Protein Kinase C Activation Inhibits Rat and Human Hyperpolarization Activated Cyclic Nucleotide Gated Channel (HCN)1 - Mediated Current in Mammalian Cells
Protein Kinase C Activation Inhibits Rat and Human Hyperpolarization Activated Cyclic Nucleotide Gated Channel (HCN)1 - Mediated Current in Mammalian Cells
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DOI：
10.1159/000350074
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发表时间：
2013-04
影响因子：
--
通讯作者：
Olivia Reetz;U. Strauss
中科院分区：
文献类型：
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作者：
Olivia Reetz;U. Strauss
背景/目的：超极化激活的环核苷酸门控1（HCN 1）通道决定了几个脑区的神经元兴奋性。与HCN 2和HCN 4相比，HCN 1对cAMP不太敏感，并且其他已知调节剂的数量有限。其中之一，蛋白激酶C（PKC），表现出相反的效果，在爪蟾卵母细胞中表达的小鼠HCN 1通道。研究方法：为了研究PKC对哺乳动物环境中HCN 1介导电流的影响，我们在人胚肾（HEK 293）细胞中表达大鼠HCN 1或人HCN 1，在鼠神经母细胞瘤（N1 E-115）细胞中表达大鼠HCN 1。我们记录了在膜片钳技术的细胞贴附模式下应用膜渗透性非代谢性PKC激活剂4βPMA之前和期间产生的Ih，使细胞内环境保持完整。结果：4βPMA使HCN 1介导的最大电流降低约60- 70%，并使其激活减慢，但其电压敏感性不变。该效应既不是由于种属相关差异，也不限于HEK 293细胞，因为HEK 293中的人和大鼠HCN 1以及N1 E-115细胞中的大鼠HCN 1具有可比性。PKC阻断剂GF 109203 X可阻断4βPMA诱导的Ih变化。通过以全细胞模式记录来破坏细胞内环境显著降低了4βPMA效应。结论：如果细胞内内容物保持完整，则PKC活化减少并减缓用大鼠或人HCN 1转染的非神经元和神经元哺乳动物细胞中的Ih。
Background/Aims: Hyperpolarization activated cyclic nucleotide gated 1 (HCN1) channels determine neuronal excitability in several brain regions. In contrast to HCN2 and HCN4, HCN1 is less sensitive to cAMP and the number of other known modulators is limited. One of those, the protein kinase C (PKC), showed opposing effects on mouse HCN1 channels expressed in Xenopus oocytes. Methods: In order to study PKC effects on HCN1 mediated currents in a mammalian environment we expressed rat HCN1 or human HCN1 in human embryonic kidney (HEK293) cells and rat HCN1 in murine neuroblastoma (N1E-115) cells. We recorded the resulting Ih before and during the application of the membrane permeable non-metabolizable PKC-activator 4βPMA in cell-attached mode of the patch-clamp technique, leaving the intracellular environment intact. Results: 4βPMA reduced maximal HCN1 mediated currents to about 60-70 % and slowed its activation, but left its voltage sensitivity unchanged. The effect was neither due to species-related differences nor restricted to HEK293 cells, because it was comparable for human and rat HCN1 in HEK293 and for rat HCN1 in N1E-115 cells. However, pre-treatment with the PKC blocker GF109203X abolished 4βPMA induced Ih changes. Disrupting the intracellular environment by recording in whole-cell mode drastically reduced the 4βPMA effect. Conclusion: PKC activation reduces and slows Ih in non-neuronal and neuronal mammalian cells transfected with rat or human HCN1 if the intracellular content remains intact.