model of sepsis was established via cecal ligation and puncture, and myocardial injury was assessed by serum Creatine Kinase-MB Isoenzyme/lactate dehydrogenase levels, histology, and cardiac function via echocardiography. In vitro , H9C2 cardiomyocytes were exposed to conditioned media from lipopolysaccharide-stimulated macrophages. The expression of 5'tiRNA-32-LysCTT-11 was measured by quantitative reverse transcription polymerase chain reaction. Functional assays, including Cell Counting Kit-8, lactate dehydrogenase release, propidium iodide staining, 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine (JC-1), adenosine triphosphate, reactive oxygen species detection, and MitoTracker staining, were performed. Necroptosis was evaluated via mixed lineage kinase domain-like phosphorylation; mitochondria-associated endoplasmic reticulum membranes (MAMs) formation was assessed by dual-label immunofluorescence and phosphofurin acidic cluster sorting protein 2 expression. Bioinformatics analysis identified mitofusin 2 (Mfn2) as a putative target, validated by western blot, mRNA stability assay (actinomycin D), and rescue experiments. FINDINGS:5'tiRNA-32-LysCTT-11 was significantly upregulated insepsis-induced cardiomyopathy. In vivo , its overexpression improved cardiac function and reduced injury biomarkers. In vitro , 5'tiRNA-32-LysCTT-11 mimics preserved mitochondrial integrity, reduced reactive oxygen species and adenosine triphosphate depletion, suppressed mitochondria-associated endoplasmic reticulum membranes formation and necroptosis. Inhibitor transfection produced opposite effects. Mechanistically, 5'tiRNA-32-LysCTT-11 enhancedmitofusin 2 mRNA stability and protein expression. Silencing mitofusin 2 abrogated the protective effects, confirming its central role in the tsRNA's action. INTERPRETATION:5'tiRNA-32-LysCTT-11 exerts cardioprotective effects during sepsis by stabilizing Mfn2 mRNA, preserving mitochondrial function, limiting mitochondria-associated endoplasmic reticulum membranes formation, and suppressing necroptosis. These findings uncover a novel regulatory mechanism and suggest 5'tiRNA-32-LysCTT-11 as a promising therapeutic target SICM.

Chronic infected diabetic wounds pose a significant global health challenge. This study explores a novel therapeutic approach using a [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA), 3-(acrylamidophenyl)boronic acid (AAPBA) and tannic acid (TA) based pH/glucose dual-responsive drug release hydrogel loaded with gallium ions (Ga3+) (named “STAG hydrogel”) to enhance the treatment of Staphylococcus aureus (S. aureus) -infected diabetic wounds. The hydrogel was designed to respond to the acidic and hyperglycemic microenvironment typical of chronic diabetic wounds, ensuring controlled release of its antimicrobial components. In vitro, the hydrogel demonstrated significant antibacterial activity against S. aureus and Escherichia coli (E. coli), reducing bacterial viability and inhibiting biofilm formation. Furthermore, the hydrogel exhibited excellent biocompatibility, promoting fibroblast viability and migration, crucial for wound healing. In vivo studies using a diabetic mouse model confirmed the hydrogel’s efficacy in accelerating wound closure, reducing bacterial load, and enhancing collagen deposition and CD31 and VEGF levels. The dual-responsive drug release hydrogel also modulated the inflammatory response, promoting M2 macrophage polarization, which is essential for tissue repair and regeneration. These findings highlight the potential of the STAG hydrogel as an effective therapeutic strategy for managing chronic infected diabetic wounds.

