Gene Dose Influences Cellular and Calcium Channel Dysregulation in Heterozygous and Homozygous T4826I-RYR1 Malignant Hyperthermia-susceptible Muscle

Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2011; 287(4):2863-76. DOI: 10.1074/jbc.M111.307926
Source: PubMed


Malignant hyperthermia susceptibility (MHS) is primarily conferred by mutations within ryanodine receptor type 1 (RYR1). Here we address how the MHS mutation T4826I within the S4-S5 linker influences excitation-contraction coupling and resting myoplasmic Ca(2+) concentration ([Ca(2+)](rest)) in flexor digitorum brevis (FDB) and vastus lateralis prepared from heterozygous (Het) and homozygous (Hom) T4826I-RYR1 knock-in mice (Yuen, B. T., Boncompagni, S., Feng, W., Yang, T., Lopez, J. R., Matthaei, K. I., Goth, S. R., Protasi, F., Franzini-Armstrong, C., Allen, P. D., and Pessah, I. N. (2011) FASEB J. doi:22131268). FDB responses to electrical stimuli and acute halothane (0.1%, v/v) exposure showed a rank order of Hom ≫ Het ≫ WT. Release of Ca(2+) from the sarcoplasmic reticulum and Ca(2+) entry contributed to halothane-triggered increases in [Ca(2+)](rest) in Hom FDBs and elicited pronounced Ca(2+) oscillations in ∼30% of FDBs tested. Genotype contributed significantly elevated [Ca(2+)](rest) (Hom > Het > WT) measured in vivo using ion-selective microelectrodes. Het and Hom oxygen consumption rates measured in intact myotubes using the Seahorse Bioscience (Billerica, MA) flux analyzer and mitochondrial content measured with MitoTracker were lower than WT, whereas total cellular calpain activity was higher than WT. Muscle membranes did not differ in RYR1 expression nor in Ser(2844) phosphorylation among the genotypes. Single channel analysis showed highly divergent gating behavior with Hom and WT favoring open and closed states, respectively, whereas Het exhibited heterogeneous gating behaviors. [(3)H]Ryanodine binding analysis revealed a gene dose influence on binding density and regulation by Ca(2+), Mg(2+), and temperature. Pronounced abnormalities inherent in T4826I-RYR1 channels confer MHS and promote basal disturbances of excitation-contraction coupling, [Ca(2+)](rest), and oxygen consumption rates. Considering that both Het and Hom T4826I-RYR1 mice are viable, the remarkable isolated single channel dysfunction mediated through this mutation in S4-S5 cytoplasmic linker must be highly regulated in vivo.

