Expression and function of the Scn5a-encoded voltage-gated sodium channel NaV 1.5 in the rat jejunum.
Expression and function of the Scn5a-encoded voltage-gated sodium channel NaV 1.5 in the rat jejunum.
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DOI：
10.1111/nmo.12697
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发表时间：
2016-01
影响因子：
3.5
通讯作者：
Farrugia G
中科院分区：
文献类型：
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作者：
Beyder A;Gibbons SJ;Mazzone A;Strege PR;Saravanaperumal SA;Sha L;Higgins S;Eisenman ST;Bernard CE;Geurts A;Kline CF;Mohler PJ;Farrugia G
SCN5A编码的电压门控钠通道NaV1.5在人的空肠和结肠中表达。NaV1.5基因突变与胃肠动力障碍有关。大鼠胃肠道表达电压门控性钠通道，但它们在大鼠胃肠道电生理中的分子特性和作用尚不清楚。用聚合酶链式反应、Western blotting和免疫组织化学方法检测编码SCN5A基因的NaV1.5在大鼠空肠中的存在和分布。对新鲜分离的平滑肌细胞进行全细胞电生理学检查。用锌指核酸酶在大鼠体内靶向SCN5A。采用慢病毒介导的shRNA转导方法，在大鼠空肠平滑肌器官型培养中靶向SCN5A。用尖电极电生理学和RT-PCR检测器官型培养。免疫印迹法检测到大鼠空肠和结肠平滑肌中均有NaV1.5的表达。用另外两种针对NaV1.5不同部分的抗体进行免疫组织化学染色，发现大鼠空肠内存在离子通道。在分离的大鼠空肠平滑肌细胞上，全细胞电压钳表现出快速激活和失活电压依赖性内向电流，该电流可被NMDG+取代Na+所消除。组成型大鼠SCN5A基因敲除可导致宫内死亡。慢病毒介导的NaV1.5 shRNA进入大鼠空肠平滑肌器质性培养后，SCN5A基因表达缺失57%，慢波出现明显变化，峰值波幅下降40%，半宽度下降30%，膜电位超极化7 mV。SCN5A编码的NaV1.5基因在大鼠胃肠道平滑肌中表达，参与了大鼠胃肠平滑肌的电生理过程。
The Scn5a-encoded voltage-gated sodium channel NaV1.5 is expressed in human jejunum and colon. Mutations in NaV1.5 are associated with gastrointestinal motility disorders. The rat gastrointestinal tract expresses voltage-gated sodium channels, but their molecular identity and role in rat gastrointestinal electrophysiology are unknown. The presence and distribution of Scn5a-encoded NaV1.5 was examined by PCR, Western blotting and immunohistochemistry in rat jejunum. Freshly dissociated smooth muscle cells were examined by whole cell electrophysiology. Zinc finger nuclease was used to target Scn5a in rats. Lentiviral-mediated transduction with shRNA was used to target Scn5a in rat jejunum smooth muscle organotypic cultures. Organotypic cultures were examined by sharp electrode electrophysiology and RT-PCR. We found NaV1.5 in rat jejunum and colon smooth muscle by Western blot. Immunohistochemistry using two other antibodies to different portions of NaV1.5 revealed the presence of the ion channel in rat jejunum. Whole cell voltage-clamp in dissociated smooth muscle cells from rat jejunum showed fast activating and inactivating voltage-dependent inward current that was eliminated by Na+ replacement by NMDG+. Constitutive rat Scn5a knock out resulted in death in utero. NaV1.5 shRNA delivered by lentivirus into rat jejunum smooth muscle organotypic culture resulted in 57% loss of Scn5a mRNA and several significant changes in slow waves, namely 40% decrease in peak amplitude, 30% decrease in half-width and 7 mV hyperpolarization of the membrane potential at peak amplitude. Scn5a-encoded NaV1.5 is expressed in rat gastrointestinal smooth muscle and it contributes to smooth muscle electrophysiology.