FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN
KCNK5 in Physiology and Pathophysiology
- Focus on Cell Volume Control
PhD Thesis
Signe Skyum Kirkegaard Petersen
Academic advisors: Professor Else Kay Hoffmann & Dr.med. Steen Gammeltoft
Submitted: September 30th 2013 FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN
KCNK5 in Physiology and Pathophysiology
- Focus on Cell Volume Control
PhD Thesis By Signe Skyum Kirkegaard Petersen
Section for Cell and Developmental Biology Department of Biology Faculty of Science University of Copenhagen
Author: Signe Skyum Kirkegaard Petersen
Thesis title: KCNK5 in Physiology and Pathophysiology – Focus on Cell Volume Control
Academic advisors:
Professor Else Kay Hoffmann Section for Cell and Developmental Biology Department of Biology University of Copenhagen Denmark
Dr.med. Steen Gammeltoft Sleep Research Unit Diagnostic Department Glostrup Hospital Denmark
Submitted: September 30th 2013
Copenhagen, sep. 2013
Preface and acknowledgements This PhD thesis will focus on the potassium channel KCNK5 and its role in different cell types i.e. the cell lines Ehrlich Ascites Tumor cells and Ehrlich Lettré Ascites cells and in human primary T cells. More precisely I have studied the involvement of protein tyrosine kinases in swelling- induced activation of the channel, the long-term effects of hypotonicity on channel physiology and expression pattern and last but not least I have looked into its role in T cells.
The thesis is based on the following papers:
Paper I: Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation Signe Skyum Kirkegaard, Ian Henry Lambert, Steen Gammeltoft and Else Kay Hoffmann Am J Physiol Cell Physiol 299: C844–C853, 2010
Paper II: KCNK5 is functionally down-regulated upon long-term hypotonicity in Ehrlich ascites tumor cells Signe Skyum Kirkegaard, Tune Wulff, Steen Gammeltoft and Else Kay Hoffmann Submitted to “Cellular Physiology and Biochemistry”. Status 09-30-2013: The paper has been found in “principle acceptable for publication”.
Paper III: The potential role of KCNK5 in activated T cell physiology with specific focus on cell volume control Signe Skyum Kirkegaard, Pernille Dyhl Strøm, Anker Jon Hansen, Steen Gammeltoft and Else Kay Hoffmann Paper in preparation.
Through this thesis I will touch upon various themes such as the activation of the KCNK5 channel upon acute cell swelling, the physiological function and expression of the channel upon long- term hypotonicity and on its potential role in activated T cell physiology. I thus used different cells and cell systems in order to investigate these topics - a fact that will be reflected in this thesis, where the introductory part of the thesis will deal with a variety of quite different topics. I will describe how the immune system works, how a cell can regulate its volume and hereunder various sub-topics, what is known about the KCNK5 channel and about potassium channels in general. With relative many large topics to describe some details will not be addressed, thus the reader might find some topics left unmentioned but hopefully this will not cloud the greater overview, issues and facts presented here.
During my time as a PhD student at Section for Cell and Developmental Biology at the University of Copenhagen and at the Department of Clinical Biochemistry, Glostrup Hospital, Denmark I have had the pleasure of guidance from my two supervisors, Else Kay Hoffmann and Steen Gammeltoft. I have had great scientific challenges and have learned a lot about research, project
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management, teaching and about myself as a person. With this in mind I cannot begin to express my gratitude towards my two supervisors, but I will try.
First of all I would like to thank Professor Else K. Hoffmann for everything! – for giving me a chance in her laboratory, first as a master student and later on as a PhD student, for sharing her extensive expertise on the area of cell volume, ion flux and on physiology in general. On a more personal level I would like to thank her for always having time to talk, listen and give advice. One thing in particular also comes to mind when thinking of the opportunities given to me by Else - I have attended a great deal of meetings and conferences and have met so many interesting scientists (probably more than most other PhD student), which is due to the fact that conference attendance and networking is highly prioritized by Else. You are indeed a very social person Else, and that fact has always had a huge impact on the great working environment in the lab. During the last couple of years we have visited Slovenia, USA, Canada and Japan besides the many meetings in Denmark. I have enjoyed every chance of presenting my work and have found the participation in all the conferences and meetings very beneficial. Thank you so much!
I am also extremely grateful to Dr. Steen Gammeltoft for giving me the opportunity to be a part of his group, first as a master student, since as a research assistant and last but not least as a PhD student. The project might never have been without his financial and scientific support. I would also like to thank Dr. Ian Lambert for always having his door open for me, whenever I needed advise or practical help and for including me in the little group of “Friends of the Coulter counter”.
I would very much like to express my gratitude to Dr. Anker Jon Hansen from Novo Nordisk, for giving me a chance to work in his lab, for giving me the opportunity to see how the industry works and for challenging my knowledge and ability to explain my work and ideas. I have very much appreciated his comments and thoughts.
A special thank should also be given to technicians Birthe Juul Hansen, Pia Birn, Dothe Nielsen and Birte Kofoed for their most appreciated help and friendship.
Last but not least I would like to thank friends and family for their everlasting support and patience, especially I would like to thank my husband Mikkel, for whom without I would not have been able to do this. I would also like to thank my son Oskar for making me learn how to get the most out of, the now more limited time, I could spend at work.
Signe Skyum Kirkegaard Petersen
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Abbreviations
2+ 2+ [Ca ]i Intracellular Ca concentration PCR Polymerase chain reaction
AA Arachidonic azid PIP2 Phosphatidylinositol 4,5-biphosphate APC Antigen presenting cell PLCγ Phospholipase C-γ AVD Apoptotic volume decrease PTK Protein tyrosine kinase B cell B lymphocyte (“B” from bursa of RA Rheumatoid arthritis Fabricius or bone marrow) RT-PCR Reverse transcription polymerase BCR B cell receptor chain reaction cDNA cyclic ADP-ribose RVD Regulatory volume decrease cADPr Complementary DNA RVI Regulatory volume increase CNS Central nervous system STIM Stromal interacting molecule 2+ 2+ CRAC Ca release-activated Ca channel T cell T lymphocyte (“T” from thymus) + ChTX Charybdotoxin TALK TWIK-related alkaline pH activated K channel DAG Diacylglycerol TWIK-related acid-sensitive K+ EAT cells Ehrlich ascites tumor cells TASK channel ELA cells Ehrlich lettré ascites cells TCR T cell receptor ER Endoplasmatic reticulum TEA Tetraethylammonium FAK Focal adhesion kinase THIK Tandem pore domain halothane- FLAP 5-LOX activating protein inhibited K+ channel
fMLP N-formyl-methionine-leucine- TM Transmembrane phenylalanine TRAAK TWIK-related arachidonic acid- - ICl, vol Swelling activated Cl current stimulated K+ channel
IK Intermediate conductance TREK TWIK-related K+ channel + IK, vol Swelling activated K current TWIK Tandem of p domains in a weak + IP inwardly rectifying K channel 3 1,4,5-inositol triphosphate JAK Janus kinase VRAC Volume-regulated anion channel + K2P Two-pore domain K channel qPCR Real-Time quantitative PCR KCa3.1 Ca2+-activated K+ channel 3.1 KCNK5 Two-pore domain channel 5.1 Kv1.3 + Voltage-gated K channel 1.3
LTC4 Leukotriene C4
LTD4 Leukotriene D4 MHC Major histocombatibility complex mRNA Messenger RNA NFAT Nuclear factor of activated T cells
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Indhold
Preface and acknowledgements ...... 1 Abbreviations ...... 3 Purpose ...... 6 Abstract ...... 7 Resumé ...... 8 1. Introduction ...... 9 1.2 K+ channels in general ...... 9 1.2.1 Potassium channel grouping by topology ...... 10 1.2.2 The 2TM and 6TM families...... 10 1.2.3 The 4TM family – the two pore-domain potassium channels ...... 11 1.3 KCNK5 – a two-pore domain potassium channel ...... 13 1.3.1 KCNK5 history and tissue distribution ...... 14 1.3.2 KCNK5 characteristics ...... 14 1.3.3 KCNK5 activation and gating ...... 16 1.3.4 KCNK5 in lymphocytes ...... 16 1.4 Cellular volume ...... 17 1.4.1 Cell volume regulation ...... 17 1.4.1.1 Pathophysiological conditions ...... 18 1.4.1.2 Regulatory mechanisms in response to volume changes ...... 18 1.4.1.3 The volume set-point ...... 19 1.4.1.4 RVD...... 19 1.4.1.5 K+ channels in volume regulation ...... 20 1.4.1.6 Volume regulation in Ehrlich cells ...... 20 1.4.1.7 Long-term effects of anisotonicity ...... 21 1.4.1.8 Volume regulation in lymphocytes ...... 22 1.4.2 When changes in cell volume act as signals ...... 23 1.4.2.1 Programmed cell death ...... 23 1.4.2.2 Proliferation ...... 24 1.4.2.3 Transepithelial transport ...... 24 1.4.2.4 Migration and invasion ...... 24 1.5 The immune system ...... 26
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1.5.1 B and T lymphocytes ...... 26 1.5.2 T cell activation ...... 27 1.5.2.1 T cell subtypes ...... 27 1.5.3 When the immune system attacks the body ...... 29 1.5.3 Ion channels in T cell activation ...... 29 1.5.3.1 Ca2+ signaling ...... 29 1.5.3.2 K+ channels in Ca2+ signaling ...... 30 1.5.3.3 Other ion channels described in T cells ...... 31 1.5.4 T cell proliferation ...... 32 1.5.4.1 K+ channels in proliferation ...... 32 2 Summary and conclusions ...... 34 3 Experimental procedures ...... 35 3.1 cDNA quantification instead of reference genes ...... 35 3.2 T cell subtypes ...... 35 References ...... 36 4 Paper I ...... 51 5 Paper II ...... 63 6 Paper III ...... 83 7 Final thesis discussion, future work and perspectives ...... 103 8 Appendix ...... 108
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Purpose
The purpose of this thesis can be divided into three sub-purposes all centered around the two pore-domain potassium channel KCNK5 (or TASK-2).
1) To study the potential role of protein tyrosine kinases in the swelling-mediated KCNK5 activation in Ehrlich Ascites Tumor (EAT) and Ehrlich Lettré Ascites (ELA) cells
2) To study the long-term effect of hypotonicity on KCNK5 physiology and expression in EAT and ELA cells
3) To study the expression pattern and potential role of KCNK5 in activated human T cell physiology
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Abstract
The KCNK5 potassium channel (also known as TASK-2 or K2p5.1) belongs to the latest discovered potassium channel family - the two-pore domain potassium channels. KCNK5 was first described in 1998 by Lazdunski and co-workers and has since been given a role in e.g. volume regulation, and recent been proposed to play a role in T cell activation and in multiple sclerosis.
When a cell experiences swelling do to osmotic changes in the extracellular or intracellular environment most mammalian cells will regulate their volume back towards the starting point – an important feature in keeping cellular homeostasis. This swelling-induced response is called Regulatory Volume Decrease (RVD) and involves the efflux of KCl and organic osmolytes through specific volume sensitive channels, with a concomitant water efflux and shrinkage back towards starting volume as a result. KCNK5 has been shown to be an important player in RVD in different cell types and tissue e.g. in Ehrlich cells. It has been speculated that tyrosine phosphorylation is necessary for the swelling-activation of the channel and in “Paper I” we show how acute hypotonic swelling induces a time-dependent tyrosine phosphorylation upon the channel itself.
Acute cell swelling and its regulatory mechanisms is by far the most studied part of the cells response to hypotonic stress and little is known about the consequences of long-term hypotonic stimuli on cells, thus we induced a long-term hypotonic stimulation on EAT cells and ELA cells to study the effect on physiology and channel expression patterns (Paper II). We found that 48 h of hypotonic stimulation reduced the maximum current through the channel and likewise decreased the cells ability to perform RVD. This impairment was found to be likely due to lowered KCNK5 protein expression.
In 2010 it was suggested that KCNK5 play a pivotal role in the autoimmune disease multiple sclerosis more precise in activated T cell. Numerous studies on ion channels in T cell activation have been published and potassium channels Kv1.3 and KCa3.1 are thought to be the two main channels involved. A potential role for KCNK5 made us shift cell type and thus look closer into the channel in human T cells. We found a time-dependent massive up-regulation of KCNK5 on mRNA and protein levels in activated human T cells, but KCNK5 up-regulation did not facilitate an increased RVD, instead we found RVD in activated T cells to be impaired in comparison with non-activated control cells. This impairment, despite of up-regulation of KCNK5, we show to be due to a decreased volume activated Cl- flux, thus making the volume regulated anion channel (VRAC) the rate-limiting factor in RVD in activated human T cells (Paper III).
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Resumé
Kaliumkanalen KCNK5 (også kaldet TASK-2 eller K2P5.1) tilhører den senest opdagede familie af K+ kanaler og tilhører således gruppen af to-pore-domæne K+ kanaler. KCNK5 blev første gang beskrevet af Lazdunski et al. i 1998. KCNK5 er vist at være involveret i volumenregulering i en række celletyper og er relativt for nylig foreslået at være af vigtig betydning i T celle aktivering og i den autoimmune sygdom multipel sklerose.
