G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to ytosolic and luminal Ca 2+

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

Research output: Contribution to journalArticle

1 Citation (Scopus)

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 Ca 2+ 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 conductCa 2+ in single-channelmeasurements. Gly-4941 replacement with Lys resulted in channels having reduced Kμ conductance and reduced selectivity for Ca 2+ compared with wildtype. RyR1-G4941K did not fully close at nanomolar cytosolic Ca 2+ concentrations and nearly fully opened at 2 μM cytosolic or sarcoplasmic reticulum luminal Ca 2+ , and Ca 2+ -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 Ca 2+ at low cytosolic Ca 2+ levels. A model suggested that luminal Ca 2+ activates RyR1 by accessing a recently identified cytosolic Ca 2+ -binding site in the open channel as the Ca 2+ 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 - Jan 1 2018

Fingerprint

S 6
Ryanodine Receptor Calcium Release Channel
Linings
Muscle
Skeletal Muscle
Substitution reactions
HEK293 Cells
Sarcoplasmic Reticulum
Computational methods
Caffeine
Ion Channels
Glycine
Purification
Salts
Binding Sites
Ions
Amino Acids

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Xu, Le ; Mowrey, David D. ; Chirasani, Venkat R. ; Wang, Ying ; Pasek, Daniel A. ; Dokholyan, Nikolay ; Meissner, Gerhard. / G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to ytosolic and luminal Ca 2+ In: Journal of Biological Chemistry. 2018 ; Vol. 293, No. 6. pp. 2015-2028.
@article{5497d737021c4ae6a4379e538db83f3b,
title = "G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to ytosolic and luminal Ca 2+",
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 Ca 2+ 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 conductCa 2+ in single-channelmeasurements. Gly-4941 replacement with Lys resulted in channels having reduced Kμ conductance and reduced selectivity for Ca 2+ compared with wildtype. RyR1-G4941K did not fully close at nanomolar cytosolic Ca 2+ concentrations and nearly fully opened at 2 μM cytosolic or sarcoplasmic reticulum luminal Ca 2+ , and Ca 2+ -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 Ca 2+ at low cytosolic Ca 2+ levels. A model suggested that luminal Ca 2+ activates RyR1 by accessing a recently identified cytosolic Ca 2+ -binding site in the open channel as the Ca 2+ ions pass through the pore.",
author = "Le Xu and Mowrey, {David D.} and Chirasani, {Venkat R.} and Ying Wang and Pasek, {Daniel A.} and Nikolay Dokholyan and Gerhard Meissner",
year = "2018",
month = "1",
day = "1",
doi = "10.1074/jbc.M117.803247",
language = "English (US)",
volume = "293",
pages = "2015--2028",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "6",

}

G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to ytosolic and luminal Ca 2+ . / Xu, Le; Mowrey, David D.; Chirasani, Venkat R.; Wang, Ying; Pasek, Daniel A.; Dokholyan, Nikolay; Meissner, Gerhard.

In: Journal of Biological Chemistry, Vol. 293, No. 6, 01.01.2018, p. 2015-2028.

Research output: Contribution to journalArticle

TY - JOUR

T1 - G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to ytosolic and luminal Ca 2+

AU - Xu, Le

AU - Mowrey, David D.

AU - Chirasani, Venkat R.

AU - Wang, Ying

AU - Pasek, Daniel A.

AU - Dokholyan, Nikolay

AU - Meissner, Gerhard

PY - 2018/1/1

Y1 - 2018/1/1

N2 - 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 Ca 2+ 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 conductCa 2+ in single-channelmeasurements. Gly-4941 replacement with Lys resulted in channels having reduced Kμ conductance and reduced selectivity for Ca 2+ compared with wildtype. RyR1-G4941K did not fully close at nanomolar cytosolic Ca 2+ concentrations and nearly fully opened at 2 μM cytosolic or sarcoplasmic reticulum luminal Ca 2+ , and Ca 2+ -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 Ca 2+ at low cytosolic Ca 2+ levels. A model suggested that luminal Ca 2+ activates RyR1 by accessing a recently identified cytosolic Ca 2+ -binding site in the open channel as the Ca 2+ ions pass through the pore.

AB - 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 Ca 2+ 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 conductCa 2+ in single-channelmeasurements. Gly-4941 replacement with Lys resulted in channels having reduced Kμ conductance and reduced selectivity for Ca 2+ compared with wildtype. RyR1-G4941K did not fully close at nanomolar cytosolic Ca 2+ concentrations and nearly fully opened at 2 μM cytosolic or sarcoplasmic reticulum luminal Ca 2+ , and Ca 2+ -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 Ca 2+ at low cytosolic Ca 2+ levels. A model suggested that luminal Ca 2+ activates RyR1 by accessing a recently identified cytosolic Ca 2+ -binding site in the open channel as the Ca 2+ ions pass through the pore.

UR - http://www.scopus.com/inward/record.url?scp=85041956147&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85041956147&partnerID=8YFLogxK

U2 - 10.1074/jbc.M117.803247

DO - 10.1074/jbc.M117.803247

M3 - Article

VL - 293

SP - 2015

EP - 2028

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 6

ER -