Two rings of negative charges in the cytosolic vestibule of type-1 ryanodine receptor modulate ion fluxes

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 (D-4938, D-4945, D-4953) with asparagine and four glutamate residues (E-4942, E-4948, E-4952, E-4955) 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 ((DN)-N-4938, (DN)-N-4945) showed a reduced K+ ion conductance, with (DN)-N-4938 also exhibiting a reduced selectivity for Ca2+ compared to K+. The cytosolic location of D-4938 and D-4945 was confirmed using the polycation neomycin. Both (DN)-N-4938 and (DN)-N-4945 exhibited an attenuated block by neomycin to a greater extent from the cytosolic than lumenal side. By comparison, charge neutralization of lumenal loop residues (D(4899)Q, (EN)-N-4900) 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 D-4938 and D-4945 in the cytosolic vestibule determine RyR ion fluxes.