Reeve H L, Vaughan P F, Peers C
Department of Pharmacology, Leeds University, UK.
Eur J Neurosci. 1994 Jun 1;6(6):943-52. doi: 10.1111/j.1460-9568.1994.tb00588.x.
Ca2+ channel currents were recorded in undifferentiated human neuroblastoma (SH-SY5Y) cells with the whole-cell patch-clamp technique, using 10 mM Ba2+ as charge carrier. Currents were only evoked by depolarizations to -30 mV or more positive (holding potential -80 mV), inactivated partially during 200 ms depolarizing steps, and were abolished by 150 microM Cd2+. Currents could be enhanced by Bay K-8644 and partially inhibited by nifedipine, suggesting that they arose in part due to activation of L-type Ca2+ channels. Currents were also inhibited by the marine snail peptide omega-conotoxin GVIA (omega-CgTx). At a concentration of 10 nM inhibition by omega-CgTx was reversible, but at higher concentrations blockade was always irreversible. Although current inhibition by nifedipine was maximal at 1 microM, supramaximal concentrations reduced the inhibitory actions of omega-CgTx in a concentration-dependent manner. Ca2+ channel currents evoked from a holding potential of -50 mV showed no inactivation during 200 ms depolarizations but declined in amplitude with successive depolarizing steps (0.2 Hz). Current amplitudes could be restored by returning the holding potential to -80 mV. Currents evoked from -50 mV were inhibited by nifedipine and omega-CgTx to a similar degree as those evoked from -80 mV. Our results indicate that undifferentiated SH-SY5Y cells possess L- and N-type Ca2+ channels which can be distinguished pharmacologically but cannot be separated by using depolarized holding potentials. Furthermore, these data suggest that nifedipine has a novel action to inhibit blockade of N-type channels by omega-CgTx.
采用全细胞膜片钳技术，以10 mM Ba2+作为电荷载体，在未分化的人神经母细胞瘤（SH-SY5Y）细胞中记录Ca2+通道电流。电流仅在去极化至-30 mV或更正电位（钳制电位-80 mV）时被诱发，在200 ms去极化步骤中部分失活，并被150 microM Cd2+阻断。电流可被Bay K-8644增强，被硝苯地平部分抑制，表明它们部分源于L型Ca2+通道的激活。电流也被海蜗牛肽ω-芋螺毒素GVIA（ω-CgTx）抑制。在10 nM浓度下，ω-CgTx的抑制作用是可逆的，但在更高浓度下，阻断总是不可逆的。尽管硝苯地平在1 microM时对电流的抑制作用最大，但超最大浓度会以浓度依赖的方式降低ω-CgTx的抑制作用。从-50 mV的钳制电位诱发的Ca2+通道电流在200 ms去极化期间没有失活，但随着连续去极化步骤（0.2 Hz）振幅下降。通过将钳制电位恢复到-80 mV，电流振幅可以恢复。从-50 mV诱发的电流被硝苯地平和ω-CgTx抑制的程度与从-80 mV诱发的电流相似。我们的结果表明，未分化的SH-SY5Y细胞具有L型和N型Ca2+通道，它们在药理学上可以区分，但不能通过使用去极化钳制电位来分离。此外，这些数据表明硝苯地平具有抑制ω-CgTx对N型通道阻断的新作用。
