The tetrameric ryanodine receptor calcium release channels (RyRs) are cation-selective channels that have pore architecture similar to that of K⁺ channels. We recently identified, in close proximity to the selectivity filter motif GGGIG, a conserved lumenal DE motif that has a critical role in RyR ion permeation and selectivity. Here, we substituted three aspartate residues (D⁴⁹³⁸, D⁴⁹⁴⁵, D⁴⁹⁵³) with asparagine and four glutamate residues (E⁴⁹⁴², E⁴⁹⁴⁸, E⁴⁹⁵², E⁴⁹⁵⁵) with glutamine hypothesized to line the cytosolic vestibule of the skeletal muscle RyR (RyR1). Mutant single channel properties were determined using the planar lipid bilayer method. Two mutants (D⁴⁹³⁸N, D⁴⁹⁴⁵N) showed a reduced K⁺ ion conductance, with D⁴⁹³⁸N also exhibiting a reduced selectivity for Ca²⁺ compared to K⁺. The cytosolic location of D⁴⁹³⁸ and D⁴⁹⁴⁵ was confirmed using the polycation neomycin. Both D⁴⁹³⁸N and D⁴⁹⁴⁵N exhibited an attenuated block by neomycin to a greater extent from the cytosolic than lumenal side. By comparison, charge neutralization of lumenal loop residues (D⁴⁸⁹⁹Q, E⁴⁹⁰⁰N) eliminated the block from the lumenal but not the cytosolic side. The results suggest that, in addition to negatively charged residues on the lumenal side, rings of four negative charges formed by D⁴⁹³⁸ and D⁴⁹⁴⁵ in the cytosolic vestibule determine RyR ion fluxes.