Co-existence of MH and CCD

The co-existence of MH and CCD is observed phenotypically and diagnostically but is still controversial [Robinson, et al., 2002]. Functional studies for RYR1 mutations sometimes provide inconsistent data. The R163C mutation, for example, is one of the most common MH mutations but it is also associated with CCD. Previous studies using rabbit RYR1 cDNA containing the rabbit R164C mutation have shown hypersensitivity of the mutant to drugs including halothane and 4-CmC [Tong, et al., 1997; Yang, et al., 2003]. Another study, however, has reported that the rabbit RYR1 mutant with R164C shows low Ca2+ release because of leakage in myotubes [Avila and Dirksen, 2001]. The reason for this inconsistency is unclear: the results may differ in different experimental conditions or methodology; the R163C mutation may give the channel variable characteristics; or hypersensitivity and leakage can simply co-exist. This study using human RYR1 cDNA and [3H]ryanodine binding assay showed hypersensitivity for the R163C mutation but no evidence for leakage was found for this mutation. A previous report has tried to explain a mechanism of co-existence suggesting that a “compensated” leaky channel causes both hypersensitivity and leakage and a “decompensated” leaky channel causes Ca2+ store depletion and insufficient Ca2+ release leading to CCD [Dirksen and Avila, 2004]. The level of Ca2+ store depletion may depend on the level of the leakage. The mutant channel which has a low level of Ca2+ leakage and low sensitivity to triggering agents may show both leakage and hypersensitivity leading to MH, and the mutant which has large Ca2+ leakage and Ca2+ store depletion may show little Ca2+ release triggered by signals leading to CCD although more functional and detailed data are required to support this hypothesis. Transient transfection was performed using only the R163C mutant cDNA clone, and hence the expressed protein was a homozygous mutant. The channel function may differ in homozygous and heterozygous conditions. A previous report using Y522S knock-in mice has shown that heterozygous mutant mice were hypersensitive to triggering agents and in contrast, homozygous knock-in mice were hyposensitive probably because of the leaky Y522S mutant channel and Ca2+ store depletion [Chelu,

et al., 2006]. Although the R163C knock-in mouse has shown hypersensitivity to triggering agents in both homozygous and heterozygous conditions [Yang, et al., 2006], results are likely to differ between homozygous and heterozygous mutant proteins and hence the genotype of the mutation may be important for the phenotype and channel functions.

The Y522S mutation is associated with both MH and CCD, and another study has reported that heterozygous Y522S knock-in mouse have shown MH-like symptoms (muscle contracture upon overheating) as well as CCD-like symptoms (swollen and misshapen mitochondria) [Durham, et al., 2008]. Previous observations suggest that the mutant RYR1 with Y522S is associated with both MH and CCD, and the single mutation leads to a leaky and hypersensitive channel.

In EC coupling, a depolarizing signal from nerves is detected by a voltage sensor protein, the dihydropyridine receptor (DHPR) and the DHPR transmits this signal to the RYR1 through a conformational change. Although a defined region of the DHPR plays a critical role in transmission of the signal [Kugler, et al., 2004], it is still not clear which RYR1 region is important in receiving the signalling that is allosteric from the DHPR. The N-terminal region of the RYR1 including hotspot region 1 is exposed in the sarcoplasm and some amino acids in this region may be important for contact with the DHPR (or other protein/s) and signal detection. Some N-terminal mutations such as Y522S may cause the functional defect in signal detection or transmission and hence this may cause hyposensitivity to the signal and insufficient Ca2+ release leading to CCD. These mutations may also cause hypersensitivity to triggering drugs such as halothane and abnormal Ca2+ release leading to MH because the pore region of the channel and the ability of channel opening is normal and Ca2+ stores are not completely depleted if the leakage is reasonably small.

There are some previous functional studies showing co-existence of leakage and hypersensitivity but there is no report so far showing co-existence of hypersensitivity and hyposensitivity. It is unlikely that the mutant RYR1 is sensitive to drugs releasing Ca2+ and insensitive without Ca2+ release at the same time. This study showed that CCD mutations I4898T and G4899R result in the RYR1 protein having no sensitivity to 4-CmC and the mutant channels may lose the ability of channel opening with less

[3H]ryanodine binding. If the mutant RYR1 cannot open the channel pore and cannot release sufficient Ca2+ leading to CCD, then these mutations would be unlikely to cause MH reactions releasing abnormal Ca2+ release through the channel. A previous study, however, has reported a CCD family which has the I4898T mutation co-segregating with the phenotype of affected family members. Some members have the mutation, have shown CCD symptoms and been diagnosed as MHS by the IVCT [Lynch, et al., 1999]. It is not clear whether the mutation is responsible for both MH and CCD or the patients have another trait for MH susceptibility. Alternatively, the IVCT is not specific for MH so an RYR1 mutation may result in a positive IVCT but not cause clinical MH. Further analyses are required to understand functional effects of CCD mutations and the mechanism of co-existence of MH and CCD.