7 Reads
  • Source
    • "), and sudden death (Laitinen et al. 2004). Recent studies demonstrate that specific RYR mutations con­ fer sex– and gene–dose­dependent susceptibil­ ity to pharmacological (halogenated anesthetic) and environmental (heat) stressors that trigger malignant hyperthermia and muscle damage in otherwise asymptomatic individuals (Barrientos et al. 2012; Yuen et al. 2012). Importantly, PCB­95 is significantly more potent and effica­ cious in disrupting cation regulation of mutant R615C­RYR1 compared with wild type RyR1 (Ta and Pessah 2007). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Aroclor 1254 (A1254) interferes with normal dendritic growth and plasticity in the developing rodent brain, but the mechanism(s) mediating this effect have yet to be established. Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) enhance the activity of ryanodine receptor (RyR) calcium ion (Ca(2+)) channels, which play a central role in regulating the spatiotemporal dynamics of intracellular Ca(2+) signaling. Ca(2+) signaling is a predominant factor in shaping dendritic arbors, but whether PCB potentiation of RyR activity influences dendritic growth is not known. We determined whether RyR activity is required for PCB effects on dendritic growth. Golgi analysis of hippocampi from weanling rats confirmed that developmental exposure via the maternal diet to NDL PCB-95 (2,2',3,5'6-pentachlorobiphenyl), a potent RyR potentiator, phenocopies the dendrite-promoting effects of A1254. Dendritic growth in dissociated cultures of primary hippocampal neurons and in hippocampal slice cultures is similarly enhanced by PCB-95 but not by PCB-66 (2,3,4',4-tetrachlorobiphenyl), a congener with negligible effects on RyR activity. The dendrite-promoting effects of PCB-95 are evident at concentrations as low as 2 pM and are inhibited by either pharmacologic blockade or siRNA knockdown of RyRs. Our findings demonstrate that environmentally relevant levels of NDL PCBs modulate neuronal connectivity via RyR-dependent effects on dendritic arborization. In addition, these findings identify RyR channel dysregulation as a novel mechanism contributing to dysmorphic dendritogenesis associated with heritable and environmentally triggered neurodevelopmental disorders.
    Environmental Health Perspectives 04/2012; 120(7):997-1002. DOI:10.1289/ehp.1104832 · 7.98 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Malignant hyperthermia (MH) susceptibility has been attributed to a leaky sarcoplasmic reticulum (SR) caused by missense mutations in RYR1 or CACNA1S, and the MH crisis has been attributed solely to massive self-sustaining release of Ca(2+) from SR stores elicited by triggering agents. Here, we show in muscle cells from MH-RyR1(R163C) knock-in mice that increased passive SR Ca(2+) leak causes an enlarged basal influx of sarcolemmal Ca(2+) that results in chronically elevated myoplasmic free Ca(2+) concentration ([Ca(2+)](i)) at rest. We discovered that Gd(+3) and GsMTx-4 were more effective than BTP2 or expression of the dominant-negative Orai1(E190Q) in reducing both Ca(2+) entry and [Ca(2+)](i), implicating a non-STIM1/Orai1 SOCE pathway in resetting resting [Ca(2+)](i). Indeed, two nonselective cationic channels, TRPC3 and TRPC6, are overexpressed, and [Na](i) is chronically elevated in MH-RyR1(R163C) muscle cells. [Ca(2+)](i) and [Na(+)](i) are persistently elevated in vivo and further increased by halothane in MH-RyR1(R163C/WT) muscle. These increases are markedly attenuated by local perfusion of Gd(+3) or GsMTx-4 and completely suppressed by dantrolene. These results contribute a new paradigm for understanding MH pathophysiology by demonstrating that nonselective sarcolemmal cation channel activity plays a critical role in causing myoplasmic Ca(2+) and Na(+) overload both at rest and during the MH crisis.-Eltit, J. M., Ding, X., Pessah, I. N., Allen, P. D., Lopez, J. R. Nonspecific sarcolemmal cation channels are critical for the pathogenesis of malignant hyperthermia.
    The FASEB Journal 11/2012; 27(3). DOI:10.1096/fj.12-218354 · 5.04 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Evidence implicates environmental factors in the pathogenesis of Autism Spectrum Disorders (ASD). However, the identity of specific environmental chemicals that influence ASD risk, severity or treatment outcome remains elusive. The impact of any given environmental exposure likely varies across a population according to individual genetic substrates, and this increases the difficulty of identifying clear associations between exposure and ASD diagnoses. Heritable genetic vulnerabilities may amplify adverse effects triggered by environmental exposures if genetic and environmental factors converge to dysregulate the same signaling systems at critical times of development. Thus, one strategy for identifying environmental risk factors for ASD is to screen for environmental factors that modulate the same signaling pathways as ASD susceptibility genes. Recent advances in defining the molecular and cellular pathology of ASD point to altered patterns of neuronal connectivity in the developing brain as the neurobiological basis of these disorders. Studies of syndromic ASD and rare highly penetrant mutations or CNVs in ASD suggest that ASD risk genes converge on several major signaling pathways linked to altered neuronal connectivity in the developing brain. This review briefly summarizes the evidence implicating dysfunctional signaling via Ca(2+)-dependent mechanisms, extracellular signal-regulated kinases (ERK)/phosphatidylinositol-3-kinases (PI3K) and neuroligin-neurexin-SHANK as convergent molecular mechanisms in ASD, and then discusses examples of environmental chemicals for which there is emerging evidence of their potential to interfere with normal neuronal connectivity via perturbation of these signaling pathways.
    Neurotoxicology and Teratology 12/2012; 36. DOI:10.1016/ · 2.76 Impact Factor
Show more