克隆的人神经母细胞瘤细胞株鲀毒素抵抗型钠通道的功能分析

Functional analysis of cloned tetrodotoxin-resistant sodium channel α-subunit in human neuroblastoma cell line

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。

AIM We have cloned the tetrodotoxin-resistant （TTX-R） sodium channel α-subunit and a splicing variant from hunman neuroblastoma cell lines（ NB-1 ） and designated them as hNbR1 and hNbR1-2, respectively. The electrophysiological properties of these two sodium channel isoforms were investigated in this study. METHODS Whole cell patch clamp was used to record the sodium currents produced bv these two clones transfected to human embryonic kidney cells （HEK293）. RESULTS These two isoforms were found to be a little differences in the kinetics of steady-state inactivation and activation, the 1/1/2 of steady-state activation in hNbR1 and hNbR1-2was - （37.8±1.3） and - （43.5±1.7）mV, respectively, the V1/2 of steady-state inactivation in hNbR1 and hNbR1-2 was -（74.4±1.1） and -（81.5±1.1） mV, respectively, shifted in 5.7 mV and 7. 1 mV the negative direction with in hNbR1-2. The IC50 of TIN blocking hNbR1 and hNbR1-2 was （7.9 ± 2.3 ） and （8.3 ± 2.5）μmol·L^-1, respectively, which indicated that there is no significant differenee in TFX sensitivity between the two clones. CONCLUSION hNbR1 anti hNbtR1-2 can be functionally expressed in HEK-293 cells and produce sodium currents. There is a little difference in the kinetics of steady-state activation and inactivation and no significant difference in the TTX sensitivity between the two isofonns.