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浙江大学学报（医学版）
浙江大学学报（医学版）  2019, Vol. 48 Issue (1): 12-18    DOI: 10.3785/j.issn.1008-9292.2019.02.03
专题报道     
I1363T突变致人骨骼肌电压门控钠通道快失活受损的机制
唐思阳1(),叶佳1,李月舟1,2()
1. 浙江大学医学院生物物理学系， 浙江 杭州310058
2. 浙江大学医学院附属儿童医院实验检验中心， 浙江 杭州310052
I1363T mutation induces the defects in fast inactivation of human skeletal muscle voltage-gated sodium channel
TANG Siyang1(),YE Jia1,LI Yuezhou1,2()
1.Department of Biophysics, Zhejiang University School of Medicine, Hangzhou 310058, China
2.Department of Laboratory, the Children’s Hospital, Zhejiang University School of Medicine, Hangzhou 310052, China
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摘要： 目的

探究人源骨骼肌电压门控钠通道hNav1.4 I1363T突变体导致患者出现先天性副肌强直症状的机制。

 方法

利用氨基酸序列比对，检测hNav1.4 I1363位点的保守性；将hNav1.4蛋白的羧基端融合荧光蛋白mCherry，利用共聚焦显微镜观察hNav1.4野生型与I1363T突变体蛋白的表达量与分布情况；通过全细胞电生理技术记录野生型与I1363T突变体的稳态激活及快失活参数，并进一步分析野生型与I1363T突变体的窗电流。

 结果

hNav1.4 I1363位点在各类钠通道中高度保守。野生型与I1363T突变体均能正常上膜，且表达量无明显差异。野生型与I1363T突变体的50%激活电压V0.5 分别为（-29.08±0.24）mV和（-28.79±0.21）mV，斜率因子k分别为5.06±0.21和4.73±0.18（均P＞0.05）；野生型与I1363T突变体的50%失活电压V0.5 分别为（-68.03±0.34）mV和（-59.01±0.26）mV，斜率因子k分别为4.55±0.21和5.24±0.23（均P＜0.05），I1363T突变体的失活电压向去极化方向移动，且更为平缓。I1363T突变体形成的窗电流大于野生型的窗电流。

 结论

I1363T突变会导致hNav1.4慢失活受损，增加肌肉细胞兴奋性，导致肌强直的发生；而增大的窗电流使得钠离子在细胞内缓慢聚集，最终导致细胞兴奋性下降，引发肌无力。
关键词： 骨骼肌/病理生理学;  先天性肌强直/遗传学;  基因表达;  电压门控钠通道/遗传学;  突变;  转染    
Abstract: Objective

To investigate the mechanism of congenital paramyotonia caused by human skeletal muscle voltage-gated sodium channel hNav1.4 mutant I1363T.

 Methods

The conservation of the mutant site were detecled by using amino acid sequence alignment; the C-terminal mCherry fusion hNav1.4 was constructed, and the expression and distribution of wild type and hNav1.4 mutant I1363T were determined by confocal microscopy; the steady-state activation, fast inactivation and window current of wild type and hNav1.4 mutant I1363T were examined by whole-cell patch clamp.

 Results

Alignment of the amino acid sequences revealed that Ile1363 is highly conserved in human sodium channels. There was no significant difference in expression level and distribution between wild type and I1363T. Although no significant differences were observed between I1363T mutant and wild type in the activation upon channel gating, the V0.5 of voltage-dependence of fast inactivation of I1363T mutant [(-59.01±0.26) mV] shifted 9 mV towards depolarization as compared with wild type [(-68.03±0.34) mV], and the slope factor of voltage-dependence curve increased to (5.24±0.23) mV, compared with (4.55±0.21) mV of the wild type. Moreover, I1363T showed the larger window current than that of the wild type.

 Conclusions

I1363T causes the defect in fast inactivation of hNav1.4, which may increase the excitability of muscle cells and be responsible for myotonia. The increased window current of I1363T may result in an increase of inward Na+ current, could subsequently inactivate the channels and lead to loss of excitability and paralysis.
Key words: Muscle, skeletal/physiopathology    Myotonia congenita/genetics    Gene expression    Voltage-gated sodium channels/genetics    Mutation    Transfection
收稿日期: 2018-08-21 出版日期: 2019-05-10
:  Q71  
基金资助: 国家重点基础研究发展计划(2014CB910302)
通讯作者: 李月舟     E-mail: 21218314@zju.edu.cn;yuezhou-li@zju.edu.cn
作者简介: 唐思阳（1989—），男，博士，助理研究员，主要从事离子通道及相关疾病研究；E-mail： 21218314@zju.edu.cn； https://orcid.org/0000-0001-9361-7111|李月舟（1972—），男，博士，研究员，主要从事离子通道及相关疾病研究；E-mail： yuezhou-li@zju.edu.cn； https://orcid.org/0000-0003-0582-4255
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引用本文:

唐思阳,叶佳,李月舟. I1363T突变致人骨骼肌电压门控钠通道快失活受损的机制[J]. 浙江大学学报（医学版）, 2019, 48(1): 12-18.

TANG Siyang,YE Jia,LI Yuezhou. I1363T mutation induces the defects in fast inactivation of human skeletal muscle voltage-gated sodium channel. J Zhejiang Univ (Med Sci), 2019, 48(1): 12-18.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2019.02.03    或    http://www.zjujournals.com/med/CN/Y2019/V48/I1/12

图1  I1363T突变体序列分析
图2  共聚焦显微镜下观察野生型和I1363T突变体在HEK293细胞中的表达及定位
图3  I1363T突变体的电压依赖性
图4  I1363T突变体的窗电流
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