Higure Yoko, Katayama Yoshimi, Takeuchi Keita, Ohtubo Yoshitaka, Yoshii Kiyonori
Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Fukuoka, Japan.
J Physiol. 2003 Jul 1;550(Pt 1):159-67. doi: 10.1113/jphysiol.2003.040733.
Lucifer Yellow CH (LY), a membrane-impermeant fluorescent dye, has been used in electro-physiological studies to visualize cell morphology, with little concern about its pharmacological effects. We investigated its effects on TTX-sensitive voltage-gated Na+ channels in mouse taste bud cells and hippocampal neurons under voltage-damp conditions. LY applied inside cells irreversibly slowed the inactivation of Na+ currents upon exposure to light of usual intensities. The inactivation time constant of Na+ currents elicited by a depolarization to -15 mV was increased by fourfold after a 5 min exposure to halogen light of 3200 Ix at source (3200 Ix light), and sevenfold after a 1-min exposure to 12,000 Ix light. A fraction of the Na+ current became non-inactivating following the exposure. The non-inactivating current was approximately 20 % of the peak total Na+ current after a 5 min exposure to 3200 Ix light, and approximately 30 % after a 1 min exposure to 12,000 Ix light. Light-exposed LY shifted slightly the current-voltage relationship of the peak Na+ current and of the steady-state inactivation curve, in the depolarizing direction. A similar light-dependent decrease in kinetics occurred in whole-cell Na+ currents of cultured mouse hippocampal neurones. Single-channel recordings showed that exposure to 6500 Ix light for 3 min increased the mean open time of Na+channels from 1.4 ms to 2.4 ms without changing the elementary conductance. The pre-incubation of taste bud cells with 1 mM dithiothreitoL a scavenger of radical species, blocked these LY effects. These results suggest that light-exposed LY yields radical species that modify Na+ channels.
荧光黄CH（LY）是一种不能透过细胞膜的荧光染料，已被用于电生理研究以观察细胞形态，而很少关注其药理作用。我们在电压钳制条件下研究了其对小鼠味蕾细胞和海马神经元中对河豚毒素敏感的电压门控钠通道的影响。在细胞内施加LY后，在通常强度的光照下，不可逆地减缓了钠电流的失活。在光源处暴露于3200勒克斯的卤素光5分钟后，由去极化至-15 mV引发的钠电流的失活时间常数增加了四倍，在暴露于12000勒克斯光1分钟后增加了七倍。暴露后，一部分钠电流变得不再失活。在暴露于3200勒克斯光5分钟后，非失活电流约为总钠电流峰值的20%，在暴露于12000勒克斯光1分钟后约为30%。光照下的LY使峰值钠电流和稳态失活曲线的电流-电压关系在去极化方向上略有偏移。在培养的小鼠海马神经元的全细胞钠电流中也出现了类似的光依赖性动力学下降。单通道记录显示，暴露于6500勒克斯光3分钟可使钠通道的平均开放时间从1.4毫秒增加到2.4毫秒，而不改变单通道电导。用1 mM二硫苏糖醇（一种自由基清除剂）对味蕾细胞进行预孵育，可阻断这些LY的作用。这些结果表明，光照下的LY产生自由基，从而修饰钠通道。
