Probing the role of negatively charged amino acid residues in ion permeation of skeletal muscle ryanodine receptor.
Sequence comparison suggests that the ryanodine receptors (RyRs) have pore architecture similar to that of the bacterial K+ channel KcsA. The lumenal loop linking the two most C-terminal transmembrane spanning segments in the RyRs has a predicted pore helix and an amino acid motif (GGGIG) similar to the selectivity filter (TVGYG) of KcsA identified by x-ray analysis. The RyRs have many negatively charged amino acid residues in the two regions linking the GGGIG motif and predicted pore helix with the two most C-terminal transmembrane spanning segments. We tested the role of these residues by generating single-site mutants, focusing on amino acid residues conserved among the mammalian RyRs. Replacement of two acidic residues immediately after the GGGIG motif in skeletal muscle ryanodine receptor (RyR1-D4899 and -E4900) with asparagine and glutamine profoundly affected ion permeation and selectivity. By comparison, mutagenesis of aspartate and glutamate residues in the putative linker regions showed a K+ conductance and selectivity for Ca2+ compared to K+ (P(Ca)/P(K)) close to wild-type. The results show that the negatively charged carboxyl oxygens of D4899 and E4900 side chains are major determinants of RyR ion conductance and selectivity.
Wang
Ying
Y
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
Xu
Le
L
Pasek
Daniel A
DA
Gillespie
Dirk
D
Meissner
Gerhard
G
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002412:Cations; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D005822:Genetic Vectors; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007477:Ions; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012433:Ryanodine / Q000737:chemistry; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D000596:Amino Acids; D002412:Cations; D018076:DNA, Complementary; D007477:Ions; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D001224:Aspartic Acid; D018698:Glutamic Acid; D011188:Potassium; D010100:Oxygen; D002118:Calcium