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Reduced inhibitory effect of Mg2+ on ryanodine receptor-Ca2+ release channels in malignant hyperthermia
D R Laver1, V J Owen, P R Junankar
1John Curtin School of Medical Research, Australian National University, Canberra.
Abstract:
Malignant hyperthermia (MH) is a potentially fatal, inherited skeletal muscle disorder in humans and pigs that is caused by abnormal regulation of Ca2+ release from the sarcoplasmic reticulum (SR). MH in pigs is associated with a single mutation (Arg615Cys) in the SR ryanodine receptor (RyR) Ca2+ release channel. The way in which this mutation leads to excessive Ca2+ release is not known and is examined here. Single RyR channels from normal and MH-susceptible (MHS) pigs were examined in artificial lipid bilayers. High cytoplasmic (cis) concentrations of either Ca2+ or Mg2+ (>100 microM) inhibited channel opening less in MHS RyRs than in normal RyRs. This difference was more prominent at lower ionic strength (100 mM versus 250 mM). In 100 mM cis Cs+, half-maximum inhibition of activity occurred at approximately 100 microM Mg2+ in normal RyRs and at approximately 300 microM Mg2+ in MHS RyRs, with an average Hill coefficient of approximately 2 in both cases. The level of Mg2+ inhibition was not appreciably different in the presence of either 1 or 50 microM activating Ca2+, showing that it was not substantially influenced by competition between Mg2+ and Ca2+ for the Ca2+ activation site. Even though the absolute inhibitory levels varied widely between channels and conditions, the inhibitory effects of Ca2+ and Mg2+ were virtually identical for the same conditions in any given channel, indicating that the two cations act at the same low-affinity inhibitory site. It seems likely that at the cytoplasmic [Mg2+] in vivo (approximately 1 mM), this Ca2+/Mg2+-inhibitory site will be close to fully saturated with Mg2+ in normal RyRs, but less fully saturated in MHS RyRs. Therefore MHS RyRs should be more sensitive to any activating stimulus, which would readily account for the development of an MH episode.
Insights
Malignant hyperthermia (MH) susceptibility in pigs is linked to a RyR mutation. This mutation reduces inhibition by Ca2+ and Mg2+ in the ryanodine receptor (RyR) channel, increasing sensitivity to stimuli and potentially causing MH episodes.
Area of Science:
- Biochemistry
- Molecular Biology
- Physiology
Background:
- Malignant hyperthermia (MH) is a severe inherited skeletal muscle disorder.
- MH in pigs is linked to a specific mutation (Arg615Cys) in the ryanodine receptor (RyR) Ca2+ channel.
- The mechanism by which this mutation causes excessive Ca2+ release remains unclear.
Purpose of the Study:
- To investigate the functional consequences of the Arg615Cys mutation in porcine ryanodine receptors (RyRs).
- To elucidate the altered regulation of Ca2+ release by the mutated RyR channel in MH-susceptible pigs.
Main Methods:
- Single RyR channels from normal and MH-susceptible pigs were reconstituted into artificial lipid bilayers.
- The effects of cytoplasmic Ca2+ and Mg2+ on channel activity were measured under varying ionic strengths.
- Inhibition kinetics, including half-maximal inhibitory concentrations and Hill coefficients, were determined.
Main Results:
- MH-susceptible RyRs exhibited reduced inhibition by high cytoplasmic Ca2+ and Mg2+ compared to normal RyRs.
- This difference in inhibition was more pronounced at lower ionic strength (100 mM vs. 250 mM).
- Ca2+ and Mg2+ acted at the same inhibitory site, with Mg2+ inhibition being less complete in MHS RyRs at physiological Mg2+ concentrations.
Conclusions:
- The Arg615Cys mutation in porcine RyR leads to impaired Ca2+/Mg2+ inhibition of the channel.
- This impaired inhibition likely results in increased channel sensitivity to activating stimuli.
- The findings provide a molecular mechanism explaining the predisposition to MH episodes in susceptible pigs.