Use Dependent Attenuation of Rat HCN1-Mediated i h in Intact HEK293 Cells

Abstract

Background/Aims: Cationic currents (I h ) through the fast activating and relatively cAMP insensitive subtype of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, HCN1, are limited by cytosolic factors in mammalian cells. This cytosolic HCN1 break is boosted by changes in membrane voltage that are not characterized on a biophysical level, yet. Methods: We overexpressed rat (r)HCN1 in human embryonic kidney cells (HEK293) and recorded pharmacologically isolated I h in cell-attached or whole-cell mode of the patch-clamp technique. Results: Recurring activation of rHCN1 reduced and slowed I h in intact HEK293 cells (cell-attached mode). On the contrary, sustained disruption of the intracellular content (whole-cell mode) ceased activity dependence and partially enables voltage dependent hysteresis. The activity induced I h attenuation in intact cells was independent of the main external cation, depended on the number of previous forced activations and was - at least in part - due to a shift in the voltage dependence of activation towards hyperpolarization as estimated by an adapted tail current analysis. Intracellular elevation of cAMP could not reverse the changes in I h . Conclusion: Reduction of rHCN1 mediated I h is use dependent and may involve the coupling of voltage sensor and pore.