title: Probing the role of negatively charged amino acid residues in ion permeation of skeletal muscle ryanodine receptor.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Le
authors.initials: L
authors.lastname: Xu
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Pasek
authors.forename: Dirk
authors.initials: D
authors.lastname: Gillespie
authors.forename: Gerhard
authors.initials: G
authors.lastname: Meissner
mesh_terms: 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
chemical_list: 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