Når mammale celler oplever cellesvulmning som følge af osmotiske ændringer i det intra- eller ekstracellulære miljø, vil de fleste være i stand til at regulere deres volumen tilbage mod udgangspunktet – en vigtig evne i forhold til at bevare den cellulære homeostase. Volumenreguleringen som følge af svulmning kaldes ”Regulatory Volume Decrease” (RVD) og involverer efflux af KCl og organiske osmolytter via specifikke volumenaktiverede kanaler. Aktiveringen af disse kanaler og den efterfølgende KCl efflux resulterer i et osmotisk vandtab og celleskrumpning. KCNK5 er vist at være vigtig i RVD i flere celletyper og væv bl.a. i Ehrlich celler. Det er blevet foreslået, at tyrosin-fosforylering er vigtig i den svulmningsinducerede aktivering af KCNK5 og i ”Paper I” viser vi, hvordan akut hypoton cellesvulmning og kanalaktivering resulterer i en tidsafhængig tyrosin-fosforylering af selve kanalen.
Akut cellesvulmning, og de regulatoriske mekanismer involveret i korrelation af dette, er uden sammenligning den mest studerede og bedst beskrevne effekt af hypotont stress og der er således en begrænset viden om konsekvenserne af langtids-hypoton stimuli af celler. Vi undersøgte derfor effekterne af langtids-hypoton påvirkning af EAT og ELA celler med henblik på KCNK5 kanalfysiologi og ekspressionsmønster (Paper II). Vi fandt at 48 timers hypoton påvirkning reducerede den maksimale svulmningsaktiverede strøm og ligeledes sås et hæmmet RVD. Denne fysiologiske hæmning ser ud til at skyldes en nedsat KCNK5 protein-ekspression.
Der har været utallige studier af ionkanaler i T celle aktivering og K+-kanalerne Kv1.3 og KCa3.1 menes, at være de to vigtigste kanaler i denne proces. Da det er blevet foreslået, at KCNK5 spiller en vigtig rolle i sygdommen multipel sklerose og også er involveret i T celle aktivering besluttede vi, at kigge nærmere på en potentiel rolle for KCNK5 i aktiveringen af humane T celler. Vi fandt, at KCNK5 blev kraftigt opreguleret i aktiverede T celler både på mRNA- og proteinniveau. Denne opregulering medførte dog ikke et øget RVD, tværtimod fandt vi at RVD i aktiverede T celler var hæmmet i forhold til RVD i ustimulerede kontrolceller. Vi har evidens for, at denne hæmning af RVD skyldes en nedsat volumenaktiveret Cl- flux, hvilket gør VRAC (volume regulated anion channel) til den begrænsende faktor for KCl efflux under RVD i aktiverede T celler (Paper III).
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1. Introduction
Potassium channels are ubiquitously distributed in all living organisms and are thus found in cellular membranes in such different organisms as e.g. bacteria, plants and mammals. To this day more than 75 known mammalian potassium channel subunit coding genes are known, which makes this family of ion channels the most diverse of all ion channel families. They are found in almost all cell types and hold a variety of cellular functions. This thesis will focus on the potassium channel KCNK5 and its activation in response to acute hypotonicity, its physiology when experiencing hypotonicity over a longer period of time and its role in T cell physiology. The following sections will thus (in broad terms) deal with potassium channels in general, KCNK5, volume regulation and T cell physiology.
1.2 K+ channels in general
K+ channels are protein complexes made of subunits that allow the passive transport of K+ across the membrane which, if not for ion channels and transporters, would be functional impermeable to ions. The passive transport follows the electrochemical gradient. K+ serves a wide range of functions including setting the membrane potential, controlling the action potential in excitable cells and being involved in cell proliferation, cell volume control, muscle contraction and secretion of various hormones and transmitters. As potassium channels are implicated in such various physiological functions, many diseases or pathological conditions can be contributed to an altered K+ channel function. Of diseases and pathological conditions implicating K+ channels can be mentioned arrhythmia, diabetes, neuronal diseases and renal diseases, for further information see e.g. (60; 140). Several K+ channels are also found to be up- regulated in cancer cells (see (125)), though one way of cancer cells to survive is to avoid apoptosis, which can be obtained by the down-regulation of K+ channels involved in apoptotic volume decrease (see section 1.4.2.1).
As stated above, potassium channels hold a range of different functions and so they differ both in topology and regulation. The K+ current running through the channel can be activated in different ways reflecting the physiology of the cell or tissue in mind. There are different ways of grouping the potassium channels e.g. by activation and type of current or by topology (see section 1.2.1). There are four main types of potassium channels based on activation and current; i) voltage-gated K+ channels which are sensitive to changes in membrane potentials and opens or closes accordingly, ii) Ca2+-activated K+ channels which, as the name implies, has a Ca2+ dependent activation, iii) Inwardly-rectifying K+ channels that favors K+ fluxes in an inward
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direction and last but not least iiii) two-pore domain K+ channels which are constitutively active and carries currents designated leak-currents see (110).
The potassium channels consist of various transmembrane (TM) spanning regions (or TM domains) together with a highly conserved pore-forming domain, that can transport between 106 to 108 K+ ions across the membrane pr. sec, while at the same time rejecting other cations (110; 140).The pore region of the channel is a feature shared by all potassium channels and thus, all potassium channel subtypes poses the very conserved amino acid sequence threonine, valine, glycine, tyrosine and glycine (TVGYG). This amino acid sequence make up the selective filter of the pore (60) and is called the signature sequence of potassium channels (48). To make a functional K+ pore, four pore-domains have to come together.
Scientists are always on the look for new discoveries including new ion channel families, thus the pore sequence was used to search DNA sequence databases for new potassium channels. This approach resulted in the discovery of the two-pore domain potassium channel family further described in the following sections (83).
1.2.1 Potassium channel grouping by topology One way to group the many different K+ channels is by activation pattern and another is by topology and thus the number of TM segments. Dividing the potassium channels by topology gives three main groups, the 2TM family, the 4TM family and the 6TM family which also include a 7TM spanning member (the BK family of Kca potassium channels (21; 60)) (see Figure 1).
1.2.2 The 2TM and 6TM families
In the 2TM family we find the KIR channels which are inward rectifying channels that include the
ATP- sensitive channels as well as G-protein coupled channels (fig. 1). KIR channels are found in e.g. skeletal and heart muscle and in neurons see e.g. (21). The family contains 7 members and is found as a tetramer in the membrane, since the generation of a functional pore requires four pore domains to come together and in the 2TM family each subunit holds one pore-forming domain.
The voltage-gated, the Na+-activated and the Ca2+-activated K+ channels all belong to the 6TM family which in spite of the name also include a 7TM channel namely the large (big) conductance (BK) Ca2+-activated potassium channel (60) (see fig.1). The functional channel is, as for the 2TM family found as a tetramer with four pore domains generating the pore.
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Figure 1: Overview of potassium channels and potassium channel families There are three potassium channel families based on membrane topology namely the 2TM family with one pore domain and two transmembrane segments containing the inward rectifying KIR channels, the 4TM family of two- pore domain channels including KCNK5 and the 6TM family with one pore domain and 6 (or 7) transmembrane segments including the voltage–gated and the Ca2+- activated K+ channels. Own figure.
1.2.3 The 4TM family – the two pore-domain potassium channels + The 4TM family comprises the latest discovered family of K channels, namely the two-pore domain channels in which we find the channel of special interest for this thesis – the KCNK5 K+ channel (also known as TASK-2 or K2p5.1). The 4TM family has an interesting structure that varies from the other two families since members of the 4TM family have four TM segments and two pore-forming domains (43; 82). Because of the two pore domains in each subunit the functional channel is found as a dimer and not as a tetramer as seen in the other two families (84) see also (83).
+ The KCNK5 channel belongs to the TALK (TWIK -related alkaline pH activated K channel) subfamily and besides TALK the 4TM family consists of five other subfamilies namely the TWIK (tandem of p domains in a weak inwardly rectifying K+ channel) subfamily, the THIK (tandem pore domain halothane-inhibited K+ channel) subfamily , the TREK (TWIK-related K+ channel) subfamily, the TRAAK (TWIK-related arachidonic acid-stimulated K+ channel) subfamily and the
TASK (TWIK-related acid-sensitive K+ channel) subfamily (9) (fig. 2).
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The idea of potassium leak-currents in the resting membrane was proposed a long time before it was possible to measure ion currents across the membrane and before cloning techniques made channel identity possible. Thus Bernstein had in 1912 proposed that the resting membrane was more permeable to potassium than other ions, which was described and further analyzed by Hodgkin and Huxley (49; 50). Though the idea of leak- Figure 2: Phylogenetic tree Phylogenetic tree for the known two-pore domain K+ currents came to light long ago, it was channels which shows how KCNK5 is related to the other not until 1995 that the identity of a members based on sequence similarities. Model based on (41). background channel was known by the cloning of the TOK1 channel in Saccharomyces cerevisicae (66). The TOK1 channel differs from the mammalian two pore- + domain channels since it has eight TM domains, whereas all other two pore-domain K has the characteristic four TM segments and it further more differs in functionality. The first mammalian two pore-domain potassium channel with the characteristic morphology of two pore-domains and four TM domains to be cloned , was TWIK-1 which was cloned in 1996 (82).
Despite a relatively poor sequence similarity between some of the members of this relatively newly described family, they share not only the distinct topology, but are also similar in regard to electrophysiological The background channels or potassium leak channels all share the same morphology with four membrane spanning domains, two of them being pore forming and they are special in the way that they are constitutively active at resting membrane potential (41). The potassium channel signature sequence is conserved in the first pore-forming loop, but is slightly different in the second pore-forming loop, where GYG is substituted by GFG or GLG (140). Since this group of K+ channels posses two pore domains, only two subunits are required to make a functional channel. Thus the channel is formed by the dimerization of two subunits (83; 84). The potassium leak-channels are defined by the lack of voltage and time-dependencies, which means that they are open even at resting membrane potentials leaking K+ ions, and thus Page 12 of 117 Copenhagen, sep. 2013 bringing the plasma membrane potential towards the equilibrium potential for K+. So they serve an important function in maintaining the negative resting membrane potential by constitutively leaking K+ ions down the concentration gradient, which in turn helps maintaining the Na+/K+ pump function see ref. (42). To this day there are 15 known members of the family of human two-pore domain potassium channels see refs. (20; 41). For more about two-pore domain potassium channels see e.g. refs. (8; 32; 41; 42; 66; 115). 1.3 KCNK5 – a two-pore domain potassium channel In the 4TM family one of the subfamilies was named TASK (TWIK-related acid-sensitive K+ channel) and started out containing the channels TASK-1 (which was the first TASK channel and the third K2P channel to be cloned (31)), TASK-2, TASK-3, TASK-4 and TASK-5, but as seen from fig. 2 the TASK-2 channel or KCNK5 together with TASK-4 (also known as TALK-2) are now found in the TALK subfamily (fig. 2). This subfamily re-arrangement was partly due to the poor molecular relation between TASK-2 and TASK-4/TALK-2 against TASK-1, thus the sequence similarity between TASK-1 and TASK-2 is only about 30% (110). In 2001 TALK-1 and TALK-2 were described (26; 39) and they were named as TWIK-related alkaline pH-activated K+ channels due to the high extracellular pH needed for activation. TASK-2 and TASK-4 were now regarded as TALK channels due to their alike alkaline pH sensitivity and because of their molecular relations mentioned above. Furthermore TASK-channels are gated only by extracellular pH (32) and thus not affected by intracellular pH as seen in the TALK channels KCNK5 and TALK-2, which are indeed sensitive to intracellular pH changes (105). Since TASK-2 is now regarded as a TALK channel and though the name TASK-2 is still in use, the TASK-2 channel will be designated KCNK5 throughout the rest of this thesis. I should though be Figure 3: KCNK5 pH sensitivity noted that the channel is called TASK-2 in “Paper I” Current-voltage relationship recorded from a TASK-expressing oozyte (31). (69) and KCNK5 in “Paper II” and “Paper III”. Page 13 of 117 Copenhagen, sep. 2013 1.3.1 KCNK5 history and tissue distribution KCNK5 was as the first member of the TALK subfamily to be cloned, which was done from human kidney in 1998 by Reyes and co-workers (128). KCNK5 Northern blot analysis have shown KCNK5 to be present in human kidney, pancreas, liver, placenta, lung and small intestine (97; 128) whereas RT-PCR from on mouse tissue showed abundant KCNK5 presence in liver, kidney, small intestine, lung and uterus (97; 128). Some studies including the KCNK5 channel is strictly expression studies and do not deal with physiological importance and between the various studies there is some species variation in regard to KCNK5 expression (see table 1). In that regard some tissue expression is up for discussion, while there is a broad agreement on the expression and importance in kidney cells where many of the functional studies on the channel has been conducted. Since the initial studies on KCNK5 it has (besides being described as a K+ leak-channel) been reported to be implicated in RVD in several cell types including Ehrlich Ascites Tumor (EAT) cells (69; 104), mouse proximal tubules (7), human and murine spermatozoa (4; 5) and murine T lymphocytes (12) but has also been reported to hold other functions in different cell types (see table 1). 