Functional Changes in RYR1 Associated with Malignant Hyperthermia Mutations

Altered SR Ca²⁺ channel gating kinetics appear to underlie the uncontrolled skeletal muscle metabolism associated with administration of halogenated anesthetics or depolarizing agents. The sustained elevation of the Ca²⁺ level in the sarcoplasm results in abusive stimulation of aerobic and glycolytic metabolism, which accounts for combined acidosis, rigidity, altered permeability, and hyperkalemia. Although studies using Ca²⁺ -selective microelectrodes have indicated that MH-affected muscle has a higher level of resting Ca²⁺ , the findings have not been confirmed by radiometric fluorescent Ca²⁺ dyes. Study of the chronologic relationship of the biochemical and clinical development of porcine MH demonstrates that the increase in intracellular Ca²⁺ concentration precedes the increase in expired carbon dioxide and the classic first sign, tachycardia. ^[35]

Extensive study of the porcine model has defined the biochemical and functional changes in SR Ca²⁺ transport and RYR1 function underlying MH. The validity of the single-point porcine RYR1 mutation^[36] for defining MH malfunction is affirmed by use of the fluorescent calcium

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Although active SR Ca²⁺ accumulation appears to be normal in MH-affected pig muscle, significant abnormalities in the process of Ca²⁺ release have been documented in several types of in vitro bioassays. In skinned muscle fiber preparations, the rate and the extent of Ca²⁺ release from SR were higher in fibers with MH abnormalities. These results in skinned fibers correlate well with those obtained from isolated SR membrane preparations enriched in RYR1 protein. Although initial studies revealed a difference in the Ca²⁺ threshold for activation for SR Ca²⁺ release, later studies using rapid-quench methods found no apparent difference in the sensitivity of SR Ca²⁺ release with respect to Ca²⁺ . O'Brien and Li^[37] developed a microassay that revealed functional differences in Ca²⁺ transport in SR membranes isolated from normal or from MHS pigs. They found that SR from MHS swine had normal maximal Ca²⁺ -ATPase pumping but that the activity of RYR1 after addition of a bolus of Ca²⁺ was 50% greater in heterozygotes and 100% greater in homozygotes for the mutation. Hypersensitivity to receptor agonists, such as caffeine, and an associated hyposensitivity to inhibition with magnesium (Mg²⁺ ) was also demonstrated. There has been some controversy about whether changes exist in the sensitivity of MH-affected muscles to inhibition by Mg²⁺ compared with normal fibers. Owen and colleagues^[38] demonstrated that fibers from pigs heterozygous or homozygous for the RYR1 MH allele needed only a smaller reduction in the free concentration of Mg²⁺ to induce Ca²⁺ release from SR. Dantrolene counteracted the effect of reduced Mg²⁺ inhibition in MH-affected muscle. The abnormal responsiveness of MH-affected muscle to various stimuli may result from the reduced ability of myoplasmic Mg²⁺ to inhibit Ca²⁺ release from SR.

Reconstitution of channels isolated from MHS pigs studied in bilayer lipid membranes has revealed significantly reduced sensitivity to inactivating concentrations of Ca²⁺ , whereas the sensitivity of channels to activating Ca²⁺ remains unchanged. The Ca²⁺ channels from MHS pigs exhibit a significantly higher open probability compared with wild-type channels across a broad range of Ca²⁺ concentrations (7 µM to 10 µM) on the cytoplasmic face when measured at pH 6.8. Whether Ca²⁺ channels reconstituted in bilayer lipid membranes from pigs with MH susceptibility exhibit an altered response to inhibition by Mg²⁺ remains controversial. In porcine MH, Mg²⁺ inhibition of RYR1 channels was not altered, whereas there is a threefold lower potency for Mg²⁺ inhibition of MH-affected Ca²⁺ channels. Similar findings were seen in human MHS skinned fibers.^[39] The underlying cause of these various porcine experimental results is unclear, but the concentration of monovalent ions in measuring channel activity might have influenced the inhibitory potency of Mg²⁺ . Channels isolated from pigs heterozygous for the MH mutation suggest that the heterozygous porcine population of Ca²⁺ release channels contains heterotetramers with properties distinct from those of MH homozygote or normal channels. The data also imply that the population of Ca²⁺ release channels in humans with MHS who are heterozygous for a dominant mutation in this protein also contains heterotetrameric channels. In contrast to Ca²⁺ uptake and release studies with isolated SR, single-channel measurements have failed to reveal an altered sensitivity of channels to activation by caffeine.

Radioligand-receptor binding studies with nanomolar concentrations of [³ H]ryanodine have determined differences between SR isolated from normal humans and pigs and those with MH susceptibility. [³ H]Ryanodine binding assays represent a sensitive means of assessing functional anomalies regulating the channel pore by use of a simple tube assay. This is possible because [³ H]ryanodine binds to a conformationally sensitive site within or near the channel pore. Mickelson and coworkers^[17] first demonstrated that the binding of [³ H]ryanodine to MH homozygotic porcine heavy SR exhibited an altered Ca²⁺ dependence at the low-affinity (inhibitory) Ca²⁺ site and a lower affinity for ryanodine compared with normal porcine SR. However, the maximum capacity of SR to bind [³ H]ryanodine was the same in both tissues, indicating an altered structure or function, or both, of the receptor rather than a change in expression associated with the disease. Studies performed with RYR1 isolated from human biopsies revealed a higher affinity for [³ H]ryanodine and a higher sensitivity to activation by caffeine with MHS preparations. Surprisingly, it was the activation, not the inactivation, of the binding of [³ H]ryanodine by Ca²⁺ that was abnormal in human MH SR.