Mutations to Gly2370, Gly2373 or Gly2375 in malignant hyperthermia domain 2 decrease caffeine and cresol sensitivity of the rabbit skeletal-muscle Ca2+-release channel (ryanodine receptor isoform 1)

Biochem J. 2001 Nov 15;360(Pt 1):97-105. doi: 10.1042/0264-6021:3600097.


Mutations G2370A, G2372A, G2373A, G2375A, Y3937A, S3938A, G3939A and K3940A were made in two potential ATP-binding motifs (amino acids 2370-2375 and 3937-3940) in the Ca(2+)-release channel of skeletal-muscle sarcoplasmic reticulum (ryanodine receptor or RyR1). Activation of [(3)H]ryanodine binding by Ca(2+), caffeine and ATP (adenosine 5'-[beta,gamma-methylene]triphosphate, AMP-PCP) was used as an assay for channel opening, since ryanodine binds only to open channels. Caffeine-sensitivity of channel opening was also assayed by caffeine-induced Ca(2+) release in HEK-293 cells expressing wild-type and mutant channels. Equilibrium [(3)H]ryanodine-binding properties and EC(50) values for Ca(2+) activation of high-affinity [(3)H]ryanodine binding were similar between wild-type RyR1 and mutants. In the presence of 1 mM AMP-PCP, Ca(2+)-activation curves were shifted to higher affinity and maximal binding was increased to a similar extent for wild-type RyR1 and mutants. ATP sensitivity of channel opening was also similar for wild-type and mutants. These observations apparently rule out sequences 2370-2375 and 3937-3940 as ATP-binding motifs. Caffeine or 4-chloro-m-cresol sensitivity, however, was decreased in mutants G2370A, G2373A and G2375A, whereas the other mutants retained normal sensitivity. Amino acids 2370-2375 lie within a sequence (amino acids 2163-2458) in which some eight RyR1 mutations have been associated with malignant hyperthermia and shown to be hypersensitive to caffeine and 4-chloro-m-cresol activation. By contrast, mutants G2370A, G2373A and G2375A are hyposensitive to caffeine and 4-chloro-m-cresol. Thus amino acids 2163-2458 form a regulatory domain (malignant hyperthermia regulatory domain 2) that regulates caffeine and 4-chloro-m-cresol sensitivity of RyR1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives*
  • Adenosine Triphosphate / metabolism
  • Adenosine Triphosphate / pharmacology
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Caffeine / pharmacology*
  • Calcium / metabolism
  • Calcium Channels / metabolism*
  • Cell Line
  • Cells, Cultured
  • Central Nervous System Stimulants / pharmacology
  • Cresols / pharmacology*
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation
  • Fever
  • Glycine / chemistry*
  • Immunoblotting
  • Kinetics
  • Malignant Hyperthermia / genetics*
  • Malignant Hyperthermia / metabolism
  • Molecular Sequence Data
  • Muscle, Skeletal / metabolism*
  • Mutation*
  • Oligonucleotides / pharmacology
  • Plasmids / metabolism
  • Protein Binding
  • Protein Isoforms
  • Protein Structure, Tertiary
  • Rabbits
  • Ryanodine / metabolism
  • Ryanodine Receptor Calcium Release Channel / metabolism*
  • Spectrometry, Fluorescence
  • Transfection


  • Calcium Channels
  • Central Nervous System Stimulants
  • Cresols
  • Oligonucleotides
  • Protein Isoforms
  • Ryanodine Receptor Calcium Release Channel
  • Ryanodine
  • 5'-adenylyl (beta,gamma-methylene)diphosphonate
  • Caffeine
  • Adenosine Triphosphate
  • cresol
  • Calcium
  • Glycine