BACKGROUND:Transfer RNA-derived fragments (tRFs) are emerging as critical post-transcriptional regulators in stress responses and tissue repair; meanwhile, endothelial cells orchestrate burn-wound vascular recovery by sprouting new vessels, restoring perfusion, and guiding epithelial regeneration. However, the role of tRFs in thermal injury-induced endothelial cell dysfunction remains unexplored. Here, we identify a novel stress-induced tRF, tRF-Ser-GCT-108, as a key suppressor of endothelial cell recovery following thermal damage. METHODS:Small-RNA sequencing was performed on paired normal and burn-injured human dermis(n = 5). Differentially expressed tRFs were validated by qPCR in both tissues and heat-injured human umbilical vein endothelial cells (HUVECs, 52 °C, 35 s). Functional assays included CCK-8 proliferation, scratch wound closure, Transwell migration, and Annexin V/PI apoptosis profiling. Mechanistic studies integrated in silico target prediction, and Western blotting to confirm direct binding of tRF-Ser-GCT-108 to the 3'-UTR of MAPK1. RESULTS:Small RNA sequencing identified 172 differentially expressed tRFs in burn-injured tissues, with tRF-Ser-GCT-108 exhibiting the most significant upregulation. Functional assays demonstrated that overexpression of tRF-Ser-GCT-108 in heat-injured HUVECs markedly reduced cell proliferation and migration capabilities while inducing a significant increase in apoptosis. Conversely, inhibition of tRF-Ser-GCT-108 normalized these parameters. Mechanistically, Western blot analysis confirmed that tRF-Ser-GCT-108 directly interacts with the 3'-UTR of MAPK1 mRNA, leading to reduced MAPK1 protein levels. This decrease in MAPK1 protein was consistent with reduced mRNA levels and may contribute to disrupted VEGF signaling. CONCLUSION:tRF-Ser-GCT-108 functions as a stress-responsive endothelial brake that impairs post-burn vascular repair by silencing MAPK1 within the VEGF axis. Therapeutic inhibition of tRF-Ser-GCT-108 represents a novel RNA-based strategy to accelerate burn-wound healing by reactivating endothelial regeneration.

TMEM63B, a mechanosensitive ion channel (MSC), is associated with severe neurodevelopmental disorders, including severe early-onset developmental and epileptic encephalopathy. Structure-function studies have shown that TMEM63B pathogenic variants, including V44M in transmembrane helix 0 (TM0) and T481N in TM4, cluster near the hydrophobic neck region of the ion permeation pathway, a region critical for gating and permeation. Notably, V44M and T481N convert TMEM63B into constitutive phospholipid scramblases without obvious effects on their MSC activity, revealing an unexpected channel-to-scramblase switch in these variants. To further define the mechanistic basis of this phenomenon, here we characterized I475del, a TM4 deletion variant near V44M and T481N. Unlike V44M and T481N, the I475del channel exhibited basal leak currents, enhanced mechanically activated currents, and elevated mechanosensitivity, altogether supporting its classification as a bona fide gain-of-function MSC variant. Like V44M and T481N, I475del also enabled constitutive phospholipid scramblase activity. However, unlike these variants, I475del uniquely displayed further potentiation of scramblase activity under hypotonic osmotic stress. In addition, mutating the key residues that control gating conformational changes abolished gain-of-function ion and lipid transport through I475del. Together, our results support a stratified molecular model of TMEM63B channelopathies in which the pathogenic variants progressively destabilize the hydrophobic gate, permitting lipid permeation at the resting state followed by force-induced ion and lipid cotransport. These findings advance the mechanistic understanding of TMEM63B function and TMEM63B-associated disease and provide a framework for developing therapeutic strategies targeting variant-specific pathologies.

