Universität Tübingen
Regulation of Potassium Channels through mTOR and PDK1 in Dendritic and Mast Cells
Abstract
Dendritic cells are antigen-presenting cells, central for the development of optimal T cell immunity, that are able to initiate primary immune responses and to establish immunological memory. Mast cells are tissue-based effector cells in allergic diseases, playing a central role in the propagation of IgE-dependent allergic reactions, such as allergic rhinitis, asthma, anaphylaxis and delayed hypersensitivity reactions. Upon stimulation of IgE receptors , mast cells release granules containing several mediators including histamine and cytokines, which regulate responses of other inflammatory cells. Dendritic and mast cell functions are regulated by phosphatidylinositol-3 (PI3) kinase signalling pathway.. The PI3 kinase is partially effective in dendritic and mast cells through alteration of their ion channel activity. Both PI3 kinase on the one hand and ion channels on the other hand are important for the regulation of mast and dendritic cell functions. However, little is known about downstream elements of the PI3 kinase that regulate ion channels in those cells. In the present project the question was addressed whether two PI3 kinase downstream targets, phosphoinositide-dependent kinase 1 (PDK1) and mammalian target of rapamycine (mTOR), regulate ion channels and ion channel-dependent functions in dendritic and mast cell. We showed that treatment of dendritic cells with rapamycine, the mTOR inhibitor, led to inhibition of the currents through voltage-gated K+ channels, which in dendritic cells belong to Kv 1.3 and Kv 1.5 families. Analysis of the time constants of activation and inactivation demonstrated that rapamycin caused faster Kv channel inactivation. To test the hypothesis that Kv1.3 and/or Kv1.5 channels could be regulated by mTOR, cRNA encoding Kv1.3 or Kv1.5 was injected into Xenopus oocytes with or without additional injection of cRNA encoding mTOR. The Kv1.3 and Kv1.5 currents were significantly increased by additional coexpression of mTOR, an effect abolished by rapamycin. Analysis of activation and inactivation time constants of Kv1.3 and Kv1.5 revealed that mTOR affected tau activation and tau inactivation of Kv1.3, but not of Kv1.5. Coexpression with mTOR resulted in a decreased tau activation and an increased tau inactivation of Kv1.3, suggesting that mTOR causes faster Kv1.3 channel activation and slower Kv1.3 channel inactivation. We were interested to analyze whether rapamycin has also effects on non-voltage gated K+ channels. We performed experiments in mouse bone marrow-derived mast cells (BMMCs), which are known to express Ca2+-activated K+ channels KCa3.1. Our observations demonstrated that though rapamycine did not influence KCa3.1 channel activation directly, it impaired antigen-dependent increase of cytosolic Ca2+ in BMMCs and that secondary led to the blunted activation of the KCa3.1 channels. In BMMC we also studied the effect of another kinase of the PI3 kinase pathway, PDK1, on ion channels and cell functions. The present study demonstrated that the stimulation of Ca2+ entry, but not Ca2+ release from intracellular stores, following exposure to antigen was significantly less pronounced in mast cells from PDK1 hypomorphic mice (pdk1hm) than in mast cells from their wild type littermates (pdk1wt). PDK1 deficiency thus blunted Ca2+ entry and consequently indirectly also the currents through Ca2+-activated K+ channels KCa3.1. Partial PDK1 deficiency did not abrogate degranulation despite the reduced Ca2+ entry in pdk1hm BMMCs. Both ß-hexosaminidase release and histamine release were not significantly different between pdk1wt and pdk1hm BMMCs. We provided a possible mechanism explaining unaffected degranulation in pdk1hm BMMCs by showing that PDK1 led to the activation of two downstream kinases, SGK1 and PKCdelta;, positive and negative regulators of mast cell degranulation, respectively.
Author and committee
dc:creator, dc:contributor.*- Author
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- Tyan, Leonid
Identifiers
dc:identifier.*- Identifier
- hdl:10900/49719