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.

Highlights

  • Skeletal muscle ryanodine receptor (RyR1) releases Ca2ϩ ions from sarcoplasmic reticulum through Ca2ϩgated ion channels

  • The results indicate that both glycines are involved in RyR1 channel gating, with RyR1-G4941A altering the kinetics of channel gating to a greater extent than RyR1-G4934A

  • Expression and Function of RyR1-G4934 and -G4941 Mutants—RyR1-with zero (WT) and G4934 and G4941 mutants were expressed in HEK293 cells, and expression levels and function were examined by immunoblot analysis and cellular Ca2ϩ release in response to the Ca2ϩ-releasing drug caffeine, respectively

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Summary

Introduction

Skeletal muscle ryanodine receptor (RyR1) releases Ca2ϩ ions from sarcoplasmic reticulum through Ca2ϩgated ion channels. Results: Mutagenesis of two glycine residues in the pore-lining helix altered channel gating and ion conductance. Conclusion: Pore-lining helix glycines provide flexibility and minimize clashes between amino acid residues. Type 1 ryanodine receptors (RyR1s) release Ca2؉ 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. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca2؉ release in HEK293 cells, low [3H]ryanodine binding levels, and channels that were not regulated by Ca2؉ and did not conduct Ca2؉ in single channel measurements. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes

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