BACKGROUND:Our previous study found that nucleolin expression exerted anti-cardiac injury effects by promoting mitochondrial biogenesis; however, it could not explain the increase in mitochondrial fragmentation during myocardial injury. Mitochondrial fragmentation is associated with mitochondrial fission, but it is unknown whether nucleolin regulates mitochondrial fission. Therefore, this study aims to investigate the mechanism by which nucleolin regulates mitochondrial fission in endotoxemia-induced myocardial dysfunction. METHODS:Nucleolin myocardial-specific knockout mice were used to construct an endotoxemia-induced myocardial dysfunction model. Mitochondrial membrane potential (MMP), ATP production, Mitotracker Red, Transmission Electron Microscope were measured to assess mitochondrial function. Mitochondria were isolated to observe Drp1 translocation to mitochondria. The expression of pGSK-3β-Tyr216, GSK-3β, pDrp1-Ser637, nucleolin and dynamin-related protein 1 (DNM1L, Drp1) were detected using qRT-PCR and western blot. RESULTS:Following cecum ligation and puncture (CLP) model, cardiac function was impaired, myocardial mitochondrial function declined, mitochondrial morphology became disorganized and fragmented, nucleolin and Drp1 expression was elevated. Myocardial injury and mitochondrial dysfunction were further exacerbated after nucleolin myocardium-specific knockout. Meanwhile, after cellular-level nucleolin interference, it further led to LPS and TNF-α-induced mitochondrial dysfunction and cardiomyocyte damage. Mechanically, nucleolin interference inhibited Drp1 phosphorylation at Ser637 and promoted Drp1 translocation to mitochondria. Myocardial injury caused by nucleolin knockdown was alleviated by the use of P110, an inhibitor of Drp1 mitochondrial translocation. CONCLUSION:Endotoxemia-induced myocardial dysfunction is accompanied by increased mitochondrial fragmentation. Nucleolin alleviates endotoxemia-induced myocardial dysfunction by enhancing Drp1 phosphorylation at Ser637, inhibiting Drp1 translocation to the mitochondria and mitochondrial fission.

A deeper understanding of sickle cell disease (SCD) pathophysiology is critical for identifying novel therapeutic targets. A hallmark of SCD is abnormal phosphatidylserine (PS) exposure on sickle red blood cells (RBCs), which contributes to anemia, thrombosis, and vaso-occlusive crises (VOC). However, the mechanisms underlying this excessive PS exposure remain unclear. Here, we identify TMEM16F, a Ca2+-activated lipid scramblase, as a key mediator of PS exposure downstream of Ca2+ influx through the mechanosensitive channel PIEZO1 in sickle RBCs. Electrophysiology, imaging, and flow cytometry reveal that deoxygenation-induced sickling activates PIEZO1, triggering Ca2+ entry, TMEM16F activation, and PS exposure. This cascade promotes PS+ microparticle release, thrombin generation, and RBC adhesion to endothelial cells. Notably, partial PIEZO1 inhibition with benzbromarone, an anti-gout drug, suppresses these effects. Our findings define a previously unrecognized mechanotransduction pathway in sickle RBCs and propose a unique therapeutic strategy to mitigate hypercoagulability and vaso-occlusion associated with SCD.

This study aimed to assess the impact of human amniotic epithelial stem cells-conditioned medium (hAECs-CM) on the healing of burn wounds and the reduction of inflammation in a mouse model of deep second degree burns. The findings indicated that hAECs-CM markedly enhanced the primary metrics of burn wound healing within a brief duration of 14 days. The rate of wound healing was substantially greater than that of the control group following 21 days of treatment with daily administration of hAECs-CM. The CD31 concentration was markedly elevated in burn tissues treated with hAECs-CM, whereas the myeloperoxidase (MPO) level was reduced. Simultaneously, the antibacterial efficacy of hAECs-CM was assessed, indicating that hAECs-CM may alleviate infections generated by burns. HE and Masson staining results indicated that the treatment markedly diminished inflammation and collagen deposition relative to the model group, while both connective tissue and neovascularization were enhanced in the dermis of the hAECs-CM-BI group. In conclusion, the utilization of hAECs-CM may expedite the wound healing process and mitigate burn infections.

