IF1 restrains excessive elevation of the mitochondrial membrane potential and safeguards the epithelial state of human iPSCs

Human pluripotent stem cells (hPSCs) rely on glycolysis and exhibit low mitochondrial respiration, conditions under which F1Fo ATP synthase tends to run in reverse, hydrolyzing ATP. ATP synthase inhibitory factor subunit 1 (IF1) inhibits F1Fo-mediated ATP hydrolysis, but its significance in glycolytic hPSCs remains unclear. Here, we show that stable IF1 knockdown (IF1-KD) in human induced pluripotent stem cells (hiPSCs) enhanced mitochondrial ATP hydrolysis and elevated the mitochondrial membrane potential ({Delta}{Psi}m). Markers of the undifferentiated state were largely maintained, whereas IF1-KD cells exhibited reduced epithelial characteristics and a partial epithelial-mesenchymal transition (EMT)-like state. IF1-KD cells also showed enhanced store-operated Ca2 entry (SOCE) and increased nuclear NFATc3. Pharmacological reduction of {Delta}{Psi}m attenuated SOCE, whereas NFATc3 overexpression reproduced key features of the EMT-like expression pattern. Endogenous IF1 therefore acts as a constitutive restraint on reverse-mode F1Fo activity, limiting excessive {Delta}{Psi}m and downstream SOCE-NFATc3 signaling and safeguarding the epithelial state of hPSCs.