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Background. The selectivity of biological cation channels is defined by a short, narrow selectivity filter, having a negative net fixed charge Qf. Voltage gated bacterial channels (NaChBac and some others) are frequently used in biophysics as simplified models of mammalian calcium and sodium channels. We report an experimental, analytic and numerical study of the effects of Qf and bulk ionic concentrations of Ca2+ and Na+ on conduction and selectivity of NaChBac channels, wild type and Qf-varied mutants. Methods. Site-directed mutagenesis and voltage clamp recordings were used to investigate the Na+/Ca2+ selectivity, divalent blockade and anomalous mole fraction effect (AMFE) for different NaChBac wild type/ mutants channels and the properties dependence on Qf. Experimental results were compared with Brownian dynamics simulations and with analytic predictions of the ionic Coulomb blockade (ICB) model, which was extended to encompass bulk concentration effects. Results. It was shown that changing of Qf from -4e (for LESWAS wild type) to -8e (for LEDWAS mutant) leads to strong divalent blockade of the Na+ current by micromolar amounts of Ca2+ ions, similar to the effects seen in mammalian calcium channels. The BD simulations revealed a concentration-related logarithmic shift of the conduction bands. These results were shown to be consistent with ICB model predictions. Conclusions. The extended ICB model explains the experimental (divalent blockade and AMFE) and simulated (multi-ion bands and their concentration-related shifts) selectivity phenomena of NaChBac channel and its charge-varied mutants. These results extend the understanding of ion channel selectivity and may also be applicable to biomimetic nanopores with charged walls. [ABSTRACT FROM AUTHOR]