Pancrazio J J, Ma W, Grant G M, Shaffer K M, Kao W Y, Liu Q Y, Manos P, Barker J L, Stenger D A
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375, USA.
J Neurobiol. 1999 Mar;38(4):466-74.
The whole-cell patch-clamp technique was used to assess the current carried by inwardly rectifying K+ channels (K(ir)) and the resting membrane potential (RMP) during long-term culture of NG108-15 cells. Culture of this cell line in serum-free medium triggers differentiation of a type I, neuron-like cell type followed by an eventual predominance of a type II, proliferative cell type. NG108-15 K(ir) currents, which strongly resemble currents carried by human ether-a-go-go related gene (HERG) K+ channels, exhibited significantly smaller current density for the more depolarized undifferentiated cells in growth media (GM) and type II cells compared to the neuron-like type I cells. Detailed examination of the transition from undifferentiated GM cells to type I cells revealed a shift in the voltage dependence of K(ir) activation which paralleled the more hyperpolarized RMP, neurite outgrowth, and biochemical differentiation characteristic of type I cells. Reverse-transcription polymerase chain reaction experiments using primers for the rat variant of HERG, RERG, revealed a a nearly twofold increase in RERG mRNA as cells differentiate from GM to type I, a finding entirely consistent with the increased K(ir) current density derived from patch-clamp recordings. Administration of CsCl(5 mM) blocked K(ir) currents and depolarized the RMP of type I cells. Furthermore, culture of NG108-15 cells in serum-free medium but with CsCl added significantly prevented neurite extension, an effect which was entirely reversible upon subsequent removal of CsCl. In contrast, other K+ channel inhibitors (4-aminopyridine and tetraethylammonium), at concentrations without marked effects on K(ir), failed to affect neurite extension. These results suggest an important role of the K(ir) channels in determining the RMP and triggering morphological differentiation of the cell line.
采用全细胞膜片钳技术评估内向整流钾通道（K(ir)）携带的电流以及NG108-15细胞长期培养过程中的静息膜电位（RMP）。将该细胞系培养于无血清培养基中会引发I型神经元样细胞类型的分化，随后最终以II型增殖细胞类型为主。NG108-15 K(ir)电流与人类醚 - 去极化相关基因（HERG）钾通道携带的电流非常相似，与生长培养基（GM）中未分化的去极化细胞和II型细胞相比，I型神经元样细胞的电流密度明显更小。对从未分化的GM细胞向I型细胞转变的详细检查显示，K(ir)激活的电压依赖性发生了变化，这与I型细胞更超极化的RMP、神经突生长和生化分化特征平行。使用针对HERG大鼠变体RERG的引物进行的逆转录聚合酶链反应实验表明，随着细胞从GM分化为I型，RERG mRNA增加了近两倍，这一发现与膜片钳记录得出的K(ir)电流密度增加完全一致。给予氯化铯（5 mM）可阻断K(ir)电流并使I型细胞的RMP去极化。此外，在无血清培养基中添加氯化铯培养NG108-15细胞可显著阻止神经突延伸，在随后去除氯化铯后，这种作用完全可逆。相比之下，其他钾通道抑制剂（4-氨基吡啶和四乙铵）在对K(ir)无明显影响的浓度下，未能影响神经突延伸。这些结果表明K(ir)通道在决定RMP和触发该细胞系的形态分化中起重要作用。
