Functional and structural characterization of a novel malignant hyperthermia-susceptible variant of DHPR-β1a subunit (CACNB1).
Functional and structural characterization of a novel malignant hyperthermia-susceptible variant of DHPR-β1a subunit (CACNB1).
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DHPR-β1a 亚基 (CACNB1) 的新型恶性高热敏感变体的功能和结构特征。
DOI：
10.1152/ajpcell.00187.2017
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Casarotto,MarcoG
中科院分区：
文献类型：
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作者：
Perez,ClaudioF;Eltit,JoseM;Lopez,JoseR;Bodnár,Dóra;Dulhunty,AngelaF;Aditya,Shouvik;Casarotto,MarcoG
恶性高热（MH）易感性最近被认为与二氢吡啶受体（DHPR）β 1亚基的一种新变体有关，DHPR是骨骼肌中Ca 2+调节所必需的通道。本研究旨在探讨突变体V156 A对DHPR β 1亚基结构/功能的影响及其对培养肌管钙代谢的影响。利用差示扫描荧光法，我们发现突变V156 A导致β 1亚基Src同源3/鸟苷酸激酶核心结构域的热稳定性显著降低。在β1-null小鼠肌管中变体亚基的表达导致对咖啡因刺激的敏感性增加。对表达β 1a-V156 A的肌管进行全细胞膜片钳分析发现，通道激活的电压依赖性发生了−2 mV的偏移，但未观察到Ca 2+电导、电流动力学或肌浆网Ca 2+负荷的变化。静息游离Ca 2+和Na+浓度的测量表明，在表达β 1a-V156 A的肌管中，两种阳离子均显著升高，并且这些变化与质膜Ca 2+通过Na+/Ca 2+交换器和/或瞬时受体电位典型通道进入的速率增加有关。总之，我们的数据表明，突变体V156 A导致β 1亚基蛋白亚结构域的不稳定性，导致Ca 2+失调的表型，部分类似于DHPR α 1 S亚基的其他MH连锁突变。这些数据证明，变异体β 1a-V156 A的纯合表达具有成为病理变异体的潜力，尽管它可能需要其他基因缺陷才能导致完全的MH表型。
Malignant hyperthermia (MH) susceptibility has been recently linked to a novel variant of β1asubunit of the dihydropyridine receptor (DHPR), a channel essential for Ca2+regulation in skeletal muscle. Here we evaluate the effect of the mutant variant V156A on the structure/function of DHPR β1asubunit and assess its role on Ca2+metabolism of cultured myotubes. Using differential scanning fluorimetry, we show that mutation V156A causes a significant reduction in thermal stability of the Src homology 3/guanylate kinase core domain of β1asubunit. Expression of the variant subunit in β1-null mouse myotubes resulted in increased sensitivity to caffeine stimulation. Whole cell patch-clamp analysis of β1a-V156A-expressing myotubes revealed a −2 mV shift in voltage dependence of channel activation, but no changes in Ca2+conductance, current kinetics, or sarcoplasmic reticulum Ca2+load were observed. Measurement of resting free Ca2+and Na+concentrations shows that both cations were significantly elevated in β1a-V156A-expressing myotubes and that these changes were linked to increased rates of plasmalemmal Ca2+entry through Na+/Ca2+exchanger and/or transient receptor potential canonical channels. Overall, our data show that mutant variant V156A results in instability of protein subdomains of β1asubunit leading to a phenotype of Ca2+dysregulation that partly resembles that of other MH-linked mutations of DHPR α1Ssubunit. These data prove that homozygous expression of variant β1a-V156A has the potential to be a pathological variant, although it may require other gene defects to cause a full MH phenotype.