Carnitine palmitoyltransferase 1 (CPT1) serves as a critical gatekeeper in mitochondrial fatty acid oxidation and plays a central role in systemic energy homeostasis. The CPT1 family comprises three isoforms-CPT1A, CPT1B, and CPT1C-which exhibit distinct tissue distributions and regulatory features, enabling specialized metabolic functions in the liver, heart, skeletal muscle, and brain. CPT1 activity is tightly controlled through multiple mechanisms, including inhibition by malonyl-CoA, epigenetic modifications, and protein-protein interactions, all of which coordinate nutrient sensing and energy adaptation. Dysregulation of CPT1 has been implicated in the development of various metabolic disorders, including obesity, metabolic (dysfunction)-associated fatty liver disease (MAFLD), diabetic cardiomyopathy, and metabolic syndrome. This review summarizes recent advances in understanding the regulatory landscape and pathological roles of CPT1 and further discusses emerging therapeutic strategies. While CPT1-targeted interventions hold promise, challenges such as isoform specificity, off-target effects, and tissue-selective delivery must be addressed to achieve precision metabolic modulation.

Mitochondria are key organelles that perform and coordinate various metabolic processes in the cell, and their homeostasis is essential for the maintenance of eukaryotic life. To maintain mitochondrial homeostasis and cellular health, close communication between noncoding RNAs (ncRNAs) and proteins is required. For example, there are numerous crosstalk between ncRNAs and the sirtuin (SIRT1-7) family, which is a group of nicotinamide adenine dinucleotides (NAD(+))-dependent Type III deacetylases. NcRNAs are involved in the regulation of gene expression of sirtuin family members, and deacetylation of sirtuin family members can also influence the generation of ncRNAs. This review focuses on the relationship between the two mentioned above and summarizes the impact of their interactions on mitochondrial metabolism, oxidative stress, mitochondrial apoptotic pathways, mitochondrial biogenesis, mitochondrial dynamics, and other mitochondria-related pathophysiological processes. Finally, the review also describes targeted and appropriate treatment strategies. In conclusion, we provide an overview of the ncRNA-sirtuins/mitochondria relationship that could provide a reference for related research in the mitochondrial field and help the future development of new biomedical applications in this area.

Background We revealed for the first time that the expression of 158 tRNA-derived small RNAs (tsRNAs) was altered in septic cardiomyopathy (SCM) by microarray analysis, and we selected 5'tiRNA-33-CysACA-1, which was the most significantly up-regulated, as a representative to explore the roles and mechanisms of tsRNAs in SCM. Methods We constructed a sepsis model by cecum ligation and puncture (CLP) in mice and detected the expression of 5'tiRNA-33-CysACA-1 using quantitative real-time PCR (qRT-PCR). The supernatant generated after LPS stimulation of macrophages was used as the conditional medium (CM) to stimulate H9C2 and established the injured cell model. CCK-8 and LDH release assays were used to detect cell viability and cell death. Mitochondrial membrane potential (MMP), ATP production, ROS production, and Mitotracker Red mitochondrial morphology were assayed to assess mitochondrial function. Expression of mRNA for molecules related to the mitochondrial quality control system was verified by qRT-PCR. The mechanism by which 5'tiRNA-33-CysACA-1 regulates peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) expression was examined by western blot, mRNA stability analysis, and rescue experiments. Results Expression of 5'tiRNA-33-CysACA-1 was elevated in cardiac tissue and H9C2 cells during septic myocardial injury. Stimulation of the CM resulted in cardiomyocyte injury and impaired mitochondrial function. Transfection of 5'tiRNA-33-CysACA-1 mimic in CM further downregulated PGC-1α expression, inhibited mitochondrial biogenesis thereby impairing mitochondrial function and leading to decreased cardiomyocyte activity and increased cell death. In contrast, transfection of the inhibitor ameliorated the above biological processes. In addition, mRNA stability assay and bioinformatics analysis showed that 5'tiRNA-33-CysACA-1 led to a decrease in the stability of PGC-1α mRNA, which in turn downregulated the expression of PGC-1α and promoted the development of SCM. Conclusions 5'tiRNA-33-CysACA-1 expression is upregulated in SCM and inhibits mitochondrial biogenesis by targeting PGC-1α and decreasing the stability of PGC-1α mRNA, leading to mitochondrial dysfunction and promoting the development of SCM.