1.3.2 KCNK5 characteristics KCNK5 has been studied in a variety of cell types and it has been found to be highly sensitive to external pH, thus open probabilities increases with alkalization and decreases with acidification (31; 64; 104) (see fig. 3) with a 90% maximum current measured at pH 8.8 and only 10% at pH 6.5 (31; 128). At pH 6.0 the KCNK5 activity is almost non-existing (107). The pH sensing mechanism has been proposed to be located in the extracellular loop between TM1 and channel pore domain one (99), but in 2007 Niemeyer and co-workers showed how an arginine residue (R224) located near the second channel pore Figure 4: KCNK5 characteristics domain acts as the pH sensor and that it See the text for further details. Own figure. functions by applying an electrostatic effect on the pore domain (107). Page 14 of 117 Copenhagen, sep. 2013 Table 1 Reported mammalian KCNK5 expression and function Species Tissue Function Reference Mouse Ehrlich ascites tumor cells Volume sensitive (69; 104) Mouse Proximal tubules Volume sensitive (7) Human Spermatozoa Volume sensitive (4) Mouse Spermatozoa Volume sensitive (5) Mouse T lymphocytes Volume sensitive (12) Mouse Proximal kidney cells AVD (73) Human T lymphocytes Possible T cell activation (10) Human T lymphocyte Volume sensitive (3) Rat CNS Possible cell excitability & signal transduction (37) - Mouse Proximal tubules Stabilizing HCO3 transport (151) Human MCF-7 and T47D Regulating proliferation (1) Mouse WEHI-231 cells BCR-ligation-dependent apoptosis (100) Mouse Retrotrapezoid nucleus neurons CO2 and O2 chemo-sensing (38) Rat Proximal neurons in inner retina Neuronal excitability (157) Rat Neurons in hippocampus Contribution to properties of epilepsy (68) Human Peripheral blood and T cells of RA Correlation between expression and disease (11) patients activity Mouse Gastrointestinal smooth muscles Background current (137) cells Mouse Gastroenstesinal tract Contribution to resting potential (156) Rat Carotid body Regulating K+ currents and acid sensing (154) Rat Taste receptor cells Setting resting potential and sour taste (89) transduction Rat Cerebellar astrocytes Not speculated on (136) Human Cerebellar Purkinje cells Not speculated on (136) Component of standing-outward potassium Rat Cerebellar granule neurons (23) conductance Furthermore the channel has proven to be insensitive to Ca2+ and a wide range of known potassium channel blockers e.g. tetraethylammonium (TEA) (128) and charybdotoxin (ChTX) but can be blocked by e.g. clofilium (104) and quinine (128) (fig. 4). For a recent review on KCNK5 see ref. (20). Page 15 of 117 Copenhagen, sep. 2013 1.3.3 KCNK5 activation and gating Different studies have been made to elucidate the activation mechanisms of KCNK5 and as mentioned earlier, KCNK5 has proven to be volume sensitive and thus activated by cell swelling. In addition we have demonstrated how protein tyrosine kinase activity is involved in RVD (Paper 1, fig. 1) and how cell swelling elicits a time-dependent tyrosine phosphorylation of the channel itself upon activation (Paper I, fig. 5). We have also shown how inactivation of the swelling- activated channel involves protein tyrosine phosphatases (Paper I, fig. 3). Furthermore swelling-activation of KCNK5 has in kidney cells been proposed to be caused by an - - - extracellular alkalinization due to Cl /HCO3 exchanger mediated Cl influx (72). LTD4 has likewise been implicated in the swelling-activation of KCNK5, since hypotonicity and cell swelling is + followed by LTD4 release (74; 76) and since addition of LTD4 results in activation of a K current similar to the swelling-activated K+ current (57). In addition it was shown that desensitization of the LTD4 receptor impairs RVD in EAT cells (61). Of other gating mechanisms can be mentioned gating by intracellular pH and also extracellular pH both working in the same pH range though they are independent of each other. A recent study have also shown how KCNK5 is inhibited by heterotrimeric G protein subunits Gβγ an effect that could be eliminated by mutation of lysine residues of the C terminus (2) (also see (20)). Despite all of these findings there are still many questions to be answered in regard to KCNK5 channel activation, thus we have yet to identify the tyrosine sites on KCNK5 being phosphorylated upon swelling-mediated activation (see section 7 and discussion in Paper I) and the direct connection between KCNK5 and LTD4 has still not been shown. Furthermore though the effect of Gβγ G protein subunits has been established, the answer to how exactly it works and through which signaling pathway also remains to be answered. 1.3.4 KCNK5 in lymphocytes KCNK5 was initially described in kidney cells and thorough studies have also been done in Ehrlich cells. The presence of KCNK5 in lymphocytes on the contrary is a relatively new observation. The first evidence of a background channel (TREK-2) present in lymphocytes was described in WEHI- 231 cells, which is a murine cell line equivalent to immature B cells (101; 158). Further studies have revealed the presence of two-pore domain channels in lymphocytes e.g. TASK- 1 and TASK- 3 in human T cells (98) and in regard to lymphocytes KCNK5 has so far been described in human T cells (10) and in WEHI-231 immature B cells (100). Page 16 of 117 Copenhagen, sep. 2013 In B cells KCNK5 has been shown to be involved in apoptosis which is initiated by B cell receptor stimulation (100). In T cells Bittner and co-workers has proposed that KCNK5 holds a role in multiple sclerosis since an up-regulation of the channel was found in T cells purified from multiple sclerosis patients. The study from Bittner et al. led us to look into the potential role of KCNK5 in activated human T cells. We found that KCNK5 is indeed expressed in T cells and that activation resulted in a time-dependent expression pattern with an initial down-regulation followed by a strong up-regulation of the channel on protein level (Paper III, fig. 2). For more on KCNK5 in lymphocytes see (20; 35). 1.4 Cellular volume The permeability of water across the plasma membrane is much larger than the permeability of potassium, sodium and chloride, thus the water permeability was measured to be 105 times higher than sodium and potassium (51) and 106 times higher than chloride (75) in EAT cells. A shift in intracellular and/or in extracellular osmolarity can be observed in response to e.g. accumulation of metabolic waste products, protein synthesis and transport over epithelia. The extracellular content has very little variation under physiological conditions, thus changes in the intracellular osmolyte composition are the main determinant for volume changes. The cell is very vulnerable to changes in cell volume which will affect cellular homeostasis e.g. is the actin cytoskeleton very susceptible to volume changes and it is seen how cell swelling results in a decrease in F-actin whereas cell shrinkage result in an increased amount of F-actin (117; 139). For more on volume regulation see e.g. refs. (56; 78). 1.4.1 Cell volume regulation Maintaining a steady cell volume is crucial for keeping cellular homeostasis, thus a fundamental ability in mammalian cells, with only some exceptions, is the ability to volume regulate. Cells are constantly osmotically challenged by extracellular and especially intracellular changes in osmolyte content and since most mammalian plasma membranes contain aquaporins water will rapidly move across the membrane in response to the osmotic changes. These osmotic changes are in general met by activation of regulatory mechanisms bringing the cell volume (almost) back to normal see e.g. refs. (56; 78). There are though exceptions where the changed volume does not result in volume regulation (see section 1.4.2 and (56)). Page 17 of 117 Copenhagen, sep. 2013 Figure 5: scanning electron images of cells kept under normal, hypotonic and hypertonic conditions Scanning electron microscopy images of Ehrlich ascites tumor cells kept under various osmolarities. B: cells kept in isotonic media shows invaginations of the membrane which are unfolded upon cell swelling (A). Cell shrinkage will on the other hand generate even more pronounced membrane-folding as seen on image C (51). 1.4.1.1 Pathophysiological conditions An altered cell volume is found in a number of pathophysiological conditions such as ischemia/hypoxia and epilepsy where an increase in intracellular osmolarity is seen. Hepatitis cirrhosis, nephrosis and hyponatremia are all examples of conditions that include a decreased plasma osmolarity, whereas increased plasma osmolarity is seen in diarrhea, uremia and diabetes mellitus/insipidus see (96; 111). Of other pathophysiological states can be mentioned cerebral edema (see (96)). 1.4.1.2 Regulatory mechanisms in response to volume changes As stated above, osmotic challenged cells will often respond to the resulting swelling or shrinkage by initiating a regulatory response thereby returning their volume back towards the starting point. The cell uses different regulatory mechanisms in regard to swelling contra shrinkage. Regulatory Volume Decrease (RVD) is the cells response to swelling and consists of mechanisms excluding osmolytes and with concomitant water efflux as result, whereas Regulatory Volume Increase (RVI) is seen in response to shrinkage and involves the uptake of osmolytes and water (fig. 6). As seen from the scanning electron images in fig. 5 EAT cells have clear invaginations of the membrane under isotonic conditions, letting them tolerate some swelling by unfolding of the surface thereby not disrupting the cell membrane. Shrinkage on the other hand causes more prominent invaginations (51). This is not only true in Ehrlich cells but also in lymphocytes (18) and certainly in a lot of other cell types. Since the mechanisms of RVI is not of direct interest to this work I will not go more into detail on the matter, but merely refer to fig. 6 and the extensive review by Hoffmann et al. (56). Page 18 of 117 Copenhagen, sep. 2013 1.4.1.3 The volume set-point Most cells have a fine tuned volume regulation which occurs instantly upon osmotic changes making volume changes almost undetectable and the cells are said to be isovolumetric. Mammalian cells are very sensitive to volume changes and as little as a 3.5% (44) volume change can result in the activation of regulatory mechanisms – the volume set-point of the cell is reached. The volume set-point defines the boarder for cell volume regulation and volumes higher or below this threshold will result in volume regulation. The volume set-point is not to be viewed as a definite size, but rather a dynamic mechanism by which the cell adapts to its environment and function (52) and it is thus seen that cells that undergo RVD or RVI often will have an altered volume set point (53; 103). The volume set-point can also change accordingly to cellular function, thus proliferating cells experience a shift in the volume set-point since volume regulation in this case is not beneficial for the growing cell (see section 1.4.2.2). 1.4.1.4 RVD When subjected to cell swelling the cell will release KCl and organic osmolytes (52) through specific channels and co-transporters which will then drive water to leave the cell and subsequently lead the cell volume back towards its starting point. Besides the volume sensitive K+ and Cl- channels KCl can also leave the cell through transporters and so the + - + - K -Cl co-transporters, the H /HCO3 anion exchanger and the K+/H+ exchanger (fig. 6) are likewise important players in a RVD response (see (111)). The exact composition and influence of the Fig. 6: Ion movement during RVD and RVI different transporters and channels involved Cell swelling results in KCl efflux through specific K+ (varies from cell type to cell type) and Cl- channels in RVD differs from cell type to cell type, thus + - + - (VRAC), the K -Cl co-transporter (KCC), the H /HCO3 different K+ channels have been shown to be anion exchanger (AE), the K+/H+ exchanger together + with an organic osmolytes efflux (not shown here). The implicated in the swelling induced K efflux. ion efflux drives osmotic obliged water to leave the cell Whereas the K+ channel picture is very and let it reach a volume close to its starting point. RVI on the other hand rely on ion up-take through diverse, it is commonly agreed that the transporters such as AE, the Na+/H+ exchanger NHE, the + + - + + swelling activated Cl- channel holds one Na /K /2Cl co-transporter (NKCC), the Na /Cl co- transporter and organic osmolytes up-take (not shown identity namely that of VRAC (volume here). Own figure. regulated anion channel), though it’s Page 19 of 117 Copenhagen, sep. 2013 molecular identity for the time being remains unknown. The molecular identity of VRAC is very much a hot topic and it has been suggested to belong to various chloride channel families though no definite answer to the puzzle has been found. A further discussion of that subject is outside the scope of this thesis and will not be further dealt with here. 1.4.1.5 K+ channels in volume regulation As mentioned previously the K+ channels picture is quite diverse and so far, and to my knowledge members of 9 different potassium channels subfamilies has been shown to be volume sensitive (see (56)). It should be noted that the discrepancies between the number stated here and the number of channel families listed as including volume sensitive members in ref. (56) is due to the fact that the volume sensitive channels KCNK5 was recently moved from the TASK subfamily to the TALK subfamily as described previously in this thesis (see section 1.3). Volume sensitive potassium channels can e.g. be found amongst the family of Ca2+-activated K+ channels (47) in the BK subfamily (see (56; 111)), the IK (63; 67; 150) and SK (63; 132) subfamilies and amongst the voltage-gated K+ channels has e.g. Kv1.3 (28) and Kv1.5 (4; 34) been shown to contribute to RVD in lymphocytes. Of special interest to this thesis the KCNK5 has been shown to be the volume regulating K+ channel in EAT cells (104; 106) and mouse proximal tubules (7) see table 1 and (20). 