Heterologous expression of a glial Kir channel (KCNJ10) in a motoneuron cell line: a novel candidate for neuronal silencing?

Introduction: Reducing the excitability of specific neurons will provide insight into global neuronal functions as well as offer novel approaches to protect neurons from excess neuronal activity. Inwardly-rectifying potassium (Kir) channels contribute to the resting membrane potential (RMP) of neurons and glial cells and thereby regulate firing activity, transmembrane gradients of transmitter molecules and energy state of the cell. The Kir4.1 subunit (KCNJ10) is expressed predominantly in glial cells of the CNS. Genetic inactivation of Kir4.1 in mice has revealed a prominent role in establishing and maintaining the RMP of astrocytes and oligodendrocytes.

Methods: We investigated a putative role of Kir4.1 in reducing neuronal excitability by plasmid transfection of motoneurons with green-fluorescent protein (EGFP)-tagged Kir4.1. The NSC34 cell line, a motoneuron-like cell line was transiently transfected with EGFP/Kir4.1. Original NSC34 cells, EGFP-transfected control cells and Kir4.1-transfected cells were voltage-clamped in whole-cell mode. RMP, K+ current, K+ conductance and Na+ inward currents were studied. Cell death was determined by counting DAPI-labeled apoptotic nuclei under control and experimental conditions.

Results: Transient overexpression of Kir4.1 channels in motoneurons did neither induce obvious morphological changes nor increased cell death. In Kir4.1 transfected motoneurons (n=13), RMP was significantly hyperpolarized by ˜30 mV compared to non-transfected (n=12) and EGFP-transfected controls (n=12). Furthermore, upon change of extracellular K+ to 50 mmol, Ba2±blockable K+ currents were significantly higher than in controls. Na+ inward currents that were observed in all NSC34 control cells (n=24) upon depolarization, were not detected in Kir4.1 transfected motoneurons (0/13 cells).

Conclusion: Our results demonstrate that transfection of a predominantly glial Kir channel in motoneurons leads to functional Kir channel expression and modifies neuronal electrophysiological characteristics. Kir4.1 channels significantly hyperpolarize motoneurons, increase K+ current density and reduce Na+ influx upon depolarization without affecting neuronal survival. This study demonstrates the feasibility of using glia-specific Kir channels to modulate neuronal activity in an in vitro model.