Heterozygous R163C mice represent a valid model for studying the mechanisms that cause the human malignant hyperthermia syndrome.

No abstract

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder of skeletal muscle that segregates with >60 mutations within the MHS-1 locus on chromosome 19 coding for ryanodine receptor type 1 (RyR1). Although some MH RyR1s have been shown to enhance sensitivity to caffeine and halothane when expressed in non-muscle cells, their influence on EC coupling can only be studied in skeletal myotubes. We therefore expressed WT RyR1, six of the most common human MH RyR1s (R163C, G341R, R614C, R2163C, V2168M, and R2458H), and a newly identified C-terminal mutation (T4826I) in dyspedic myotubes to study their functional defects and how they influence EC coupling. Myotubes expressing any MH RyR1 were significantly more sensitive to stimulation by caffeine and 4-CmC than those expressing WT RyR1. ] i typical of normal myotubes at rest are key defects that contribute to the initiation of MH episodes. Malignant hyperthermia (MH)1 is a rare potentially fatal pharmacological disorder of skeletal muscle that can be triggered by commonly used volatile anesthetic agents and depolarizing muscle relaxants. It is clinically characterized by masseter spasm, tachycardia, increased end-tidal CO 2 , lactic acidosis, and hyperthermia, and if untreated progresses to death. The ryanodine receptor isoform-1 gene (ryr1) on chromosome 19q13.1 clearly represents a primary molecular locus for MH in humans, termed the MHS-1 locus, as mutations in ryr1 have been linked to more than 50% of all MH families and most central core disease (CCD) families (1). The ryr1 gene codes for a large conductance channel (RyR1) essential for release of SR Ca 2ϩ during skeletal muscle excitation contraction (EC) coupling (2, 3). Molecular genetic studies have shown that RyR1 mutations R615C and R614C co-segregate with porcine and human MH, respectively (4, 5). Functional analysis of skeletal muscle expressing either of these analogous mutations has revealed that a causative defect in MH is hypersensitive gating of the Ca 2ϩ release channel. However abnormalities in SR Ca 2ϩ release function have also been indicated even in the absence of RyR1 mutations, suggesting other loci, possibly in genes coding for RyR1 accessory proteins, may be involved in producing a common MH phenotype. To date, about 60 missense and deletion mutations (6) have been associated with an abnormal in vitro contracture test (CHCT/IVCT) and/or clinical MH or CCD. CCD is a non-progressive autosomal dominant myopathy that is characterized by hypotonia and mild proximal weakness affecting mainly the lower limbs. However the relationship between MH and CCD is not clear. Interestingly, all known MH-and CCD-related mutations found in the ryr1 gene are located in one of three "hot spots." The first hot spot is in the N-terminal region clustered between amino acid residues 35 and 614 (MH/CCD region 1); the second between amino acid residues 2163 and 2458 (MH/CCD region 2); and the third in the C-terminal transmembrane region, between amino acid residues 4643 and 4898.Functional analysis o...

Homer proteins are adapters that physically bind and functionally couple target proteins (1, 2). Homer1, Homer2, and Homer3 are encoded by three mammalian genes whose expression is dynamically regulated by cellular activity (2) and can attain high levels of protein in the nervous system where their functional regulation of excitatory signaling has been studied (3-5). A common element of structure of all Homer proteins is an N-terminal Enabled/Vasp homology domain (EVH1) essential for binding Homer ligands (6 -7). Crystallographic analysis of the EVH1 domain binding surface has revealed its specific association with polyproline ligands (7) that have previously been defined within plasmalemmal glutamate receptors including mGluR1a 1 and mGluR5a/b (1), inositol 1,4,5-trisphosphate receptors (IP 3 R) localized within endoplasmic and sarcoplasmic reticulum (SR) membranes (1), and cytoplasmic Shank proteins that are part of the N-methyl-D-aspartate receptor-associated PSD-95 complex (8, 9). A C-terminal coiled-coil domain is responsible for Homer self-multimerization (10 -13). Although full-length Homer proteins are constitutively expressed in a number of tissues, immediate-early gene products of the Homer1 gene including Homer1a and Ania 3 lack the CC domain (3, 10). "Short form" Homer1a is rapidly and transiently induced by physiological synaptic stimuli that evoke long term potentiation in the hippocampus (3, 10) or in striatum by the addition of dopaminergic drugs (3). Thus, a use-dependent exchange of multimeric and protomeric short forms of Homer appears to be responsible for dynamic regulation of context-dependent signaling in neurons. The functional influences of Homer adaptors on targeted protein have not been identified. Multi-PDZ domain proteins have been reported to cluster membrane ion channels with the result that the channel become active, but this effect is mimicked by agents that otherwise cross-link the channel (14). Homer binding to mGluR1 was recently reported to define agonist-independent activity of the receptor (15); however, this report did not define a molecular mechanism. The direct consequence of forming Homer-IP 3 R complexes on the dynamics of endoplasmic reticulum/SR Ca 2ϩ release and its possible contribution to temporal and spatial aspects of Ca 2ϩ signals remain unclear.Recently all three Homer mRNAs have also been detected in striated muscle, and Homer protein has been identified within skeletal and cardiac muscle (4, 5). Moreover, putative Homer ligand sequences are found within both the type 1 ryanodine receptor (RyR1) and the ␣ 1S -subunit of the dihydropyridine receptor (DHPR; CACNA1S) of skeletal muscle (7). RyR1 assembles as tetrameric structures within junctional regions of SR where it forms large organized arrays (16). RyR1 forms physical associations with ␣ 1S -DHPR that are essential for engaging reciprocal signaling units that are essential for excitation-contraction (E-C) coupling, a process whereby depolarization in the T-tubules triggers Ca 2ϩ release from SR resulting i...

Mutation T4825I in the type 1 ryanodine receptor (RYR1(T4825I/+)) confers human malignant hyperthermia susceptibility (MHS). We report a knock-in mouse line that expresses the isogenetic mutation T4826I. Heterozygous RYR1(T4826I/+) (Het) or homozygous RYR1(T4826I/T4826I) (Hom) mice are fully viable under typical rearing conditions but exhibit genotype- and sex-dependent susceptibility to environmental conditions that trigger MH. Hom mice maintain higher core temperatures than WT in the home cage, have chronically elevated myoplasmic[Ca(2+)](rest), and present muscle damage in soleus with a strong sex bias. Mice subjected to heat stress in an enclosed 37°C chamber fail to trigger MH regardless of genotype, whereas heat stress at 41°C invariably triggers fulminant MH in Hom, but not Het, mice within 20 min. WT and Het female mice fail to maintain euthermic body temperature when placed atop a bed whose surface is 37°C during halothane anesthesia (1.75%) and have no hyperthermic response, whereas 100% Hom mice of either sex and 17% of the Het males develop fulminant MH. WT mice placed on a 41°C bed maintain body temperature while being administered halothane, and 40% of the Het females and 100% of the Het males develop fulminant MH within 40 min. Myopathic alterations in soleus were apparent by 12 mo, including abnormally distributed and enlarged mitochondria, deeply infolded sarcolemma, and frequent Z-line streaming regions, which were more severe in males. These data demonstrate that an MHS mutation within the S4-S5 cytoplasmic linker of RYR1 confers genotype- and sex-dependent susceptibility to pharmacological and environmental stressors that trigger fulminant MH and promote myopathy.