1.4.1.6 Volume regulation in Ehrlich cells Volume regulation in Ehrlich cells (EAT and ELA cells) have been intensely studied over the years and much knowledge has come from that. The main pathways for KCl efflux in Ehrlich cells are through specific volume sensitive K+ and Cl- channels and the currents through these channels are designated IK,vol and ICl,vol see (55) ICL,vol The outward rectified chloride current activated by cell swelling runs through the volume regulated anion channel (VRAC). It has been described as being Ca2+ independent, tamoxifen inhibited, relative insensitive to DIDS and niflumic acid (118) and to hold a permeability - - - - sequence of SCN >I >NO3>Br>Cl F (see ref. (53)). IK,vol Initially an intermediate-conductance K+ (IK) channel was shown to be activated in cell attached patch studies on EAT cells (19) but later studies e.g. showing how IK,vol was poorly affected by clotrimazol, apamin, kalitoxin, margatoxin, TEA and ChTX which are all known inhibitors of K+ 2+ + channels (including Ca -activated K channels), together with the fact that IK,vol was activated despite buffering [Ca2+]i (57) led to the conclusion that RVD in Ehrlich cells were dominated by a Page 20 of 117 Copenhagen, sep. 2013 2+ Ca -independent channel (62; 129). It was further more demonstrated how IK,vol had a specific selection profile favoring entry of Rb+ and K+ which eliminated the stretch-activated K+ channel since it was proven to be non-specific (19; 108). For further information see (53). IK,vol did not belong to the family of voltage-gated K+ channels either (108). One drug clofilium, did though inhibit IK,vol hence evidence pointed towards the family of two-pore domain channels (106). PH sensitivity with potentiation of the current with alkalization and inhibition at acidification narrowed the field to – at that time - a TASK channel (see section 1.3) (54; 71). It was later shown that KCNK5 (TASK-2) but neither TASK-1 nor TASK-3 was present in Ehrlich cells (104). Activation of IK,vol in EAT cells When swollen phospholipase cPLA2α is translocated to the nucleus (120) where it is phosphorylated and activated by a G-coupled process. This activation results in a release of arachidonic acid (AA) which is 3.3 times higher than seen in cells kept isotonic (145). AA which is a precursor for leukotriene C4 (LTC4) requires the action of FLAP (5-LOX activating protein), 5- LOX and LTC4 synthase to be generated. LTC4 is subsequently transported out of the cell where it is converted to leukotriene D4 (LTD4) by γ-Glutamyl transpeptidase, here it binds to its receptor which in turn activates IK,vol (57; 61; 74) (also see (53; 56)). The direct mechanisms between LTD4 and channel activation still remains to be studied. Protein tyrosine kinase activation has been shown to play a role during RVD in various cell lines (6; 22; 25; 88) and it was speculated that tyrosine phosphorylation was also of importance in the RVD response in Ehrlich cells somewhere in the signaling pathway just described. In “Paper I” we show how tyrosine phosphorylation indeed is vital for RVD, since protein tyrosine kinase phosphorylation of the volume sensitive K+ channel (which in Ehrlich cells is KCNK5) is part of the activation mechanisms upon cell swelling. Thus we found a swelling-induced and time- dependent tyrosine phosphorylation on the channel itself in response to acute hypotonicity (Paper I, fig. 5). 1.4.1.7 Long-term effects of anisotonicity During long-term exposure to an anisotonic environment cells respond with late-phase volume regulatory mechanisms in contrary to the acute phase response (described in section 1.4.1.2) which takes place within minutes of cell swelling. The late-phase mechanisms are often connected with an altered gene expression and protein synthesis changes. Organic osmolytes makes good late-phase regulators since accumulation of organic osmolytes compared to e.g. electrolytes are harmless, thus an accumulation of electrolytes can upset various metabolic processes e.g. denature macromolecules and influence membrane potential (see (142)). Page 21 of 117 Copenhagen, sep. 2013 Long-term anisotonicity can be both hypo- or hypertonic and hypertonicity is by far the most studied of the two conditions. It has been shown how long-term hypertonicity results in an increased gene transcription of various volume regulatory genes (14) while the number of studies on long-term hypotonicity is scarce. Thus we studied the KCNK5 function and expression on various levels in Ehrlich cells upon long-term exposure to hypotonicity (Paper II). We found that the maximum current through KCNK5 in ELA cells were inhibited (Paper II, fig. 1) as were the EAT cells ability to perform RVD (Paper II, fig. 2). We further more found a decreased KCNK5 protein expression in both cell lines upon stimulation (Paper II, fig. 4) and our results thus show how a functional down-regulation of KCNK5 upon long-term hypotonicity is probably due to a decreased KCNK5 protein synthesis (see section 7 and the discussion in Paper II). 1.4.1.8 Volume regulation in lymphocytes Lymphocytes like most other mammalian cells are subject to changing intracellular environment causing potential volume changes and subsequent regulation. T cells are of course not static in the sense that they are in constant movement around the body and thereby eligible to extracellular osmotic changes e.g. when passing around the kidney. Lymphocytes are as most other mammalian cells capable of performing RVD in response to cell swelling (18; 29; 135) and as seen in many cells the higher the osmotic challenge the lesser the recovery (18). T cells are only able to perform RVI if previously experiencing hypotonically elicited RVD – also called post- RVD RVI (29). As seen in other volume regulating cells VRAC is responsible for the swelling activated Cl- current in lymphocytes (17; 81; 85) (also see (16)), whereas the K+ channel picture is more diverse and different regulatory volume pathways and K+ channels have been suggested to play a role in T cell volume regulation including the voltage-gated K+ channel Kv1.3 (17; 28; 80), the Ca2+- activated KCa3.1 (138), and two-pore domain potassium channels (3; 12). Kv1.3 is involved in RVD in mature T cells whereas KCa3.1 is so far only implicated in RVD in thymocytes (immature T cells), thus it is seen that RVD in mature T cells is Ca2+-independent (16). Kv1.3 is not directly volume sensitive, but is thought to be activated by a change in membrane potential due to activation of volume sensitive anion channels causing Cl- efflux, plasma membrane depolarizing and subsequent Kv1.3 activation (16; 29; 87). KCa3.1 and Kv1.3 is by far the most studied and described K+ channels in T cell physiology including cell volume regulation and the role of other K+ channels is still object to some discussion (see e.g. (16; 112)). Recently members of the two-pore domain potassium channel family were proposed to play a role in cell volume regulation in murine and human T cells (3; 12), thus both KCNK5 (in mouse Page 22 of 117 Copenhagen, sep. 2013 and human T cells) (3; 12) and TRESK-2 (in human T cells) (3) were given prominent roles in RVD. We have likewise studied the effect of KCNK5 in human T cell volume regulation and find that the channel is expressed in these cells and that this expression is up-regulated when the T cells are activated (Paper III, fig. 2). We find that the RVD response in activated T cells is impaired (Paper III, fig. 3), contradicting findings by Andronic et al. (see section 7 and the discussion in Paper III). We furthermore found that the Cl- permeability in activated and swollen T cells were inhibited compared to non-activated control cells (Paper III, fig. 4), thus we suggest that Cl- is the limiting factor in the RVD response in activated T cells and that the strong up-regulation of KCNK5 has another function. We speculate whether this function could be in maintaining an electrochemical gradient favoring a sustained Ca2+ influx, by extruding K+ and thus keeping the plasma membrane hyperpolarized (see paper III and section 1.5.3.2). 1.4.2 When changes in cell volume act as signals Not all cell volume changes will result in the activation of volume regulatory mechanisms and often the change in cellular volume is an important player in cell signaling. As described previously an altered cell volume is seen in various pathophysiological events, but changes in cell volume without any regulatory mechanisms activating is also seen under physiological conditions. It has for instance been shown that volume changes serves as signaling events in cell proliferation and migration, transport across an epithelia and in apoptosis (fig. 7) which will be further addressed below (also see ref. (56)). 1.4.2.1 Programmed cell death Different conditions can lead to programmed cell death (or apoptosis), thus programmed cell death is part of both cell physiology (e.g. cells lacking growth factor stimulation) and pathophysiology (e.g. cells with DNA damage). If not wanted, the cells can become suicidal by undergoing programmed cells death a process that in contrast to necrosis will not damage the surrounding cells. Apoptosis is a Figure 7: volume changes as a signaling mechanism physiological process that is essential to There are various examples of an altered cell volume keep the balance in cell number (see (79)). A acting as a signaling event and some of these examples are depicted in this figure. See the text for more details. number of characteristics can be observed Model based on (56). Page 23 of 117 Copenhagen, sep. 2013 in an apoptotic cell and one well described feature in apoptotic cell death is the initial loss of cell volume. The apoptotic loss of cell volume is called Apoptotic Volume Decrease (AVD) and will not result in RVI. The volume decrease is concomitant to a loss of K+ and Cl- ions which initially does not change the K+ concentration but later in the AVD it decreases significantly (123). The decrease in K+ is followed by an increase in the Na+ concentration. AVD was first believed to be a secondary event in apoptosis, but have shown to be an important signal in facilitating the further progress into programmed cell death (79; 93; 123). It has further more been shown that the decrease in intracellular K+ concentration is vital in apoptosis and that this decrease is to some extend due to an inhibition of Na+/K+ pump function. The role of K+ channels in apoptosis is complex and so various K+ channels have been implicated depending on cell type. It is most often seen that inhibition of K+ channel function and thus inhibited K+ loss will protect against apoptosis, but in some cell types the opposite has been seen. For more on ion channels in programmed cell death see (79). 1.4.2.2 Proliferation When looking at cell volume changes and cell proliferation it is well known that the proliferating cell undergo swelling to be able to produce two daughter cells of the same volume as the initial parental cell, thus the dividing cell has increased in size without activating RVD mechanisms. What is perhaps less well known and definitely less described is how this increase in volume probably serves as a signal for the cell to progress through the cell cycle (see (56; 70; 92)). It has thus been shown how proliferation in some cells can be stimulated in swollen cells and inhibited in shrinking cells (30; 77; 122; 134). It is well known that cancer cells often have dysregulated control of cell cycle progression involving changes in ion channels (119). How exactly these volume changes influence cell cycle progression is yet to be fully elucidated. 1.4.2.3 Transepithelial transport Keeping a steady cell volume in epithelial cells constitute a challenge for the cells, since they are subject to massive changes in osmolyte composition due to absorption and secretion of ions and nutrients which are followed by water (see (119)). Though cell volume under normal conditions are without large changes due to tight regulatory mechanisms there is evidence of epithelial cell volume as a mechanism in the physiology of absorption and secretion. In that respect cell swelling has been implicated in epithelial absorption and cell shrinkage in secretion (see (56; 78)). 