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目的作者已经克隆了人神经母细胞瘤(NB-1)细胞株毒素抵抗型(TTX-R)钠通道α亚单位及其选择性剪切体,分别命名为hNbR1和hNbR1-2,本实验观察这两个钠通道同型异构体的电生理特性。方法使用全细胞膜片钳技术在转染了hNbR1和hNbR1-2的人胚肾细胞(HEK-293)上记录这两个钠通道的钠电流。结果这两个变构体在稳态激活和失活方面略微不同,hNbR1和hNbR1-2的半数激活电压分别为-(37.8±1.3)和-(43.5±1.7)mV,半数失活电压分别为-(74.4±1.1)和-(81.5±1.1)mV,hNbR1-2的稳态激活和稳态失活曲线较hNbR1分别负向移位5.7mV和7.1mV。TTX阻断hNbR1和hNbR1-2的IC50分别为(7.9±2.3)和(8.3±2.5)μmol·L-1,二者对TTX的敏感性无显著性差异。结论两个钠通道变构体hNbR1和hNbR1-2均可在HEK-293细胞上表达并产生钠电流,二者在稳态激活和失活方面略微不同,对TTX的敏感性无显著性差异。
Objective The authors have cloned the TTX-R sodium channel α subunit of human neuroblastoma (NB-1) cells and their alternative splices, named hNbR1 and hNbR1-2, respectively The electrophysiological properties of the two sodium channel isoforms were observed. Methods Whole-cell patch-clamp techniques were used to record sodium current in both sodium channels on human embryonic kidney cells (HEK-293) transfected with hNbR1 and hNbR1-2. Results The two variants were slightly different in steady state activation and inactivation. The half activation voltages of hNbR1 and hNbR1-2 were - (37.8 ± 1.3) and - (43.5 ± 1.7) mV, respectively. The half activation voltages were - (74.4 ± 1.1) and - (81.5 ± 1.1) mV respectively. The steady-state activation and homeostasis curves of hNbR1-2 were negatively shifted by 5.7mV and 7.1mV respectively compared with hNbR1. The IC50 of TTX blocking hNbR1 and hNbR1-2 were (7.9 ± 2.3) and (8.3 ± 2.5) μmol·L-1, respectively. There was no significant difference in TTX sensitivity between TTX and TTX. Conclusion Both sodium channel variants hNbR1 and hNbR1-2 can be expressed on HEK-293 cells and produce sodium currents. The two sodium channels show slight differences in steady-state activation and inactivation, with no significant difference in TTX sensitivity.