Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616, USA.
J Biol Chem. 2012 Jan 20;287(4):2863-76. doi: 10.1074/jbc.M111.307926. Epub 2011 Dec 2.
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+)) 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+) in Hom FDBs and elicited pronounced Ca(2+) oscillations in ∼30% of FDBs tested. Genotype contributed significantly elevated Ca(2+) (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+), 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.
恶性高热易感性（MHS）主要由兰尼碱受体 1（RYR1）内的突变引起。在这里，我们研究了 S4-S5 连接子内的 MHS 突变 T4826I 如何影响来自杂合子（Het）和纯合子（Hom）T4826I-RYR1 基因敲入小鼠的屈趾短肌（FDB）和股外侧肌的兴奋-收缩偶联和静息肌浆 Ca（2+）浓度[Ca（2+）]（rest）。（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.和 Pessah，I.N.（2011）FASEB J. doi：22131268）。FDB 对电刺激和急性氟烷（0.1%，v/v）暴露的反应表现出 Hom＞Het＞WT 的顺序。肌浆网 Ca（2+）释放和 Ca（2+）内流有助于氟烷引发的 Hom FDBs 中[Ca（2+）]（rest）的增加，并在约 30%的 FDBs 中诱发明显的 Ca（2+）振荡。使用离子选择性微电极在体内测量时，基因型显著升高[Ca（2+）]（rest）（Hom＞Het＞WT）。使用 Seahorse Bioscience（马萨诸塞州比勒里卡）通量分析仪在完整肌管中测量的 Het 和 Hom 耗氧量率和使用 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。肌肉膜在 RYR1 表达和基因型之间的丝氨酸（2844）磷酸化方面没有差异。单通道分析显示，Hom 和 WT 分别有利于开放和关闭状态，而 Het 表现出异质门控行为，具有高度不同的门控行为。[（3）H]ryanodine 结合分析显示基因剂量对结合密度和 Ca（2+）、Mg（2+）和温度的调节有影响。T4826I-RYR1 通道固有的明显异常赋予 MHS 并促进兴奋-收缩偶联、[Ca（2+）]（rest）和耗氧量率的基础紊乱。考虑到 Het 和 Hom T4826I-RYR1 小鼠都是存活的，这种 S4-S5 细胞质连接子中的突变介导的显著孤立单通道功能障碍在体内必须受到高度调节。