1.4.2.4 Migration and invasion In cell migration there is likewise evidence of cell volume being an important signal and it has e.g. been shown that hypertonicity inhibits neutrophile migration in the lung (131) and that cell swelling plays a role in human polymorphonuclear leucocytes migration stimulated by fMLP Page 24 of 117 Copenhagen, sep. 2013 (130). In transformed renal epithelial cells it was seen that migration could be inhibited both by hypo- and hypertonicity, thus it seems that the type of impact cell volume changes has on migration, can vary between cell types. For more on the subject of volume changes as a signal (see (56)). Briefly, it is thought that a migrating cell is capable of cell swelling in the protruding part of the cell and cell shrinkage in the back. These volume changes are facilitated by specific shrinkage-activated transporters and swelling-activated channels resulting in cell protrusion in the leading end and retraction in the rear end of the cell respectively (139). Page 25 of 117 Copenhagen, sep. 2013 1.5 The immune system The immune system can be divided into two parts; the innate and the acquired (or adaptive) immune system. As the name implies the acquired immune system is under constant development through the exposure to pathogens, the following immune response and the subsequent activation of memory cells. The immune system is able to remember different pathogens, an ability that is acquired throughout the numerous infections and pathogenic influence an individual undergoes. This memory function makes a second exposure to a pathogen elicit a far greater and faster immune response compared to the first exposure response, even if the second exposure occurs many years after the initial one. Immunological memory can be obtained by exposing an individual to a pathogen either by disease or by vaccination. The innate immune system, on the contrary, is the part of the immune system that we have with us from birth and it constitutes the first barrier that meets an invading pathogen. Only when the innate immune system has proven to be insufficient, the more specialized acquired immune system is activated. Some infections can be defeated solely by an innate immune response and those infections will hardly give any symptoms or do much harm to the body. The innate and the adaptive immune systems, though playing very different parts in the battle against intruders, should not be viewed as two distinct features, but rather as players on the same team. The two system overlaps in different ways e.g. the dendritic cells of the innate immune system plays a crucial role in the activation of the adaptive immune system since they act as antigen presenting cells (APC’s), in fact the dendritic cells are the most important of APC’s. Two other cell types can also act as APC’c namely the macrophages and the B cells. Both the innate and the adaptive immune system relies on white blood cells or leucocytes, but whereas the major players in the innate system are the macrophages, granulocytes, mast cells and dendritic cells the highly specialized T and B lymphocytes (T and B cells) constitutes the acquired part. A small fraction of activated T and B cells end up as memory cells making a future response to the same pathogen more efficient (see above). 1.5.1 B and T lymphocytes When encountering an antigen, it is recognized by different mechanisms being a T cell or a B cell and whereas T cells recognize antigens with the T cell receptor (TCR) B cells have immunoglobulins acting as antigen receptors. Upon antigenic binding to the TCR T cells activates, proliferate and differentiates into effector cells. Page 26 of 117 Copenhagen, sep. 2013 While B cells are specialized in detecting antigens of pathogens living outside the cells, T cells are specialized against pathogens living inside the host cell, thus they will always act on another cell and not the pathogen itself. So to be activated T cells have to recognize a specific antigen which can be from viruses, intracellular bacteria or from pathogens which have been internalized, degraded by the host cell and taken up by major histocompatibility complex (MHC) molecules to be displayed on the cell surface. Whereas B cells recognize the antigen directly, T cells cannot recognize antigens unless displayed by MHC molecules. B cells when activated differentiate into plasma cells which are antibody producing (fig. 8), but since the B lymphocytes are of lesser interest in regard to this thesis they will not be further dealt with here. 1.5.2 T cell activation T cell activation – from naïve to effector T cell – requires various signals and mechanisms. The activation signaling can be divided into three sub-signals; signal 1, 2 and 3. Signal 1 comprises the antigen-specific TCR activation whereas signal 2 involves the interaction between the APC and the T cell itself, resulting in the promotion of survival and expansion of the T cell. In signal 3 differentiations of the effector T cells in response to cytokine stimulation is seen. One of the co- stimulatory molecules involved in signal 2 is B7 molecules, which act on the CD28 receptor on the T cell. Different morphology of naïve vs. activated lymphocytes Inactivated or naïve lymphocytes are small, has few organelles and most of the chromatin in the nucleus is inactive. When activated the cell undergo and extreme transformation, they swell (both the nucleus and the surroundings), the chromatin becomes less dense, nucleoli becomes visible and a massive mRNA and protein synthesis occurs – the lymphocytes goes from being an inactive naïve lymphocyte to becoming an activated lymphoblast. 1.5.2.1 T cell subtypes As mentioned previously activation of T cells result in proliferation and differentiation into different kinds of effector T lymphocytes such as Cytotoxic T cells, Helper T cells or Regulatory T cells (fig. 8). From the start (after their development in the thymus) T lymphocytes can be divided into two groups according to the cell surface proteins displayed. One group bears the surface protein CD4 the other carries CD8, both surface proteins being important for T cell function. There are two main classes of MHC molecules – MHC class I and MHC class II and whereas CD8+ T cells recognize MHC class I molecules, CD4+ T cells recognize antigens shown on the surface by MHC class II molecules. MHC class I molecules display peptides from the cytosol Page 27 of 117 Copenhagen, sep. 2013 while MHC class II molecules display peptides from intracellular vesicles, a difference reflecting the different tasks of the two T cell subtypes. CD8+ T cells Cytotoxic T cells carry the CD8 surface protein (or co-receptor) and kills virus infected cells. They recognize antigen peptides generated in the cytoplasm of a cell and presented by MHC class I molecules, which is found on almost all nucleated cells. When activated CD8+ T cells are always bound to differentiate into cytotoxic T cells. CD4+ T cells Whereas CD8+ T cells always become cytotoxic T cells, CD4+ T cells can differentiate further into the subsets TH1, TH2, TH17 and regulatory T cells (fig 8). TH1 and TH2 subsets are also known as helper T cells since they help activate B cells, TH17 are involved in activating a neutrophile response, whereas the role of regulatory T cells is to suppress the immune system. CD4+ T cells recognize MHC class II molecules and their general role is to activate other cells such as the B cell. MHC class II molecules are found on B cells, dendritic cells and macrophages which are all cells of the immune system and which is in contrast to the MHC class I molecules found on numerous cell types. Figure 8: overview of the development of T and B cells and their subsets upon activation Both T and B cells originate from the common lymphoid progenitor. A B cell will stay a B cell but will after activation differentiate into antibody secreting plasma cells. T cells on the other hand can be divided into two main groups; those carrying the CD8 surface protein and those carrying CD4. CD8+ T cells are bound to become + cytotoxic T cells upon activation while CD4 cells can further differentiate into the subsets TH1, TH2, TH17 and regulatory T cells. A small fraction activated T and B cells will become memory cells (not shown on this figure). Own figure. Page 28 of 117 Copenhagen, sep. 2013 1.5.3 When the immune system attacks the body Sometimes the immune system turns on us in a way that is not beneficial for us. Factors in the surrounding environment can be taken for antigens and elicit an unwanted immune response as seen in e.g. allergic reactions, which as its best means a lot of discomfort for the individual, but as its worst can be life-threatening. Another type of unwanted immune reaction is not seen in response to the surroundings but to the body’s own cells and tissue – a response known as autoimmunity. One of the most important features of the immune system is its ability to regulate and to generate self-tolerance. During lymphocyte development lymphocytes with affinity to the individual will be generated since the process is strictly random, but they will be removed before entering the bloodstream. In autoimmune diseases something in the process of generating self-tolerance is not working properly with potential catastrophic consequences as a result. Of T cell mediated autoimmune diseases can be mentioned e.g. type 1 diabetes mellitus and multiple sclerosis. 1.5.3 Ion channels in T cell activation When encountering an antigen presented by MHC class proteins on dendritic cells T cells get activated. This activation leads to clonal expansion of the antigen specific T cells and the (hopeful) destruction of the invading pathogen. Clonal expansion involves strong proliferation thereby amplifying the number of T cells bearing the TCR’s relevant for a given infection. The activation of T cells relies on numerous factors and ion channel function is essential in this process. Three ion channels have been well described in T cell activation namely the voltage-activated Kv1.3 channel, the Ca2+-activated KCa3.1 channel and the Ca2+ release-activated Ca2+ channel (CRAC). For more on the subject see e.g. (16; 112; 146). All of these channels and mechanisms will be further described in the following sections. 1.5.3.1 Ca2+ signaling 2+ 2+ For a sustained activation of T cells an elevated intracellular Ca concentration ([Ca ]i) is crucial (59; 152) and is generated by Ca2+ release from intracellular stores together with Ca2+ influx from the extracellular matrix. MHC class II protein presentation of an antigen and the subsequent TCR stimulation results in depletion of Ca2+ from intracellular stores to the cytoplasm through two different signaling pathways - an 1,4,5-inositol triphosphate (IP3) (58) and a cyclic ADP-ribose 2+ (cADPr) (45) mediated signaling pathway. The elevated [Ca ]i in turn activates calcineurin – a Page 29 of 117 Copenhagen, sep. 2013 Ca2+-calmodulin-dependent phosphatase which dephosphorylates the transcription factor NFAT facilitating interleukin-2 (IL-2) transcription and expression, which is important in keeping the T 2+ cell activated in an antigen independent manner. A 1-2 hour rise in [Ca ]i is needed for sufficient NFAT stimulation and IL-2 expression, thus a sustained high intracellular Ca2+ level is therefore essential for T cell activation (24; 40; 65; 102). For a review on Ca2+ signaling in T cells see (86). The IP3 mediated pathway The binding of MHC complexes (bearing antigen peptides) to the T cell receptor results in recruitment and activation of protein tyrosine kinases (PTK’s) from the families Src, Csk, Tec and Syk (see (127)). The PTK’s then phosphorylates and activates phospholipase C-γ (PLCγ) leading to the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and IP3. IP3 binds to the IP3-receptor in the membrane of the endoplasmatic reticulum (ER) causing release of Ca2+ from ER stores into the cytosol. When Ca2+ is depleted from ER-stores Ca2+ is released from of the EF-hand domain of stromal interacting molecule 1 (STIM1) located within the lumen of ER, thus STIM1 acts as a store depletion sensors (90; 133). The Ca2+ from the ER stores causes the STIM protein to oligomerize and translocate to activate Orai1 bound to the plasma membrane. This results in the forming of a Ca2+ selective pore – the CRAC channel which allows Ca2+ to enter the cell down its concentration gradient (124; 149; 155) (see fig. 9). The cADPr mediated pathway Another pathway resulting in Ca2+ release from intracellular thapsigargin insensitive stores is via cADPr – a pathway much lesser studied and described than the IP3 mediated pathway described above. The intracellular stores releasing Ca2+ through this pathway are located close to the 2+ plasma membrane (13) and the Ca release is as for the IP3 pathway initiated by tyrosine kinases in response to receptor coupling see (112). Signaling events by both pathways result in the uptake of Ca2+ through CRAC channels and 2+ results by Guse et al. (46) suggests that high [Ca ]i via the IP3 pathway is important for the initiation and cADPr signaling for the sustainment of the signal. 