PIEZO1, a mechanosensor in endothelial cells, plays a critical role in fetal vascular development during embryogenesis. However, its expression and function in placental trophoblasts remain unexplored. Here, we demonstrate that PIEZO1 is expressed in placental villus trophoblasts, where it is essential for trophoblast fusion and placental development. Mice with trophoblast-specific PIEZO1 knockout exhibit embryonic lethality without obvious vascular defects. Instead, PIEZO1 deficiency disrupts the formation of the syncytiotrophoblast layer in the placenta. Mechanistically, PIEZO1-mediated calcium influx activates TMEM16F lipid scramblase, facilitating the externalization of phosphatidylserine, a key "fuse-me" signal for trophoblast fusion. These findings reveal PIEZO1 as a crucial mechanosensor in trophoblasts and highlight its essential role in regulating trophoblast fusion and placental development, expanding our understanding of PIEZO1's functions beyond endothelial cells during pregnancy.

Cell-surface exposure of phosphatidylserine (PS) is essential for phagocytic clearance and blood clotting. Although a calcium-activated phospholipid scramblase (CaPLSase) has long been proposed to mediate PS exposure in red blood cells (RBCs), its identity, activation mechanism, and role in RBC biology and disease remain elusive. Here, we demonstrate that TMEM16F, the long-sought-after RBC CaPLSase, is activated by calcium influx through the mechanosensitive channel PIEZO1 in RBCs. PIEZO1-TMEM16F functional coupling is enhanced in RBCs from individuals with hereditary xerocytosis (HX), an RBC disorder caused by PIEZO1 gain-of-function channelopathy. Enhanced PIEZO1-TMEM16F coupling leads to an increased propensity to expose PS, which may serve as a key risk factor for HX clinical manifestations including anemia, splenomegaly, and postsplenectomy thrombosis. Spider toxin GsMTx-4 and antigout medication benzbromarone inhibit PIEZO1, preventing force-induced echinocytosis, hemolysis, and PS exposure in HX RBCs. Our study thus reveals an activation mechanism of TMEM16F CaPLSase and its pathophysiological function in HX, providing insights into potential treatment.

Objective: To investigate the clinical effect of the modified vertical rectus abdominis myocutaneous flap in repairing the skin and soft tissue defect after abdominoperineal resection for rectal cancer. Methods: This study was a retrospective observational study. From June 2019 to July 2022, five male patients with low rectal cancer who were conformed to the inclusion criteria were admitted to the Department of Basic Surgery of Xiangya Hospital of Central South University, with ages ranging from 65 to 70 years and the sizes of the perianal skin ulcers ranging from 5 cm×4 cm to 11 cm×9 cm, and all of them underwent abdominoperineal resection. The secondary skin and soft tissue defects in the perineum with an area of 8 cm×6 cm-14 cm×12 cm (with the depth of pelvic floor dead space being 10-15 cm) were repaired intraoperatively with transplantation of modified vertical rectus abdominis myocutaneous flaps with the skin area being 9 cm×7 cm-16 cm×12 cm, the volume of the muscle being 18 cm×10 cm×5 cm-20 cm×12 cm×5 cm, and the vessel pedicle being 18-20 cm in length. During the operation, most of the anterior sheath of the rectus abdominis muscle was retained, the flap was transferred to the recipient area through the abdominal cavity, the remaining anterior sheaths of the rectus abdominis muscle on both sides of the donor area were repeatedly folded and sutured, the free edge of the transverse fascia of the abdomen was sutured with the anterior sheath of the rectus abdominis muscle, and the donor area skin was directly sutured. After the operation, the survival of the transplanted myocutaneous flap was observed. The occurrence of complications in the perineal recipient area was recorded within 2 weeks after the operation. The recovery of the perineal recipient area and the abdominal donor area was observed during follow-up, and the occurrence of complications in the donor area of the abdomen as well as the recurrence of tumors and metastasis were recorded. Results: All transplanted myocutaneous flaps in 5 patients survived after surgery. One patient had dehiscence of the incision in the perineal recipient area 2 days after surgery, which healed after 7 d with intermittent dressing changes and routine vacuum sealing drainage treatment. In the other 4 patients, no complications such as incisional rupture, incisional infection, or fat liquefaction occurred in the perineal recipient area within 2 weeks after surgery. Follow-up for 6-12 months after discharge showed that the skin of the perineal recipient area had good color, texture, and elasticity, and was not bloated in appearance; linear scars were left in the perineal recipient area and the abdominal donor area without obvious scar hyperplasia or hyperpigmentation; no complications such as incisional rupture, incisional infection, intestinal adhesion, intestinal obstruction, or weakening of the abdominal wall strength occurred in the abdominal donor area, and the abdominal appearance was good with no localized bulge or formation of abdominal hernia; there was no local recurrence of tumor or metastasis in any patient. Conclusions: The surgical approach of using the modified vertical rectus abdominis myocutaneous flap to repair the skin and soft tissue defects after abdominoperineal resection for rectal cancer is relatively simple in operation, can achieve good postoperative appearances of the donor and recipient areas with few complications, and is worthy of clinical promotion.

