Glutamic acid residues in the SS2 segment of the internal repeats III and IV of the brain calcium channel BI were subjected to single point mutations. The mutant channels were tested for macroscopic current properties and sensitivities to inorganic blockers. The mutation that replaces glutamic acid 1,469 with glutamine altered ion-selection properties and strongly reduced the sensitivity to Cd2+, whereas the analogous mutation of glutamic acid 1,765 exerted smaller effects on ion-selection properties. Our results indicate that these glutamic acid residues, equivalently positioned in the aligned sequences, play different roles in the selective permeability of the calcium channel.