1.5.3.2 K+ channels in Ca2+ signaling 2+ + The sustained high [Ca ]i depends on membrane hyperpolarization facilitated by K efflux + 2+ through specific K channels, thus besides the release from ER stores [Ca ]i is also influenced by voltage sensitive Ca2+ influx from the extracellular matrix through CRAC channels (fig. 9). Ca2+ influx is governed by capacitative Ca2+ entry by which the depletion of intracellular stores facilitates Ca2+ uptake (126; 144) through CRAC channels situated in the plasma membrane (114) and once activated the electrochemical gradient determines the Ca2+ influx (159). This also Page 30 of 117 Copenhagen, sep. 2013 means that the influx will increase in response to cell membrane hyperpolarization which can be obtained by the efflux of K+ (fig. 9). One of the most well described K+ channels in T cell activation physiology is the voltage-gated Kv1.3 channel (also known as KCNA3) which was first described in 1984 (15; 27; 36; 95). The channel activates at approximately -60mV (94; 147) not far from the resting potential (148) and further depolarization causes increased conductance, thus T cell activation and the following Ca2+ influx results in Figure 9: Ion channels and T cell activation When activated Ca2+ is released from a depolarization of the membrane, Kv1.3 activation intracellular stores which in turn causes and K+ efflux. This in turn results in hyperpolarization activation CRACK channels in the plasma membrane, which in turn causes a rise in 2+ which creates the electrochemical gradient favoring [Ca ]i, depolarization, KCa3.1 and Kv1.3 2+ channel activation, K+ efflux and sustained Ca entry (16; 112). + hyperpolarization. The K efflux and following hyperpolarization favors further Ca2+ influx via the CRAC channels. All of these mechanisms The Kv1.3 channel is not alone in generating the act to gain a sustained high intracellular Ca2+ membrane potential needed for Ca2+ entry thus the needed for the T cell to stay activated. Model adapted from (112). Ca2+ activated KCa3.1 (also known as hIKCa1, IKCa1 or KCNN4) also play an important role. Activation of KCa3.1 is not dependent on membrane 2+ potential but is activated – as the name implies – with rising [Ca ]i which is detected by calmodulin bound to the C-terminus (33; 67). KCa3.1 activation level is between 200 and 300nM 2+ 2+ [Ca ]i which is below the concentration found in non-activated T cells (30-100nM Ca (147)). 2+ 2+ The channel is activated by the rise in [Ca ]i mediated by store depletion and Ca influx via CRAC channels (fig. 9). 1.5.3.3 Other ion channels described in T cells There are five main channel types described in T cells; Kv1.3, KCa3.1, ICl,vol, CRAC and TRPM7 channels but other channel types has also been proposed to play a role in T cell physiology. Amongst them are e.g. channels such as voltage-gated Na+ channel (15) and the potassium channels TASK-1 and TASK-3 has likewise been reported in T cell function (98) though their roles still need further investigations and might be by some regarded as questionable (16; 146). Besides TASK-1 and TASK-3 one of those, in T cell activation relatively recent described K+ channels, is KCNK5 (also known as TASK-2 or K2p5.1), thus in 2010 Bittner et al. published a paper giving KCNK5 a role in the autoimmune disease multiple sclerosis (10). Though the presence and functional importance is questioned by some (REF) preliminary data supported that KCNK5 was expressed in T cells and we thus investigated the expression pattern of KCNK5 Page 31 of 117 Copenhagen, sep. 2013 Figure 10: T cell subtypes KCNK5 protein expression and proliferation upon activation A: SDS-PAGE, western blotting and antibodies against KCNK5 and β-actin was used to measure KCNK5 protein expression pattern in T cell subtypes upon 72 hours stimulation and activation with CD3/CD28 beads (n=5-7). B: T cell proliferation upon 72 hours CD3/CD28 activation was measured using a NucleoCounter NC-100 (n=5). both on mRNA and protein levels and found a strong up-regulation of the channel upon T cell activation (see Paper III, fig. 1+2). Bittner et al. furthermore found a significant up-regulation of KCNK5 in the T cell subtypes CD4+ and CD8+ in multiple sclerosis patients during acute relapses and furthermore they found KCNK5 levels to be increased in CD8+ T cells. We have also looked into the KCNK5 expression pattern in various T cell subtypes, confirming an up-regulation upon activation though we find no significant difference in KCNK5 protein expression between the studied subtypes (see fig. 10, own unpublished results). 1.5.4 T cell proliferation T cell proliferation is essential for the battle against pathogen intruders, since the activation and increased number of the right T cells will result in efficient fight. Clonal expansion is one of the most important dogmas in immunology and describes how T cells will proliferate upon activation. Being able to control or ultimately stop proliferation has thus gained high interest since it might be a mean to prevent autoimmune diseases. 1.5.4.1 K+ channels in proliferation K+ channels are shown to be involved in proliferation in various cell types and though the exact mechanisms behind the involvement of ion channels in proliferation is still somewhat uncertain the importance of ion channels are not. It is believed that ion channels help regulate Page 32 of 117 Copenhagen, sep. 2013 proliferation through their potential impact on mechanisms such as membrane potential, calcium homeostasis and or cell volume regulation (see below and section 1.4.2.2). As mentioned previously ion channels and volume regulation is thought to influence cell proliferation since a key feature of the dividing cell is the gain in volume ensuring that the daughter cells do not get smaller and smaller with every division (see section 1.4.2.2). Figure 11: Progression through the cell cycle See the text for details. Figure from Clinical topics, Inc. Different K+ channels have been implicated in cell cycle-control in various cell types where they are thought to play a role in cell cycle progression by control of the membrane potential thus it is seen that the membrane potential in early G1 phase (see fig. 11) is depolarized and followed by a hyperpolarization through G1 to the S phase (see fig. 11) – a hyperpolarization believed to be governed by K+ channels (143). In MCF-7 breast cancer cells this hyperpolarization depend on the activation of ATP-sensitive K+ channels (153) and in brown fat cells (113) melanoma cells (109) and prostate cancer cells (141) voltage-gated K+ channels was shown to be involved. The same mechanisms was also true for the two pore-domain K+ channel TASK-3 in a human lung carcinoma cell line (121). For more on the subject see (116). KCNK5 has been implicated in the regulation of proliferation in human breast cancer cell lines T47D and MCF-7 where it is believed that a increase in KCNK5 is necessary for the progression of the cells from G1 to the S phase (1). Even though we cannot draw direct conclusions from our data two things are certain, T cells proliferate upon activations and KCNK5 is at the same time heavily up-regulated (see fig. 10 A+B and Paper III). Page 33 of 117 Copenhagen, sep. 2013 2 Summary and conclusions During my time as a PhD-student I have worked on the KCNK5 channel in three different set-ups. I have worked on the signaling pathway from acute cell swelling to activation of KCNK5 and RVD in Ehrlich cells, I have studied the long-term effects on channel physiology and expression pattern also in Erhlich cells and last but not least I have studied the expression pattern and physiology in activated human T cell physiology. I have learned a great deal about the channel and in the following I will try and summarize and when possible conclude on my findings. Paper I - KCNK5 is tyrosine phosphorylated upon swelling-induced activation in a time- dependent manner - As tyrosine phosphorylation seem to be involved in activation of the channel tyrosine phosphatases seem to be involved in the de-activation - This tyrosine phosphorylation does not seem to be caused by neither Src nor FAK - The JAK/STAT pathway could be up-streams of the phosphorylation and activation of KCNK5 Paper II - Long-term hypotonicity (48h) reduces the maximum current through KCNK5 - Long-term hypotonicity likewise reduces RVD performance upon cell swelling - This physiological impairment is probably due to an decreased KCNK5 protein expression Paper III - CD3/CD28 activation of human T cells result in proliferation and cell swelling - KCNK5 is heavily up-regulated both on mRNA and protein levels in activated T cells - Despite KCNK5 up-regulation activated T cells has impaired RVD - RVD impairment in activated T cells is probably due to a functional down-regulation of the volume activated anion channel VRAC in these cells Page 34 of 117 Copenhagen, sep. 2013 3 Experimental procedures Most of the experimental procedures are described in the three articles, but one procedure deserves a few comments (section 3.1) and some experiments are not published but are included in this thesis, thus the experimental procedures for these experiments will be explained in the following section (section 3.2). 3.1 cDNA quantification instead of reference genes In the search of suitable reference genes for normalization of Real-Time qPCR data numerous candidates were tested but none showed satisfactory results. Instead I looked at alternative ways of normalizing my data and such an alternative was cDNA quantification using Quant-It Oligreen (Invitrogen) which was described by Lundby et al. in 2005 (91). One could argue that since the same amount of mRNA is used for each cDNA sample generated there should be no particular need for an extra normalization procedure but besides being standard method of operation when conducting Real-Time qPCR previously results verify the need for an additional normalization step. The discrepancies between mRNA starting concentration and cDNA end result could be due partly to pipetting uncertainties or slight differences in PCR reaction in each tube. Thus cDNA was quantified in each sample as described (91) and the data normalized to that value. 3.2 T cell subtypes During my studies of KCNK5 in activated T cell physiology I also looked at the KCNK5 expression pattern in T cell subtypes besides the studies on whole T cell populations. The results are found in section 1.5.3.3 and in fig. 10. 3.2.1 Purification T cell subtypes were purified from human peripheral blood mononuclear cells (PBMC’s) using EasySep enrichment kits from StemCell Tech. PBMC’s were previously purified from human buffy coats obtained from the blood bank at Rigshospitalet as described in paper III. The following subtypes were purified in accordance to manufactures descriptions: Human CD8+ T cells, Human CD4+ T cells, Human Naïve CD8+ T cells and Human Naïve CD4+ T. T cell subtypes were kept in the same media and under the same conditions as the heterogeneous T cell population (see paper III) and were kept either quiescent or activated using Dynabeads Human T-activator CD3/CD28 (Invitrogen, Life Sciences) in the ratio 1:1. 3.2.2 Proliferation Proliferation was simply measured by cell count using the NucleoCounter® NC-100™ machine (ChemoMetech, Allerød, Denmark). Page 35 of 117 Copenhagen, sep. 2013 References 1. Alvarez-Baron CP, Jonsson P, Thomas C, Dryer SE and Williams C. The Two-Pore Domain Potassium Channel KCNK5: Induction by Estrogen Receptor and Role in Proliferation of Breast Cancer Cells. Molecular Endocrinology 25: 1326-1336, 2011. 2. Añazco C, Peña-Münzenmayer G, Araya C, Cid LP, Sepúlveda F and Niemeyer M. G protein modulation of K2P potassium channel TASK-2. Pflugers Arch - Eur J Physiol 1-12, 2013. 3. Andronic J, Bobak N, Bittner S, Ehling P, Kleinschnitz C, Herrmann AM, Zimmermann H, Sauer M, Wiendl H, Budde T, Meuth SG and Sukhorukov VL. Identification of two- pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes. Biochimica et Biophysica Acta (BBA) - Biomembranes 1828: 699- 707, 2013. 4. Barfield JP, Yeung CH and Cooper TG. Characterization of potassium channels involved in volume regulation of human spermatozoa. Molecular Human Reproduction 11: 891- 897, 2005. 5. Barfield JP, Yeung CH and Cooper TG. The Effects of Putative K+ Channel Blockers on Volume Regulation of Murine Spermatozoa. Biology of Reproduction 72: 1275-1281, 2005. 6. Barfod ET, Moore AL, Melnick RF and Lidofsky SD. Src Regulates Distinct Pathways for Cell Volume Control through Vav and Phospholipase Cgamma. J Biol Chem 280: 25548- 25557, 2005. 7. Barriere H, Belfodil R, Rubera I, Tauc M, Lesage F, Poujeol C, Guy N, Barhanin J and Poujeol P. Role of TASK2 Potassium Channels Regarding Volume Regulation in Primary Cultures of Mouse Proximal Tubules. J Gen Physiol 122: 177-190, 2003. 8. Bayliss DA and Barrett PQ. Emerging roles for two-pore-domain potassium channels and their potential therapeutic impact. Trends in Pharmacological Sciences 29: 566-575, 2008. 9. Bayliss DA, Sirois JE and Talley EM. The TASK Family: Two-Pore Domain Background K+ Channels. Mol Interv 3: 205-219, 2003. 10. Bittner S, Bobak N, Herrmann AM, Göbel K, Meuth P, Höhn KG, Stenner MP, Budde T, Wiendl H and Meuth SG. Upregulation of K2P5.1 potassium channels in multiple sclerosis. Ann Neurol 68: 58-69, 2010. 11. Bittner S, Bobak N, Feuchtenberger M, Herrmann A, Gobel K, Kinne R, Hansen A, Budde T, Kleinschnitz C, Frey O, Tony HP, Wiendl H and Meuth S. Expression of K2P5.1 Page 36 of 117 Copenhagen, sep. 2013 potassium channels on CD4+ T lymphocytes correlates with disease activity in rheumatoid arthritis patients. Arthritis Research & Therapy 13: R21, 2011. 12. Bobak N, Bittner S, Andronic J, Hartmann S, ühlpfordt F, Schneider-Hohendorf T, Wolf K, Schmelter C, Göbel K, Meuth P, Zimmermann H, Döring F, Wischmeyer E, Budde T, Wiendl H, Meuth SG and Sukhorukov VL. Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels. Biochim Biophys Acta 1808: 2036-2044, 2011. 13. Bourguignon LYW, Chu A, Jin H and Brandt NR. Ryanodine Receptor-Ankyrin Interaction Regulates Internal Ca Release in Mouse T-lymphoma Cells. J Biol Chem 270: 17917-17922, 1995. 14. Burg MB, Kwon ED and Kültz D. Regulation of gene expression by hypertonicity. Annual Review of Physiology 59: 437-455, 1997. 15. Cahalan MD, Chandy KG, DeCoursey TE and Gupta S. A voltage-gated potassium channel in human T lymphocytes. The Journal of Physiology 358: 197-237, 1985. 16. Cahalan MD and Chandy KG. The functional network of ion channels in T lymphocytes. Immunological Reviews 231: 59-87, 2009. 17. Cahalan MD and Lewis RS. Role of potassium and chloride channels in volume regulation by T lymphocytes. Soc Gen Physiol Ser 43: 281-301, 1988. 