Rise and fall of Kir2.2 current by TLR4 signaling in human monocytes: PKC-dependent trafficking and PI3K-mediated PIP2 decrease

Abstract

LPSs are widely used to stimulate TLR4, but their effects on ion channels in immune cells are poorly known. In THP-1 cells and human blood monocytes treated with LPS, inwardly rectifying K+ channel current (I-Kir,I-LPS) newly emerged at 1 h, peaked at 4 h (-119 +/- 8.6 pA/pF), and decayed afterward (-32 +/- 6.7 pA/pF at 24 h). Whereas both the Kir2.1 and Kir2.2 mRNAs and proteins were observed, single-channel conductance (38 pS) of I-Kir,(LPS) and small interfering RNA-induced knockdown commonly indicated Kir2.2 than Kir2.1. LPS-induced cytokine release and store-operated Ca2+ entry were commonly decreased by ML-133, a Kir2 inhibitor. Immunoblot, confocal microscopy, and the effects of vesicular trafficking inhibitors commonly suggested plasma membrane translocation of Kir2.2 by LPS. Both I-Kir,I-LPS and membrane translocation of Kir2.2 were inhibited by GF109203X (protein kinase C [PKC] inhibitor) or by transfection with small interfering RNA-specific PKC epsilon. Interestingly, pharmacological activation of PKC by PMA induced both Kir2.1 and Kir2.2 currents. The spontaneously decayed I-Kir,I-LPS at 24 h was recovered by PI3K inhibitors but further suppressed by an inhibitor of phosphatidylinositol(3,4,5)-trisphosphate (PIP3) phosphatase (phosphatase and tensin homolog). However, I-Kir,I-LPS at 24 h was not affected by Akt inhibitors, suggesting that the decreased phosphatidylinositol(4,5)-bisphosphate availability, that is, conversion into PIP3 by PI3K, per se accounts for the decay of I-Kir,I-LPS. Taken together, to our knowledge these data are the first demonstrations that I-Kir is newly induced by TLR4 stimulation via PKC-dependent membrane trafficking of Kir2.2, and that conversion of phosphatidylinositol(4,5)-bisphosphate to PIP3 modulates Kir2.2. The augmentation of Ca2+ influx and cytokine release suggests a physiological role for Kir2.2 in TLR4-stimulated monocytes.