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 (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.
Xu
Le
L
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Wang
Ying
Y
Gillespie
Dirk
D
Meissner
Gerhard
G
D020816:Amino Acid Motifs; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism; D002412:Cations; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D007477:Ions; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism; D009154:Mutation; D009355:Neomycin / Q000494:pharmacology; D011188:Potassium / Q000737:chemistry; D011817:Rabbits; D012433:Ryanodine / Q000737:chemistry; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
D002412:Cations; D018076:DNA, Complementary; D007477:Ions; D008051:Lipid Bilayers; D019837:Ryanodine Receptor Calcium Release Channel; D005973:Glutamine; D012433:Ryanodine; D018698:Glutamic Acid; D001216:Asparagine; D009355:Neomycin; D011188:Potassium; D002118:Calcium