Members of the transmembrane channel/scramblase (TCS) superfamily—encompassing the TMEM16, TMEM63/OSCA and TMC families—have been implicated in myriad biological processes, including anxiety, motor learning, blood clotting, cell-cell fusion, viral infection, and sensory perception. Uniquely, most TMEM16s exhibit dual functions as calcium-activated ion channel/phospholipid scramblases whereas the remaining TCS members are currently classified as ion channels. Thus, how phospholipid permeability evolved in the TCS superfamily is an intriguing and open question.

ABSTRACT TMEM16F (also known as ANO6), a Ca2+-activated lipid scramblase (CaPLSase) that dynamically disrupts lipid asymmetry, plays a crucial role in various physiological and pathological processes, such as blood coagulation, neurodegeneration, cell–cell fusion and viral infection. However, the mechanisms through which it regulates these processes remain largely elusive. Using endothelial cell-mediated angiogenesis as a model, here we report a previously unknown intracellular signaling function of TMEM16F. We demonstrate that TMEM16F deficiency impairs developmental retinal angiogenesis in mice and disrupts angiogenic processes in vitro. Biochemical analyses indicate that the absence of TMEM16F enhances the plasma membrane association of activated Src kinase. This in turn increases VE-cadherin phosphorylation and downregulation, accompanied by suppressed angiogenesis. Our findings not only highlight the role of intracellular signaling by TMEM16F in endothelial cells but also open new avenues for exploring the regulatory mechanisms for membrane lipid asymmetry and their implications in disease pathogenesis.

The calcium-activated TMEM16 proteins and the mechanosensitive/osmolarity-activated OSCA/TMEM63 proteins belong to the Transmembrane Channel/Scramblase (TCS) superfamily. Within the superfamily, OSCA/TMEM63 proteins, as well as TMEM16A and TMEM16B, are thought to function solely as ion channels. However, most TMEM16 members, including TMEM16F, maintain an additional function as scramblases, rapidly exchanging phospholipids between leaflets of the membrane. Although recent studies have advanced our understanding of TCS structure-function relationships, the molecular determinants of TCS ion and lipid permeation remain unclear. Here, we show that single mutations along the transmembrane helix (TM) 4/6 interface allow non-scrambling TCS members to permeate phospholipids. In particular, this study highlights the key role of TM 4 in controlling TCS ion and lipid permeation and offers novel insights into the evolution of the TCS superfamily, suggesting that, like TMEM16s, the OSCA/TMEM63 family maintains a conserved potential to permeate ions and phospholipids.