A key driving force for ion channel selectivity is represented by the negative charge of the Selectivity Filter carried by aspartate (D) and glutamate (E) residues. However, despite studies on T-type Ca2+ channels, the structural effects and specific properties of D and E residues have not been extensively studied. In order to investigate this issue we studied the mutants of NaChBac channel with all possible combinations of D and E in the charged rings in position 191 and 192. Whole-cell patch clamp mea- surements showed significant Ca2+ currents only when position 191 was occupied by E. Equilibrium Molecular Dynamics simulations revealed the existence of two binding sites, one at the level of the charged rings and another one, more internal, at the level of L190. The simulations showed that the ion in the innermost site can interact with the residue in position 191 only when this is glutamate. Based on the MD simulations, we suggest that a D in position 191 leads to a high affinity Ca2+ block site resulting from a significant drop in the free energy of binding for an ion moving between the binding sites and causing small (or absent) ionic currents; in contrast, the free energy change is more gradual when an E residue occupies position 191, resulting in Ca2+permeability. This scenario is consistent with the model of ion channel selectivity through stepwise changes in binding affinity proposed by Dang and McCleskey. Our study also high- lights the importance of the structure of the selectivity filter which should contributeto the development of more detailed physical models for ion channel selectivity.