Kirkegaard SS, Lambert IH, Gammeltoft S, Hoffmann EK. Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation. Am J Physiol Cell Physiol 299: C844 -C853, 2010. First published July 14, 2010; doi:10.1152/ajpcell.00024.2010.-The swelling-activated K ϩ currents (I K,vol ) in Ehrlich ascites tumor cells (EATC) has been reported to be through the two-pore domain (K2p), TWIK-related acid-sensitive K ϩ channel 2 (TASK-2). The regulatory volume decrease (RVD), following hypotonic exposure in EATC, is rate limited by IK,vol indicating that inhibition of RVD reflects inhibition of TASK-2. We find that in EATC the tyrosine kinase inhibitor genistein inhibits RVD by 90%, and that the tyrosine phosphatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) [mpV(pic)] shifted the volume set point for inactivation of the channel to a lower cell volume. Swelling-activated K ϩ efflux was impaired by genistein and the Src kinase family inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl) pyrazolo [3,4-d]pyrimidine (PP2) and enhanced by the tyrosine phosphatase inhibitor mpV(pic). With the use of the TASK-2 inhibitor clofilium, it is demonstrated that mpV(pic) increased the volumesensitive part of the K ϩ efflux 1.3 times. To exclude K ϩ efflux via a KCl cotransporter, cellular Cl Ϫ was substituted with NO 3 Ϫ . Also under these conditions K ϩ efflux was completely blocked by genistein. Thus tyrosine kinases seem to be involved in the activation of the volumesensitive K ϩ channel, whereas tyrosine phosphatases appears to be involved in inactivation of the channel. Overexpressing TASK-2 in human embryonic kidney (HEK)-293 cells increased the RVD rate and reduced the volume set point. TASK-2 has tyrosine sites, and precipitation of TASK-2 together with Western blotting and antibodies against phosphotyrosines revealed a cell swelling-induced, timedependent tyrosine phosphorylation of the channel. Even though we found an inhibiting effect of PP2 on RVD, neither Src nor the focal adhesion kinase (FAK) seem to be involved. Inhibitors of the epidermal growth factor receptor tyrosine kinases had no effect on RVD, whereas the Janus kinase (JAK) inhibitor cucurbitacin inhibited the RVD by 40%. It is suggested that the cytokine receptor-coupled JAK/STAT pathway is upstream of the swelling-induced phosphorylation and activation of TASK-2 in EATC.cell volume regulation; Janus kinase; volume-sensitive channels

Background/Aims: Regulatory volume decrease (RVD) in response to acute cell swelling is well described and KCNK5 (also known as TASK-2 or K_2P5.1) has been shown to be the volume sensitive K⁺ channel in Ehrlich cells. Very little is, on the other hand, known about the effects of long-term hypotonicity on expression and function of KCNK5, thus we have investigated the effect of long-term hypotonicity (24h - 48h) on KCNK5 in Ehrlich cells on the mRNA, protein and physiological levels. Methods: Physiological effects of long-term hypotonicity were measured using patch-clamp and Coulter counter techniques. Expression patterns of KCNK5 on mRNA and protein levels were established using real-time qPCR and western blotting respectively. Results: The maximum swelling-activated current through KCNK5 was significantly decreased upon 48h of hypotonicity and likewise the RVD response was significantly impaired after both 24 and 48h of hypotonic stimulation. No significant differences in the KCNK5 mRNA expression patterns between control and stimulated cells were observed, but a significant decrease in the KCNK5 protein level 48h after stimulation was found. Conclusion: The data suggest that the strong physiological impairment of KCNK5 in Ehrlich cells after long-term hypotonic stimulation is predominantly due to down-regulation of the KCNK5 protein synthesis.

Background/Aims: The potential role of the two-pore domain potassium channel KCNK5 (also known as TASK-2 and K_2P5.1) in activated T cell physiology has only recently been described. So far KCNK5 has been described to be up-regulated in T cells in multiple sclerosis patients and to be implicated in the volume regulatory mechanism regulatory volume decrease (RVD) in T cells. Methods: We investigated the time-dependent expression pattern of KCNK5 in CD3/CD28 activated human T cells using qPCR and Western blotting and its role in RVD using a Coulter Counter. Results: KCNK5 is highly up-regulated in CD3/CD28 activated T cells both at mRNA (after 24 h) and protein level (72 and 144 h), but despite this up-regulation the RVD response is inhibited. Furthermore, the swelling-activated Cl^- permeability in activated T cells is strongly decreased, and the RVD inhibition is predominantly due to the decreased Cl^- permeability. Conclusion: The up-regulated KCNK5 in activated human T cells does not play a volume regulatory role, due to decreased Cl^- permeability. We speculate that the KCNK5 up-regulation might play a role in hyperpolarization of the cell membrane leading to increased Ca²⁺ influx and proliferation of T cells.

The two‐pore domain potassium channel KCNK5 has been shown to play a role in activation of T cells from multiple sclerosis patients1. KCNK5 is also known to be important in the regulatory volume decrease (RVD) following cell swelling2. Our aim with the study is to elucidate a potential role for KCNK5 in T cell activation.We use T cells purified from human peripheral blood mononuclear cells. Real‐time PCR and Western blotting was used to investigate mRNA and protein levels, respectively of the three potassium channels Kv1.3, KCa3.1 and KCNK5 in non‐activated and CD3/CD28 activated T cells. Furthermore volume measurements using a coulter counter were used to study RVD. We find a strong up‐regulation of KCNK5 on protein and mRNA levels in activated T cells compared to the non‐activated cells. Surprisingly, despite this up‐regulation of the potent volume regulator KCNK5 activated T cells were larger and volume regulated less efficiently than nonactivated cells. The exact role for KCNK5 in activated T cells will be examined in future studies of the increase in membrane potential expected from KCNK5 up‐regulation.