18. Cheung RK, Grinstein S, Dosch H-M and Gelfand EW. Volume regulation by human lymphocytes: characterization of the ionic basis for regulatory volume decrease. J Cell Physiol 112: 189-196, 1982. 19. Christensen O and Hoffmann EK. Cell swelling activates K+ and Cl- channels as well as nonselective, stretch-activated cation channels in Ehrlich ascites tumor cells. J of Membr Biol 129: 13-36, 1992. 20. Cid LP, Roa-Rojas HA, Niemeyer MI, González W, Araki M, Araki K and Sepúlveda FV. + TASK-2: a K2P K channel with complex regulation and diverse physiological functions. Frontiers in Physiology 4: 1-9, 2013. 21. COETZEE WA, AMARILLO YIMY, CHIU JOAN, CHOW ALAN, LAU DAVI, McCORMACK TOM, MORENA HERM, NADAL MS, OZAITA ANDE, POUNTNEY DAVI, SAGANICH MICH, DE MIERA EV-S and RUDY BERN. Molecular Diversity of K+ Channels. Annals of the New York Academy of Sciences 868: 233-255, 1999. 22. Cohen DM. SRC family kinases in cell volume regulation. Am J Physiol Cell Physiol 288: C483-C493, 2005. Page 37 of 117 Copenhagen, sep. 2013 23. Cotten JF, Zou HL, Liu C, Au JD and Yost CS. Identification of native rat cerebellar granule cell currents due to background K channel KCNK5 (TASK-2). Molecular Brain Research 128: 112-120, 2004. 24. Crabtree GR. Contingent Genetic Regulatory Events in T Lymphocyte Activation. Science 243: 355-361, 1989. 25. de la Paz L, Lezama R, Torres-Marquez E and Pasantes-Morales H. Tyrosine kinases and amino acid efflux under hyposmotic and ischaemic conditions in the chicken retina. Pflügers Arch 445: 87-96, 2002. 26. Decher N, Maier M, Dittrich W, Gassenhuber J, Brüggemann A, Busch AE and Steinmeyer K. Characterization of TASK-4, a novel member of the pH-sensitive, two- pore domain potassium channel family. FEBS Letters 492: 84-89, 2001. 27. DeCoursey TE, Chandy KG, Gupta S and Cahalan MD. Voltage-gated K+ channels in human T lymphocytes: a role in mitogenesis? Nature 307: 465-468, 1984. 28. Deutsch C and Chen LQ. Heterologous expression of specific K+ channels in T lymphocytes: functional consequences for volume regulation. Proceedings of the National Academy of Sciences 90: 10036-10040, 1993. 29. Deutsch C and Lee SC. Cell volume regulation in lymphocytes. Renal Physiol Biochem 11: 260-276, 1988. 30. Dubois JM and Rouzaire-Dubois B. The influence of cell volume changes on tumour cell proliferation. Eur Biophys J 33: 227-232, 2004. 31. Duprat F, Lesage F, Fink M, Reyes R, Heurteaux C and Lazdunski M. TASK, a human background K+ channel to sense external pH variations near physiological pH. EMBO J 16: 5464-5471, 1997. 32. Enyedi P and Czirják G. Molecular Background of Leak K+ Currents: Two-Pore Domain Potassium Channels. Physiol Rev 90: 559-605, 2010. 33. Fanger CM, Ghanshani S, Logsdon NJ, Rauer H, Kalman K, Zhou J, Beckingham K, Chandy KG, Cahalan MD and Aiyar J. Calmodulin Mediates Calcium-dependent Activation of the Intermediate Conductance KCa Channel,IKCa1. J Biol Chem 274: 5746- 5754, 1999. 34. Felipe A, Snyders DJ, Deal KK and Tamkun MM. Influence of cloned voltage-gated K+ channel expression on alanine transport, Rb+ uptake, and cell volume. American Journal of Physiology - Cell Physiology 265: C1230-C1238, 1993. 35. Flores CA, Cid LP, Niemeyer MI and Sepúlveda FV. B lymphocytes taken to task: a role for a background conductance K2P K+ channel in B cells. Focus on "Expression of TASK- Page 38 of 117 Copenhagen, sep. 2013 2 and its upregulation by B cell receptor stimulation in WEHI-231 mouse immature B cells". American Journal of Physiology - Cell Physiology 300: C976-C978, 2011. 36. Fukushima Y, Hagiwara S and Henkart M. Potassium current in clonal cytotoxic T lymphocytes from the mouse. The Journal of Physiology 351: 645-656, 1984. 37. Gabriel A, Abdallah M, Yost CS, Winegar BD and Kindler CH. Localization of the tandem pore domain K+ channel KCNK5 (TASK-2) in the rat central nervous system. Molecular Brain Research 98: 153-163, 2002. 38. Gestreau C, Heitzmann D, Thomas J, Dubreuil V+, Bandulik S, Reichold M, Bendahhou Sd, Pierson P, Sterner C, Peyronnet-Roux J, Benfriha Cr, Tegtmeier I, Ehnes H, Georgieff M, Lesage F, Brunet JF, Goridis C, Warth R and Barhanin J. Task2 potassium channels set central respiratory CO2 and O2 sensitivity. Proceedings of the National Academy of Sciences 107: 2325-2330, 2010. 39. Girard C, Duprat F, Terrenoire C, Tinel N, Fosset M, Romey G, Lazdunski M and Lesage F. Genomic and Functional Characteristics of Novel Human Pancreatic 2P Domain K+ Channels. Biochemical and Biophysical Research Communications 282: 249-256, 2001. 40. Goldsmith MA and Weiss A. Early signal transduction by the antigen receptor without commitment to T cell activation. Science 240: 1029-1031, 1988. 41. Goldstein SAN, Bayliss DA, Kim D, Lesage F, Plant LD and Rajan S. International Union of Pharmacology. LV. Nomenclature and Molecular Relationships of Two-P Potassium Channels. Pharmacol Rev 57: 527-540, 2005. 42. Goldstein SAN, Bockenhauer D, O'Kelly I and Zilberberg N. Potassium leak channels and the KCNK family of two-p-domain subunits. Nat Rev Neurosci 2: 175-184, 2001. 43. Goldstein SA, Price LA, Rosenthal DN and Pausch MH. ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences 93: 13256-13261, 1996. 44. Grunnet M, MacAulay N, Jorgensen N, Jensen B, Olesen SP and Klaerke D. Regulation of cloned, Ca2+-activated K+ channels by cell volume changes. Pflügers Arch - Eur J Physiol 444: 167-177, 2002. 45. Guse AH, da Silva CP, Emmrich F, Ashamu GA, Potter BV and Mayr GW. Characterization of cyclic adenosine diphosphate-ribose-induced Ca2+ release in T lymphocyte cell lines. The Journal of Immunology 155: 3353-3359, 1995. 46. Guse AH, da Silva CP, Berg I, Skapenko AL, Weber K, Heyer P, Hohenegger M, Ashamu GA, Schulze-Koops H, Potter BVL and Mayr GW. Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose. Nature 398: 70-73, 1999. Page 39 of 117 Copenhagen, sep. 2013 47. Hazama A and Okada Y. Ca2+ sensitivity of volume-regulatory K+ and Cl- channels in cultured human epithelial cells. The Journal of Physiology 402: 687-702, 1988. 48. Heginbotham L, Lu Z, Abramson T and MacKinnon R. Mutations in the K+ channel signature sequence. Biophysical Journal 66: 1061-1067, 1994. 49. Hodgkin AL and Huxley AF. Potassium leakage from an active nerve fibre. The Journal of Physiology 106: 341-367, 1947. 50. Hodgkin AL and Huxley AF. A quantitative description of membrane current and its application to conduction and excitation in nerve. The Journal of Physiology 117: 500- 544, 1952. 51. Hoffmann EK. Regulation of cell volume by selective changes in the leak permeabilities of Ehrlich ascites tumor cells. Alfred Benzon Symp XI 197-417, 1978. 52. Hoffmann EK and Dunham PB. Membrane mechanisms and intracellular signalling in cell volume regulation. Int Rev Cytol 161: 173-262, 1995. 53. Hoffmann Else K. Intracellular signalling involved in volume regulatory decrease. Cellular Physiology and Biochemistry 10: 273-288, 2000. 54. Hoffmann Else K, Simonsen LO and Lambert IH. Volume-induced increase of K+ and Cl- permeabilities in Ehrlich ascites tumor cells. Role of internal Ca2+. J Membr Biol 78: 211-222, 1984. 55. Hoffmann EK. Ion Channels Involved in Cell Volume Regulation: Effects on Migration, Proliferation, and Programmed Cell Death in Non Adherent EAT Cells and Adherent ELA Cells. Cellular Physiology and Biochemistry 28: 1061-1078, 2011. 56. Hoffmann EK, Lambert IH and Pedersen SF. Physiology of Cell Volume Regulation in Vertebrates. Physiol Rev 89: 193-277, 2009. 57. Hougaard C, Niemeyer MI, Hoffmann EK and Sepúlveda FV. K+ currents activated by leukotriene D4 or osmotic swelling in Ehrlich ascites tumour cells. Pflügers Archiv European Journal of Physiology 440: 283-294, 2000. 58. Imboden JB and Stobo JD. Transmembrane signalling by the T cell antigen receptor. Perturbation of the T3-antigen receptor complex generates inositol phosphates and releases calcium ions from intracellular stores. The Journal of Experimental Medicine 161: 446-456, 1985. 59. Imboden JB, Weiss A and Stobo JD. The antigen receptor on a human T cell line initiates activation by increasing cytoplasmic free calcium. The Journal of Immunology 134: 663-665, 1985. Page 40 of 117 Copenhagen, sep. 2013 60. Jenkinson DH. Potassium channels multiplicity and challenges. British Journal of Pharmacology 147: S63-S71, 2006. 61. Jørgensen NK, Lambert IH and Hoffmann EK. Role of LTD4 in the Regulatory Volume Decrease Response in Ehrlich Ascites Tumor Cells. J Membrane Biol 151: 159-173, 1996. 62. Jørgensen NK, Christensen S, Harbak H, Brown AM, Lambert IH, Hoffmann EK and Simonsen LO. On the Role of Calcium in the Regulatory Volume Decrease (RVD) Response in Ehrlich Mouse Ascites Tumor Cells. J Membrane Biol 157: 281-299, 1997. 63. Jørgensen NK, Pedersen SF, Rasmussen HB, Grunnet M, Klaerke DA and Olesen SP. Cell swelling activates cloned Ca2+-activated K+ channels: a role for the F-actin cytoskeleton. Biochim Biophys Acta 1615: 115-125, 2003. 64. Kang D and Kim D. Single-channel properties and pH sensitivity of two-pore domain K+ channels of the TALK family. Biochemical and Biophysical Research Communications 315: 836-844, 2004. 65. Karttunen J and Shastri N. Measurement of ligand-induced activation in single viable T cells using the lacZ reporter gene. Proceedings of the National Academy of Sciences 88: 3972-3976, 1991. 66. Ketchum KA, Joiner WJ, Sellers AJ, Kaczmarek LK and Goldstein SAN. A new family of outwardly rectifying potassium channel proteins with two pore domains in tandem. Nature 376: 690-695, 1995. 67. Khanna R, Chang MC, Joiner WJ, Kaczmarek LK and Schlichter LC. hSK4/hIK1, a Calmodulin-binding KCa Channel in Human T Lymphocytes: ROLES IN PROLIFERATION AND VOLUME REGULATION. J Biol Chem 274: 14838-14849, 1999. 68. Kim JE, Kwak SE and Kang TC. Upregulated TWIK-related acid-sensitive K+ channel-2 in neurons and perivascular astrocytes in the hippocampus of experimental temporal lobe epilepsy. Epilepsia 50: 654-663, 2009. 69. Kirkegaard SS, Lambert IH, Gammeltoft S and Hoffmann EK. Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation. American Journal of Physiology - Cell Physiology 299: C844-C853, 2010. 70. Klausen TK, Preisler S, Pedersen SF and Hoffmann EK. Monovalent ions control proliferation of Ehrlich Lettre ascites cells. American Journal of Physiology - Cell Physiology 299: C714-C725, 2010. 71. Kramhoft B, Lambert IH, Hoffmann EK and Jorgensen F. Activation of Cl-dependent K transport in Ehrlich ascites tumor cells. American Journal of Physiology - Cell Physiology 251: C369-C379, 1986. Page 41 of 117 Copenhagen, sep. 2013 72. L'Hoste S, Barriere H, Belfodil R, Rubera I, Duranton C, Tauc M, Poujeol C, Barhanin J and Poujeol P. Extracellular pH alkalinization by Cl-/HCO3- exchanger is crucial for TASK2 activation by hypotonic shock in proximal cell lines from mouse kidney. Am J Physiol Renal Physiol 292: F628-F638, 2007. 73. L'Hoste S, Poet M, Duranton C, Belfodil R, Barriere H, Rubera I, Tauc M, Poujeol C, Barhanin J and Poujeol P. Role of TASK2 in the Control of Apoptotic Volume Decrease in Proximal Kidney Cells. J Biol Chem 282: 36692-36703, 2007. 74. Lambert IH, Hoffmann Else K and Christensen P. Role of prostaglandins and leukotrienes in volume regulation by Ehrlich ascites tumor cells. J Membr Biol 98: 247- 256, 1987. 75. Lambert IH, Hoffmann EK and Jørgensen F. Membrane potential, anion and cation conductances in Ehrlich ascites tumor cells. J Membr Biol 111: 113-131, 1989. 76. Lambert I. Effect of arachidonic acid, fatty acids, prostaglandins, and leukotrienes on volume regulation in Ehrlich ascites tumor cells. J Membrain Biol 98: 207-221, 1987. 77. Lang F, Ritter M, Gamper N, Huber S, Fillon S, Tanneur V, Lepple-Wienhues A, Szabo I and Bulbins E. Cell Volume in the Regulation of Cell Proliferation and Apoptotic Cell Death. Cellular Physiology and Biochemistry 10: 417-428, 2000. 78. Lang F, Busch GL, Ritter M, Völkl H, Waldegger S, Gulbins E and Häussinger D. Functional Significance of Cell Volume Regulatory Mechanisms. Physiol Rev 78: 247- 306, 1998. 79. Lang F and Hoffmann EK. Role of Ion Transport in Control of Apoptotic Cell Death. In: Comprehensive Physiology, John Wiley & Sons, Inc., 2012. 80. Lee SC, Price M, Prystowsky MB and Deutsch C. Volume response of quiescent and interleukin 2-stimulated T-lymphocytes to hypotonicity. American Journal of Physiology - Cell Physiology 254: C286-C296, 1988. 81. Lepple-Wienhues A, Szabò I, Laun T, Kaba NK, Gulbins E and Lang F. The Tyrosine Kinase p56lck Mediates Activation of Swelling-induced Chloride Channels in Lymphocytes. The Journal of Cell Biology 141: 281-286, 1998. 82. Lesage F, Guillemare E, Fink M, Duprat F, Lazdunski M, Romey G and Barhanin J. TWIK-1, a ubiquitous human weakly inward rectifying K+ channel with a novel structure. EMBO J 15: 1004-1011, 1996. 83. Lesage F and Lazdunski M. Molecular and functional properties of two-pore-domain potassium channels. Am J Physiol Renal Physiol 279: F793-F801, 2000. Page 42 of 117 Copenhagen, sep. 2013 84. Lesage F, Reyes R, Fink M, Duprat F, Guillemare E and Lazdunski M. Dimerization of TWIK-1 K+ channel subunits via a disulfide bridge. EMBO J 15: 6400-6407, 1996. 85. Lewis RS, Ross PE and Cahalan MD. Chloride channels activated by osmotic stress in T lymphocytes. J Gen Physiol 101: 801-826, 1993. 86. Lewis RS. CALCIUM SIGNALING MECHANISMS IN T LYMPHOCYTES. Annu Rev Immunol 19: 497-521, 2001. 87. Lewis RS and Cahalan MD. Potassium and Calcium Channels in Lymphocytes. Annu Rev Immunol 13: 623-653, 1995. 88. Lezama R, Ortega A, Ordaz B and Pasantes-Morales H. Hyposmolarity-induced ErbB4 phosphorylation and its influence on the non-receptor tyrosine kinase network response in cultured cerebellar granule neurons. J Neurochem 93: 1189-1198, 2005. 89. Lin W, Burks CA, Hansen DR, Kinnamon SC and Gilbertson TA. Taste Receptor Cells Express pH-Sensitive Leak K+ Channels. Journal of Neurophysiology 92: 2909-2919, 2004. 