Protein Kinase C Activation Inhibits Rat and Human Hyperpolarization Activated Cyclic Nucleotide Gated Channel (HCN)1-Mediated Current in Mammalian Cells

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 I-h before and during the application of the membrane permeable non-metabolizable PKC-activator 4 beta PMA in cell-attached mode of the patch-clamp technique, leaving the intracellular environment intact. Results: 4 beta PMA 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 4 beta PMA induced Ih changes. Disrupting the intracellular environment by recording in whole-cell mode drastically reduced the 4 beta PMA 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. Copyright (C) 2013 S. Karger AG, Basel