Zeilhofer H U, Blank N M, Neuhuber W L, Swandulla D
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Erlangen, Germany.
Neuroscience. 2000;95(1):235-41. doi: 10.1016/s0306-4522(99)00434-0.
Dephosphorylation by the Ca2+/calmodulin-dependent phosphatase calcineurin has been suggested as an important mechanism of Ca2+-dependent inactivation of voltage-gated Ca2+ channels. We have tested whether calcineurin plays a role in the inactivation process of two types of high-voltage-activated Ca2+ channels (L and N type) widely expressed in the central nervous system, using the immunosuppressive drug FK506 (tacrolimus), which inhibits calcineurin after binding to intracellular FK506 binding proteins. Inactivation of L- and N-type Ca2+ channels was studied in a rat pituitary tumor cell line (GH3) and chicken dorsal root ganglion neurons, respectively. With the use of antisera directed against the calcineurin subunit B and the 12,000 mol. wt binding protein, we show that both proteins are present in the cytoplasm of GH3 cells and chicken dorsal root ganglion neurons. Ionic currents through voltage-gated Ca2+ channels were investigated in the perforated-patch and whole-cell configurations of the patch-clamp technique. The inactivation of L- as well as N-type Ca2+ currents could be well fitted with a bi-exponential function. Inactivation was largely reduced when Ba2+ substituted for extracellular Ca2+ or when the Ca2+ chelator EGTA was present intracellularly, indicating that both types of Ca2+ currents exhibited Ca2+-dependent inactivation. Extracellular (perforated-patch configuration) or intracellular (whole-cell configuration) application of FK506 to inactivate calcineurin had no effect on the amplitude and time-course of Ca2+ channel current inactivation of either L- or N-type Ca2+ channels. In addition, we found that recovery from inactivation and rundown of N-type Ca2+ channel currents were not affected by FK506. Our results provide direct evidence that the calcium-dependent enzyme calcineurin is not involved in the inactivation process of the two Ca2+ channel types which are important for neuronal functioning, such as gene expression and transmitter release.
钙调神经磷酸酶是一种依赖于Ca2+/钙调蛋白的磷酸酶，其去磷酸化作用被认为是电压门控Ca2+通道Ca2+依赖性失活的重要机制。我们使用免疫抑制药物FK506（他克莫司）来测试钙调神经磷酸酶是否在中枢神经系统中广泛表达的两种类型的高电压激活Ca2+通道（L型和N型）的失活过程中发挥作用，该药物在与细胞内FK506结合蛋白结合后会抑制钙调神经磷酸酶。分别在大鼠垂体肿瘤细胞系（GH3）和鸡背根神经节神经元中研究了L型和N型Ca2+通道的失活情况。通过使用针对钙调神经磷酸酶亚基B和12,000道尔顿结合蛋白的抗血清，我们发现这两种蛋白都存在于GH3细胞和鸡背根神经节神经元的细胞质中。通过膜片钳技术的穿孔膜片和全细胞配置研究了通过电压门控Ca2+通道的离子电流。L型和N型Ca2+电流的失活可以很好地用双指数函数拟合。当用Ba2+替代细胞外Ca2+或细胞内存在Ca2+螯合剂EGTA时，失活在很大程度上降低，这表明两种类型的Ca2+电流都表现出Ca2+依赖性失活。在穿孔膜片配置下细胞外应用或在全细胞配置下细胞内应用FK506来使钙调神经磷酸酶失活，对L型或N型Ca2+通道的Ca2+通道电流失活的幅度和时间进程均无影响。此外，我们发现N型Ca2+通道电流从失活中的恢复和衰减不受FK506的影响。我们的结果提供了直接证据，表明钙依赖性酶钙调神经磷酸酶不参与对神经元功能（如基因表达和递质释放）很重要的两种Ca2+通道类型的失活过程。
