Gene Dose Influences Cellular and Calcium Channel Dysregulation in Heterozygous and Homozygous T4826I-RYR1 Malignant Hyperthermia-susceptible Muscle
Gene Dose Influences Cellular and Calcium Channel Dysregulation in Heterozygous and Homozygous T4826I-RYR1 Malignant Hyperthermia-susceptible Muscle
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DOI：
10.1074/jbc.m111.307926
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发表时间：
2012-01-20
影响因子：
4.8
通讯作者：
Pessah, Isaac N.
中科院分区：
文献类型：
--
作者：
Barrientos, Genaro C.;Feng, Wei;Pessah, Isaac N.
恶性高热易感性（MHS）主要由兰尼碱受体1型（RYR 1）内的突变赋予。在此，我们阐述了S4-S5接头内的MHS突变T4826 I如何影响从杂合（Het）和纯合（Hom）T4826 I-RYR 1敲入小鼠制备的趾短屈肌（FDB）和股外侧肌中的兴奋-收缩偶联和静息肌浆Ca 2+浓度（[Ca 2 +]（静息））（Yuen，B. T.，Boncompagni，S.，冯伟，杨，T.，洛佩斯，J.R.，Matthaei，K.一、Goth，S. R.，Protasi，F.，Franzini-Armstrong，C.，艾伦，警察，Pessah，I. N.（2011）FASEB J. doi：22131268）。FDB对电刺激和急性氟烷（0.1%，v/v）暴露的反应显示出Hom >> Het >> WT的等级顺序。肌浆网释放的Ca 2+和Ca 2+进入氟烷触发的增加[Ca 2 +]（休息）在Hom FDBs和引起明显的Ca 2+振荡类似于30%的FDBs测试。使用离子选择性微电极在体内测量，基因型贡献显著升高的[Ca 2 +]（静息）（Hom > Het > WT）。使用Seahorse Bioscience（Billerica，MA）通量分析仪在完整肌管中测量的Het和Hom耗氧速率和使用MitoTracker测量的线粒体含量低于WT，而总细胞钙蛋白酶活性高于WT。肌膜在RYR 1表达和Ser（2844）磷酸化方面在基因型之间没有差异。单通道分析显示出高度发散的门控行为，Hom和WT分别有利于开放和闭合状态，而Het表现出异质门控行为。[H-3]Ryanodine结合分析揭示了基因剂量对结合密度的影响，并通过Ca 2+，Mg 2+和温度进行调节。T4826 I-RYR 1通道固有的明显异常赋予MHS并促进兴奋-收缩偶联、[Ca 2 +]（静息）和耗氧率的基础紊乱。考虑到Het和HomT 4826 I-RYR 1小鼠都是存活的，通过S4-S5胞质接头中的这种突变介导的显著的孤立的单通道功能障碍必须在体内高度调节。
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 Ca2+ concentration ([Ca2+](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 Ca2+ from the sarcoplasmic reticulum and Ca2+ entry contributed to halothane-triggered increases in [Ca2+](rest) in Hom FDBs and elicited pronounced Ca2+ oscillations in similar to 30% of FDBs tested. Genotype contributed significantly elevated [Ca2+](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. [H-3] Ryanodine binding analysis revealed a gene dose influence on binding density and regulation by Ca2+, Mg2+, and temperature. Pronounced abnormalities inherent in T4826I-RYR1 channels confer MHS and promote basal disturbances of excitation-contraction coupling, [Ca2+](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.