Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels

Nematocytes, the stinging cells of Cnidarians, have a cytoplasm confined to a thin rim. The main cell body is occupied by an organoid, the nematocyst, containing the stinging tubule and venom. Exposed to hypotonic shock, nematocytes initially swell during an osmotic phase (OP) and then undergo regulatory volume decrease (RVD) driven by K⁺, Cl^- and obligatory water extrusion mechanisms. The purpose of this report is to characterize the OP. Nematocytes were isolated by the NaSCN/Ca²⁺ method from tentacles of the jellyfish Pelagia noctiluca, collected in the Strait of Messina, Italy. Isolated nematocytes were subjected to hyposmotic shock in 65% artificial seawater (ASW) for 15 min. The selective aquaporin water channel inhibitor HgCl₂ (0.1-25 µM) applied prior to osmotic shock prevented the OP and thus RVD. These effects were attenuated in the presence of 1mM dithiothreitol (DTT), a mercaptide bond reducing agent. AgNO₃ (1 µM) and TEA (tetraethylammonium, 100 µM), also reported to inhibit water transport, did not alter the OP but significantly diminished RVD, suggesting different modes of action for the inhibitors tested. Based on estimates of the nematocyte surface area and volume, and OP duration, a relative water permeability of ∼10^-7 cm/sec was calculated and the number of putative aquaporin molecules mediating the OP was estimated. This water permeability is 3-4 orders of magnitude lower in comparison to higher order animals and may constitute an evolutionary advantage for Cnidarian survival.

Section: Introductionmentioning

confidence: 99%

Evidence for Aquaporin-mediated Water Transport in Nematocytes of the Jellyfish <i>Pelagia noctiluca</i>

Marino

Morabito

Spada

et al. 2011

“…Therefore there were no changes in the extracellular ion concentration during hypotonic treatment only in osmolarity. This is different from the condition used by Bobak et al [18] and Andronic et al [23] where hypotonic solutions also had a decreased ion concentration, the possible importance of this difference has not been tested.…”

Section: Discussionmentioning

confidence: 80%

“…Reverse transcriptase PCR with SuperScript II (Invitrogen) and oligo(dT) [12][13][14][15][16][17][18] primer (Invitrogen) was used to generate cDNA from the purified mRNA and performed on a Eppendorf Mastercycler as previously described [24]. QPCR was performed using a Stratagene MX4000 real-time PCR system, Brilliant ® II SYBR ® Green QPCR Master Mix (Stratagene, Agilent Technologies) and the following primers: hKCNK5 forward: 5'-ACCACCCACTCATCTTCCAG-3', hKCNK5 reverse: 5'-AGTGCTGGTGAAGGTGGACT-3', hKv1.3 forward: 5'-CACTTCAGGTTTCAGCAGCA-3', hKv1.3 reverse: 5'-TGTCTCCCGGTGGTAGAAGT-3', hKCa3.1 forward: 5'-ACTGGGCACCTTTCAGACAC-3' and KCa3.1 reverse: 5'-ACGTGCTTCTCTGCCTTGTT-3'.…”

Section: Solution and Materialsmentioning

confidence: 99%

“…KCNK5 has previously been shown to be volume sensitive and to be implicated in the volume regulatory process of RVD in response to cell swelling in various cell types and tissues including Ehrlich Ascites Tumor (EAT) cells [13,14], mouse proximal tubules [15], human and murine spermatozoa [16,17] and murine [18] and human T lymphocytes (1). For a recent review on KCNK5 see [19].…”

mentioning

confidence: 99%

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The Volume Activated Potassium Channel KCNK5 is Up-Regulated in Activated Human T Cells, but Volume Regulation is Impaired

Kirkegaard

Strøm²,

Gammeltoft³

et al. 2016

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.

“…In EAT cells and Ehrlich lettré ascites (ELA) cells the volume sensitive channels are the two-pore domain K + channel KCNK5 (also known as TASK-2 - TWIK-related Acid-Sensitive K + channel 2 or K 2P 5.1) [5,6,7], and the volume regulated anion channel (VRAC, I Cl,vol ) [8]. KCNK5 has, besides in Ehrlich cells [5,6,7], also been shown to be involved in RVD in other cell types including mouse proximal tubules [9], T lymphocytes [10,11], murine spermatozoa [12] and retinal glial cells [13]. It has previously been shown that the rate limiting factor in RVD in EAT cells is the volume sensitive K + efflux and thus the efflux through the KCNK5 channel [14], making an altered RVD response very likely to be due to changes related to this channel.…”

Section: Introductionmentioning

confidence: 99%

KCNK5 is Functionally Down-Regulated Upon Long-Term Hypotonicity in Ehrlich Ascites Tumor Cells

Kirkegaard

Wulff

Gammeltoft

et al. 2013

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.