Cox R H, Zhou Z, Tulenko T N
Bockus Research Institute, The Graduate Hospital, Department of Physiology, University of Pennsylvania and Departments of Physiology and Surgery, Medical College of Pennsylvania, Philadelphia, Pa., USA.
J Vasc Res. 1998 Sep-Oct;35(5):310-7. doi: 10.1159/000025600.
Whole-cell, voltage clamp methods were used to study inward currents in human aortic smooth muscle cells in culture. Cells were plated on glass coverslips, cultured in supplemented M-199 media with 5% serum and studied as primary cells and at passages 2-5. Inward currents were measured with a pipette solution containing Cs+ and TEA+ to block K+ currents and with 2.5 mM [Ca2+]0 in the perfusate. Inward currents activated at about -50 mV, peaked at about -15 mV and reversed at about +30 mV. Values of peak inward current averaged 14. 7 +/- 3.3 pA/pF and cell capacitance averaged 124 +/- 10 pF (n = 35). These currents activated rapidly with a time-to-peak current of 2.4 +/- 0.3 ms at a test potential of -10 mV from a holding potential of -80 mV. The current also inactivated rapidly with a time course that could be described by two components with time constants of 1.8 +/- 0.2 and 17.8 +/- 3.5 ms at -10 mV. The currents decreased when extracellular Na+ was reduced and were completely inhibited by 50 nM tetrodotoxin (TTX), suggesting that they represented voltage-gated Na+ currents (INa). Activation curves were characterized with a V0.5 = -16.6 +/- 2.4 mV and a slope factor k = -5.2 +/- 0.2 mV while inactivation curves were characterized with a V0.5 = -60.9 +/- 1.7 mV and a slope factor k = 8.8 +/- 0.4 mV. Lowering external [Ca2+] to zero increased the maximum INa, shifted its voltage dependence in the hyperpolarizing direction and increased the rate of INa inactivation. Increasing external [Ca2+] or [Mg2+]decreased INa and slowed its rate of inactivation. These studies demonstrate the presence of voltage-gated Na+ channels with high TTX sensitivity that are modulated by extracellular divalent cations in human aortic smooth muscle cells maintained in cell culture. Window currents were found in the voltage range of -50 to -20 mV, suggesting that these channels could contribute to the resting membrane potential.
采用全细胞电压钳方法研究培养的人主动脉平滑肌细胞的内向电流。将细胞接种在玻璃盖玻片上，在添加了5%血清的M-199培养基中培养，并作为原代细胞以及传代2至5代的细胞进行研究。使用含有Cs⁺和TEA⁺的电极内液来阻断钾电流，并在灌流液中加入2.5 mM [Ca²⁺]₀来测量内向电流。内向电流在约-50 mV时激活，在约-15 mV时达到峰值，并在约+30 mV时反转。峰值内向电流平均值为14.7±3.3 pA/pF，细胞电容平均值为124±10 pF（n = 35）。在从-80 mV的钳制电位到-10 mV的测试电位下，这些电流在2.4±0.3 ms的时间内迅速激活至峰值电流。在-10 mV时，电流也迅速失活，其时间进程可用两个时间常数分别为1.8±0.2和17.8±3.5 ms的成分来描述。当细胞外Na⁺减少时电流降低，并且被50 nM河豚毒素（TTX）完全抑制，这表明它们代表电压门控钠电流（INa）。激活曲线的特征为V0.5 = -16.6±2.4 mV，斜率因子k = -5.2±0.2 mV，而失活曲线的特征为V0.5 = -60.9±1.7 mV，斜率因子k = 8.8±0.4 mV。将细胞外[Ca²⁺]降至零会增加最大INa，使其电压依赖性向超极化方向移动，并增加INa失活的速率。增加细胞外[Ca²⁺]或[Mg²⁺]会降低INa并减慢其失活速率。这些研究表明，在细胞培养条件下的人主动脉平滑肌细胞中存在对TTX高度敏感的电压门控钠通道，且这些通道受细胞外二价阳离子的调节。在-50至-20 mV的电压范围内发现了窗电流，这表明这些通道可能对静息膜电位有贡献。
