ИСТИНА

Intracellular Na+i/K+i imbalance leads to changes in the transcription of a number of genes, and most of them alter their expression even when Ca2+ signaling is turned off. A possible mechanism for this action on gene expression is an effect on the G-quadruplexes (G4) conformation. G4 are four-stranded secondary structures of nucleic acids that are ubiquitous in the genome and are stabilized by monovalent cations, with K+-bound G4 being a more stable structure than the Na+-bound form. Since G4 are located in the promoters of many genes, one can assume that they can regulate gene expression when the Na+i/K+i ratio is altered. To test our assumptions, we chose the fluorescence lifetime imaging microscopy for visualization of benzothiazole orange (BO) excited states, whose lifetimes are sensitive to binding to the G4 in gene promoters (c-Myc and c-Kit1) but not to dsDNA (DS23) or telomeric G4 (22AG). Incubation with 0.25-5 μM BO for 24 h doesn’t affect the viability of HeLa cells but increases the Na+i/K+i ratio by 10–30%. Therefore, in further experiments we used 0.25 μM BO, which has minimal effect on the ion composition of cells. The label differentially stains the cytoplasm, nucleus, and intranuclear structures that are presumed to be nucleoli. Inhibition of Na-pump with ouabain increases the Na+i/K+i ratio by up to 30 times and decreases the average BO fluorescence lifetime in the cell, which correlates with the incubation time and ranged from 0.6 to 13%. On the other hand, cells treatment with a G4-stabilizing molecule, pyridostatin, leads to an increase in the average BO fluorescence lifetime. Thus, we demonstrated the possibility of using BO in living cell G4 visualization and an evidence that G4 could be a Na+i/K+i sensor. The study was supported by Russian Science Foundation (#19-75-10009, #22-25-00161) and by the Interdisciplinary Scientific and Educational School of Moscow University «Molecular Technologies of the Living Systems and Synthetic Biology».