Abstract
Naturally occurring mutations in the skeletal muscle Ca(2+) release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca(2+) release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca(2+) release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca(2+) release during store Ca(2+) overload, a process we refer to as store overload-induced Ca(2+) release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca(2+) activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca(2+) and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca(2+) activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca(2+) regulation of RyR1 represents a primary causal mechanism of MH.
Highlights
The pig model has proven invaluable in investigating the molecular basis of malignant hyperthermia (MH), and these studies have consistently demonstrated that Ca2ϩ release from MH-susceptible (MHS) pig skeletal muscle or sarcoplasmic reticulum (SR) membrane vesicles is enhanced upon exposure to various stimuli (1, 4, 9 –14)
Some studies reported that this enhanced activity of MHS RyR1 channels was associated with changes in the apparent sensitivity of the channel to cytosolic Ca2ϩ or Mg2ϩ, whereas others found no marked difference in the apparent sensitivity to cytosolic Ca2ϩ activation between MHS and normal RyR1 channels [1, 13,14,15,16,17,18,19,20]
We propose that a reduced threshold for store overload-induced Ca2ϩ release (SOICR) as a result of augmented luminal Ca2ϩ activation of RyR1 represents a primary defect underlying the pathogenesis of MH
Summary
The pig model has proven invaluable in investigating the molecular basis of MH, and these studies have consistently demonstrated that Ca2ϩ release from MH-susceptible (MHS) pig skeletal muscle or sarcoplasmic reticulum (SR) membrane vesicles is enhanced upon exposure to various stimuli (1, 4, 9 –14). MH and Enhanced Luminal Ca2؉ Activation of RyR1 release was markedly reduced in SR membranes isolated from MHS animals, suggesting that a defect in intraluminal Ca2ϩ regulation may underlie MH. Disease-linked RyR2 mutations are located in regions corresponding to the MH/central core disease mutation regions in RyR1 [28] This similar distribution suggests that disease-linked RyR2 and RyR1 mutations may exert similar effects on the intrinsic properties of the channel. To test this hypothesis, in the present study, we assessed the impact of the MH R615C mutation and the MHtriggering agent, halothane, on the response of RyR1 to luminal Ca2ϩ and the propensity for SOICR. We propose that a reduced threshold for SOICR as a result of augmented luminal Ca2ϩ activation of RyR1 represents a primary defect underlying the pathogenesis of MH
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