G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to ytosolic and luminal Ca2+

Le Xu, David D. Mowrey, Venkat R. Chirasani, Ying Wang, Daniel A. Pasek, Nikolay V. Dokholyan, Gerhard Meissner

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

The ryanodine receptor ion channel RyR1 is present in skeletal muscle and has a large cytoplasmic N-terminal domain and smaller C-terminal pore-forming domain comprising six transmembrane helices, a pore helix, and a selectivity filter. The RyR1 S6 pore-lining helix has two conserved glycines, Gly-4934 and Gly-4941, that facilitate RyR1 channel gating by providing S6 flexibility and minimizing amino acid clashes. Here, we report that substitution of Gly-4941 with Asp or Lys results in functional channels as indicated by caffeine-induced Ca2+ release response in HEK293 cells, whereas a low response of the corresponding Gly-4934 variants suggested loss of function. Following purification, the RyR1 mutants G4934D, G4934K, and G4941Ddid not noticeably conductCa2+in single-channelmeasurements. Gly-4941 replacement with Lys resulted in channels having reduced Kμ conductance and reduced selectivity for Ca2+ compared with wildtype. RyR1-G4941K did not fully close at nanomolar cytosolic Ca2+ concentrations and nearly fully opened at 2 μM cytosolic or sarcoplasmic reticulum luminal Ca2+, and Ca2+-And voltage-dependent regulation of RyR1-G4941K mutant channels was demonstrated. Computational methods and single-channel recordings indicated that the open G4941K variant results in the formation of a salt bridge to Asp-4938. In contrast, wildtype RyR1 was closed and not activated by luminal Ca2+ at low cytosolic Ca2+ levels. A model suggested that luminal Ca2+ activates RyR1 by accessing a recently identified cytosolic Ca2+-binding site in the open channel as the Ca2+ ions pass through the pore.

Original languageEnglish (US)
Pages (from-to)2015-2028
Number of pages14
JournalJournal of Biological Chemistry
Volume293
Issue number6
DOIs
StatePublished - Feb 9 2018

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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