Membrane trafficking and PIP2‐dependent regulation of Kir2.2 by LPS in THP‐1 human monocyte (LB839)

Abstract

Lipopolysaccharide (LPS) is an effective agonist for Toll‐like receptor 4 (TLR4) in monocyte/macrophage. However, investigation on ion channels affected by LPS is not completely understood. In THP‐1 human monocyte cells, we observed decrease in voltage‐gated K+ channel current (51 %) and increase in Ca2+‐activated K+ channel (235 %) at 6 h after LPS application (1 μg/ml). Interestingly, marked increase of inwardly rectifying K+ channel (Kir) current was observed from 1‐2 h (IKir‐LPS), which peaked at 4 h and spontaneously decayed to 27 % of peak at 24 h. Single channel conductance of the IKir‐LPS was 38 pS, and their Ba2+‐sensitivity was relatively high (IC50, 1.42 μM), which were consistent with Kir2.2 isotype. Functional upregulation of IKir‐LPS was also found in primary human peripheral blood monocytes. While total Kir2.2 protein was not changed, its membrane fraction was increased time‐dependently and confocal microscopy confirmed the increased plasma membrane expression from 2 h. Also, IKir‐LPS was partially attenuated by Exo‐1 (vesicular trafficking inhibitor). However, the spontaneous decay of IKir‐LPS after 4 h was not prevented by dynamin inhibitor (Dynasore). Interestingly, the decayed IKir‐LPS at 24 h was rapidly reversed by PI3 kinase (PI3K) inhibitors (Wortmannin and LY294002). In contrast, IKir‐LPS was suppressed by bpV(Phen), an inhibitor for PTEN (PIP3 phosphatase). IKir‐LPS was not affected by Akt inhibitors, suggesting that the decay of IKir‐LPS was not due to PIP3‐dependent signaling but to decreased availability of PIP2 (conversion to PIP3 by PI3K) that is known to be critical for Kir2.2 activity. Store‐operated Ca2+ entry significantly increased in 4 h LPS treated THP‐1, which was prevented by KCl‐induced depolarization. Taken together, we firstly report that Kir2.2 is functionally upregulated by LPS via membrane trafficking in human monocytes. Subsequent hyperpolarization enhanced Ca2+ influx, implying a physiological role of K+ channels in the innate immunity.