title: Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor.
abstract: Type 1 ryanodine receptors (RyR1s) release Ca(2+) from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca(2+) release response in HEK293 cells and bound the RyR-specific ligand [(3)H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K(+) conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca(2+) release in HEK293 cells, low [(3)H]ryanodine binding levels, and channels that were not regulated by Ca(2+) and did not conduct Ca(2+) in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes.
authors.affiliation: From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: Yingwu
authors.initials: Y
authors.lastname: Mei
authors.affiliation: From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: Le
authors.initials: L
authors.lastname: Xu
authors.affiliation: From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: David D
authors.initials: DD
authors.lastname: Mowrey
authors.affiliation: From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: Raul
authors.initials: R
authors.lastname: Mendez Giraldez
authors.affiliation: From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Pasek
authors.affiliation: From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599.
authors.forename: Nikolay V
authors.initials: NV
authors.lastname: Dokholyan
authors.affiliation: From the Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599 meissner@med.unc.edu.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Meissner
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D005998:Glycine / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D008958:Models, Molecular; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D005998:Glycine
keywords: biophysics; calcium transport; computation; ryanodine receptor; sarcoplasmic reticulum (SR); skeletal muscle