Functional regulation by divalent cations is a phenomenon observed in various tetrameric ion channels. For example, NMDA receptors, which are tetrameric ion channels involved in memory formation, are inhibited by magnesium ions. This phenomenon has been known for a long time, but these detailed blocking mechanism remains unknown. NavAb, a similar tetrameric sodium channel, is one of the most structurally analyzed tetrameric channels, which is not originally inhibited by divalent cations. Therefore, by adding this blocking effect to NavAb and analyzing its structural function, we thought that we could elucidate the blocking mechanism. When the amino acid of NavAb ion pore at the corresponding position was mutated to an amino acid of the NMDR receptor, the inhibitory effect by the divalent cation was also reproduced in NavAb. 
We found that mutagenesis enhances the hydrophilicity of the channel lumen from the crystal structural analysis. The electron density in the ion transmission pathway of mutants were enhanced in the presence of calcium ions. In molecular dynamics simulations, retention of calcium ions near the lumen entrance at the bottom of the selective filter was observed in the mutant channel. Thus, calcium ions stay in the ion pore, and as a result, sodium ions are difficult to permeate the ion pore.