Abstract
BackgroundMacrophages are important cells of the innate immune system and contribute to a variety of physiological and pathophysiological responses. Monovalent and divalent ion channels have been studied in macrophage function, and while much research is still required, a role for these channels is beginning to emerge in macrophages. In addition to the plasma membrane, ion channels are also found in intracellular membranes including mitochondrial, lysosomal and nuclear membranes. While studying the function of plasma membrane located large conductance voltage- and calcium-activated potassium channels (BK channels) in a macrophage cell line RAW264.7, we became aware of the expression of these ion channels in other cellular locations.MethodsImmunofluorescence and Western blot analysis were used to identify the expression of BK channels. To demonstrate a functional role for the nuclear located channel, we investigated the effect of the lipid soluble BK channel inhibitor paxilline on CREB phosphorylation.ResultsTreatment of resting macrophages with paxilline resulted in increased CREB phosphorylation. To confirm a role for nuclear BK channels, these experiments were repeated in isolated nuclei and similar results were found. Ca2+ and calmodulin-dependent kinases (CaMK) have been demonstrated to regulate CREB phosphorylation. Inhibition of CaMKII and CaMKIV resulted in the reversal of paxilline-induced CREB phosphorylation.ConclusionsThese results suggest that nuclear BK channels regulate CREB phosphorylation in macrophages. Nuclear located ion channels may therefore be part of novel signalling pathways in macrophages and should be taken into account when studying the role of ion channels in these and other cells.Graphic abstract
Highlights
Large conductance voltage- and C a2+-activated potassium channels (BK channels) are characterized by a large single channel conductance (~ 100–300pS) and high sensitivity to intracellular Ca2+ concentration [1]
It was noted that Western blot analysis of whole cell lysates resulted in the expression of a protein band doublet for the BK channel α-subunit while in nuclear preparations, the α-subunit was seen as a single protein band (Fig. 1a)
To exclude the possibility that BK channel expression in the nuclear lysates was due to contamination, lysates were analysed for cytochrome c oxidase subunit IV (COX IV), a mitochondrial marker; GAPDH, a cytoplasmic marker; and calnexin, an endoplasmic reticulum membrane marker
Summary
Large conductance voltage- and C a2+-activated potassium channels (BK channels) are characterized by a large single channel conductance (~ 100–300pS) and high sensitivity to intracellular Ca2+ concentration [1]. In addition to the pore-gate domain, each α-subunit contains a voltage sensing domain and C a2+ and M g2+ binding domain These α-subunit domains are responsible for the channel opening in response to a rise in intracellular Ca2+ and/or membrane voltage [3,4,5]. While studying the function of plasma membrane located large conductance voltage- and calcium-activated potassium channels (BK channels) in a macrophage cell line RAW264.7, we became aware of the expression of these ion channels in other cellular locations. To demonstrate a functional role for the nuclear located channel, we investigated the effect of the lipid soluble BK channel inhibitor paxilline on CREB phosphorylation. Conclusions These results suggest that nuclear BK channels regulate CREB phosphorylation in macrophages. Nuclear located ion channels may be part of novel signalling pathways in macrophages and should be taken into account when studying the role of ion channels in these and other cells
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