90. Liou J, Kim ML, Do Heo W, Jones JT, Myers JW, Ferrell J and Meyer T. STIM Is a Ca2+ Sensor Essential for Ca2+-Store-Depletion-Triggered Ca2+ Influx. Current Biology 15: 1235-1241, 2005. 91. Lundby C, Nordsborg N, Kusuhara K, Kristensen K, Neufer P and Pilegaard H. Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies. European Journal of Applied Physiology 95: 351-360, 2005. 92. Madsen CP, Klausen TK, Fabian A, Hansen BJ, Pedersen SF and Hoffmann EK. On the role of TRPC1 in control of Ca2+ influx, cell volume, and cell cycle. American Journal of Physiology - Cell Physiology 303: C625-C634, 2012. 93. Maeno E, Ishizaki Y, Kanaseki T, Hazama A and Okada Y. Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proceedings of the National Academy of Sciences 97: 9487-9492, 2000. 94. Maltsev VA. Oscillating and triggering properties of T cell membrane potential. Immunology Letters 26: 277-282, 1990. 95. Matteson DR and Deutsch C. K+ channels in T lymphocytes: a patch clamp study using monoclonal antibody adhesion. Nature 307: 468-471, 1984. 96. McManus ML, Churchwell KB and Strange K. Regulation of Cell Volume in Health and Disease. N Engl J Med 333: 1260-1267, 1995. Page 43 of 117 Copenhagen, sep. 2013 97. Medhurst AD, Rennie G, Chapman CG, Meadows H, Duckworth MD, Kelsell RE, Gloger II and Pangalos MN. Distribution analysis of human two pore domain potassium channels in tissues of the central nervous system and periphery. Molecular Brain Research 86: 101-114, 2001. 98. Meuth SG, Bittner S, Meuth P, Simon OJ, Budde T and Wiendl H. TWIK-related Acid- sensitive K+ Channel 1 (TASK1) and TASK3 Critically Influence T Lymphocyte Effector Functions. J Biol Chem 283: 14559-14570, 2008. 99. Morton MJ, Abohamed A, Sivaprasadarao A and Hunter M. pH sensing in the two-pore domain K+ channel, TASK2. Proceedings of the National Academy of Sciences of the United States of America 102: 16102-16106, 2005. 100. Nam JH, Shin DH, Zheng H, Lee DS, Park SJ, Park KS and Kim SJ. Expression of TASK-2 and its upregulation by B cell receptor stimulation in WEHI-231 mouse immature B cells. American Journal of Physiology - Cell Physiology 300: 1013-1022, 2011. 101. Nam JH, Woo JE, Uhm DY and Kim SJ. Membrane-delimited Regulation of Novel Background K+ Channels by MgATP in Murine Immature B Cells. J Biol Chem 279: 20643-20654, 2004. 102. Negulescu PA, Shastri N and Cahalan MD. Intracellular calcium dependence of gene expression in single T lymphocytes. Proceedings of the National Academy of Sciences 91: 2873-2877, 1994. 103. Nielsen DK, Jensen AKr, Harbak H, Christensen SrC and Simonsen LO. Cell content of phosphatidylinositol (4,5)bisphosphate in Ehrlich mouse ascites tumour cells in response to cell volume perturbations in anisotonic and in isosmotic media. The Journal of Physiology 582: 1027-1036, 2007. 104. Niemeyer MI, Cid LP, Barros LF and Sepúlveda FV. Modulation of the Two-pore Domain Acid-sensitive K+ Channel TASK-2 (KCNK5) by Changes in Cell Volume. J Biol Chem 276: 43166-43174, 2001. 105. Niemeyer MI, Cid LP, Peña-Münzenmayer G and Sepúlveda FV. Separate Gating Mechanisms Mediate the Regulation of K2P Potassium Channel TASK-2 by Intra- and Extracellular pH. J Biol Chem 285: 16467-16475, 2010. 106. Niemeyer MI, Cid LP and Sepúlveda FV. K+ conductance activated during regulatory volume decrease. The channels in Ehrlich cells and their possible molecular counterpart. Comp Biochem Physiol A Mol Integr Physiol 130: 565-575, 2001. 107. Niemeyer MI, González-Nilo FD, Zúñiga L, González W, Cid LP and Sepúlveda FV. Neutralization of a single arginine residue gates open a two-pore domain, alkali- activated K+ channel. Proceedings of the National Academy of Sciences 104: 666-671, 2007. Page 44 of 117 Copenhagen, sep. 2013 108. Niemeyer MI, Hougaard C, Hoffmann EK, Jørgensen F, Stutzin A and Sepúlveda FV. Characterisation of cell swelling-activated K+ selective cunductance of ehrlich ascites tumour cells. J Physiol 524: 757-767, 2000. 109. Nilius B and Wohlrab W. Potassium channels and regulation of proliferation of human melanoma cells. The Journal of Physiology 445: 537-548, 1992. 110. O'Connell AD, Morton MJ and Hunter M. Two-pore domain K+ channels--molecular sensors. Biochimica et Biophysica Acta (BBA) - Biomembranes 1566: 152-161, 2002. 111. Okada Y. Ion channels and transporters involved in cell volume regulation and sensor mechanisms. Cell Biochem Biophys 41: 233-258, 2004. 112. Panyi G, Varga Z and Gáspár R. Ion channels and lymphocyte activation. Immunology Letters 92: 55-66, 2004. 113. Pappone PA and Ortiz-Miranda SI. Blockers of voltage-gated K channels inhibit proliferation of cultured brown fat cells. American Journal of Physiology - Cell Physiology 264: C1014-C1019, 1993. 114. Partiseti M, Le Deist F, Hivroz C, Fischer A, Korn H and Choquet D. The calcium current activated by T cell receptor and store depletion in human lymphocytes is absent in a primary immunodeficiency. J Biol Chem 269: 32327-32335, 1994. 115. Patel AJ and Honor E. Properties and modulation of mammalian 2P domain K+ channels. Trends in Neurosciences 24: 339-346, 2001. 116. Patel A and Lazdunski M. The 2P-domain K + channels: role in apoptosis and tumorigenesis. Pflügers Archiv European Journal of Physiology 448: 261-273, 2004. 117. Pedersen SF, Hoffmann EK and Mills JW. The cytoskeleton and cell volume regulation. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 130: 385-399, 2001. 118. Pedersen SF, Prenen J, Droogmans G, Hoffmann EK and Nilius B. Separate Swelling- and Ca2+-activated Anion Currents in Ehrlich Ascites Tumor Cells. J Membr Biol 163: 97- 110, 1998. 119. Pedersen, Stine Falsig, Hoffmann, Else Kay, and Novak, Ivana. Cell volume regulation in epithelial physiology and cancer. 2013. Ref Type: Unpublished Work 120. Pedersen S, Lambert IH, Thoroed SM and Hoffmann EK. Hypotonic cell swelling induces translocation of the isoform of cytosolic phospholipase A2 but not the isoform in Ehrlich ascites tumor cells. European Journal of Biochemistry 267: 5531-5539, 2000. Page 45 of 117 Copenhagen, sep. 2013 121. Pei L, Wiser O, Slavin A, Mu D, Powers S, Jan LY and Hoey T. Oncogenic potential of TASK3 (Kcnk9) depends on K+ channel function. Proceedings of the National Academy of Sciences 100: 7803-7807, 2003. 122. Pendergrass W, Angello J, Kirschner M and Norwood T. The relationship between the rate of entry into S phase, concentration of DNA polymerase alpha, and cell volume in human diploid fibroblast-like monokaryon cells. Experimental Cell Research 192: 418- 425, 1991. 123. Poulsen KA, Andersen EC, Hansen CF, Klausen TK, Hougaard C, Lambert IH and Hoffmann EK. Deregulation of apoptotic volume decrease and ionic movements in multidrug-resistant tumor cells: role of chloride channels. Am J Physiol Cell Physiol 298: C14-C25, 2010. 124. Prakriya M, Feske S, Gwack Y, Srikanth S, Rao A and Hogan PG. Orai1 is an essential pore subunit of the CRAC channel. Nature 443: 230-233, 2006. 125. Prevarskaya N, Skryma R and Shuba Y. Ion channels and the hallmarks of cancer. Trends in Molecular Medicine 16: 107-121, 2010. 126. Putney JW. Capacitative calcium entry revisited. Cell Calcium 11: 611-624, 1990. 127. Qian D and Weiss A. T cell antigen receptor signal transduction. Current Opinion in Cell Biology 9: 205-212, 1997. 128. Reyes R, Duprat F, Lesage F, Fink M, Salinas M, Farman N and Lazdunski M. Cloning and Expression of a Novel pH-sensitive Two Pore Domain K+ Channel from Human Kidney. J Biol Chem 273: 30863-30869, 1998. 129. Riquelme G, Sepúlveda FV, Jørgensen F, Pedersen S and Hoffmann EK. Swelling- activated potassium currents of Ehrlich ascites tumour cells. Biochim Biophys Acta 1371: 101-106, 1998. 130. Ritter M, Schratzberger P, Rossmann H, Wöll E, Seiler K, Seidler U, Reinisch N, Kähler CM, Zwierzina H, Lang HJ, Lang F, Paulmichl M and Wiedermann CJ. Effect of inhibitors of Na+/H+-exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes. British Journal of Pharmacology 124: 627-638, 1998. 131. Rizoli SB, Kapus A, Fan J, Li YH, Marshall JC and Rotstein OD. Immunomodulatory Effects of Hypertonic Resuscitation on the Development of Lung Inflammation Following Hemorrhagic Shock. The Journal of Immunology 161: 6288-6296, 1998. 132. Roman R, Feranchak AP, Troetsch M, Dunkelberg JC, Kilic G, Schlenker T, Schaack J and Fitz JG. Molecular characterization of volume-sensitive SKCa channels in human liver cell lines. AJP 282: G116-G122, 2002. Page 46 of 117 Copenhagen, sep. 2013 133. Roos J, DiGregorio PJ, Yeromin AV, Ohlsen K, Lioudyno M, Zhang S, Safrina O, Kozak JA, Wagner SL, Cahalan MD, Veli+ºelebi G+ and Stauderman KA. STIM1, an essential and conserved component of store-operated Ca2+ channel function. The Journal of Cell Biology 169: 435-445, 2005. 134. Rouzaire-Dubois B+, O'regan S and Dubois JM. Cell size-dependent and independent proliferation of rodent neuroblastoma x glioma cells. J Cell Physiol 203: 243-250, 2005. 135. Rusthoven JJ, Adams J, Cheung RK and Dosch HM. Volume regulation of natural killer cells under hypotonic stress: Comparison with T and B cell subpopulations. Immunology Letters 13: 203-207, 1986. 136. Rusznák Z, Pocsai K, Kovács I, Pór A, Pál B, Bíró T and Szücs G. Differential distribution of TASK-1, TASK-2 and TASK-3 immunoreactivities in the rat and human cerebellum. Cellular and Molecular Life Sciences 61: 1532-1542, 2004. 137. Sanders KM and Koh SD. Two-pore-domain potassium channels in smooth muscles: new components of myogenic regulation. The Journal of Physiology 570: 37-43, 2006. 138. Schlichter LC and Sakellaropoulos G. Intracellular Ca2+ Signaling Induced by Osmotic Shock in Human T Lymphocytes. Experimental Cell Research 215: 211-222, 1994. 139. Schwab A, Nechyporuk-Zloy V, Fabian A and Stock C. Cells move when ions and water flow. Pflugers Arch - Eur J Physiol 453: 421-432, 2007. 140. Shieh CC, Coghlan M, Sullivan JP and Gopalakrishnan M. Potassium Channels: Molecular Defects, Diseases, and Therapeutic Opportunities. Pharmacol Rev 52: 557- 594, 2000. 141. Skryma RN, Prevarskaya NB, Dufy-Barbe L, Odessa MF, Audin J and Dufy B. Potassium conductance in the androgen-sensitive prostate cancer cell line, LNCaP: Involvement in cell proliferation. Prostate 33: 112-122, 1997. 142. Strange K. Cellular volume homeostasis. Advances in Physiology Education 28: 155-159, 2004. 143. Strobl JS, Wonderlin WF and Flynn DC. Mitogenic signal transduction in human breast cancer cells. General Pharmacology: The Vascular System 26: 1643-1649, 1995. 144. Takemura H, Hughes AR, Thastrup O and Putney JW. Activation of calcium entry by the tumor promoter thapsigargin in parotid acinar cells. Evidence that an intracellular calcium pool and not an inositol phosphate regulates calcium fluxes at the plasma membrane. J Biol Chem 264: 12266-12271, 1989. Page 47 of 117 Copenhagen, sep. 2013 145. Thoroed SM, Lauritzen L, Lambert IH, Hansen HS and Hoffmann EK. Cell Swelling Activates Phospholipase A2 in Ehrlich Ascites Tumor Cells. J Membrane Biol 160: 47-58, 1997. 146. Varga Z, Hajdu P and Panyi G. Ion channels in T lymphocytes: An update on facts, mechanisms and therapeutic targeting in autoimmune diseases. Immunology Letters 130: 19-25, 2010. 147. Verheugen JA, Vijverberg HP, Oortgiesen M and Cahalan MD. Voltage-gated and Ca(2+)-activated K+ channels in intact human T lymphocytes. Noninvasive measurements of membrane currents, membrane potential, and intracellular calcium. J Gen Physiol 105: 765-794, 1995. 148. Verheugen JAH and Vijverberg HPM. Intracellular Ca2+ oscillations and membrane potential fluctuations in intact human T lymphocytes: role of K+ channels in Ca2+ signaling. Cell Calcium 17: 287-300, 1995. 149. Vig M, Beck A, Billingsley JM, Lis A, Parvez S, Peinelt C, Koomoa DL, Soboloff J, Gill DL, Fleig A, Kinet JP and Penner R. CRACM1 Multimers Form the Ion-Selective Pore of the CRAC Channel. Current Biology 16: 2073-2079, 2006. 150. Wang J, Morishima S and Okada Y. IK channels are involved in the regulatory volume decrease in human epithelial cells. AJP 284: C77-C84, 2003. 151. Warth R, Barrière H, Meneton P, Bloch M, Thomas J, Tauc M, Heitzmann D, Romeo E, Verrey F, Mengual R, Guy N, Bendahhou S, Lesage F, Poujeol P and Barhanin J. Proximal renal tubular acidosis in TASK2 K+ channel-deficient mice reveals a mechanism for stabilizing bicarbonate transport. Proceedings of the National Academy of Sciences of the United States of America 101: 8215-8220, 2004. 152. Weiss A, Imboden J, Shoback D and Stobo J. Role of T3 surface molecules in human T- cell activation: T3-dependent activation results in an increase in cytoplasmic free calcium. Proceedings of the National Academy of Sciences 81: 4169-4173, 1984. 153. Woodfork KA, Wonderlin WF, Peterson VA and Strobl JS. Inhibition of ATP-sensitive potassium channels causes reversible cell-cycle arrest of human breast cancer cells in tissue culture. J Cell Physiology 162: 163-171, 1995. 154. Yamamoto Y, Kummer W, Atoji Y and Suzuki Y. TASK-1, TASK-2, TASK-3 and TRAAK immunoreactivities in the rat carotid body. Brain Research 950: 304-307, 2002. 155. Yeromin AV, Zhang SL, Jiang W, Yu Y, Safrina O and Cahalan MD. Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature 443: 226-229, 2006. Page 48 of 117 Copenhagen, sep. 2013 156. Yun Cho S, Beckett EA, Baker SA, Han I, Park KJ, Monaghan K, Ward SM, Sanders KM and Koh SD. A pH-sensitive potassium conductance (TASK) and its function in the murine gastrointestinal tract. The Journal of Physiology 565: 243-259, 2005. 157. Zhang XM, Zhong YM and Yang XL. TASK-2 is expressed in proximal neurons in the rat retina. NeuroReport 20: 2009. 158. Zheng H, Nam JH, Pang B, Shin DH, Kim JS, Chun YS, Park JW, Bang H, Kim WK, Earm YE and Kim SJ. Identification of the large-conductance background K+ channel in mouse B cells as TREK-2. American Journal of Physiology - Cell Physiology 297: C188-C197, 2009. 159. Zweifach A and Lewis RS. Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores. Proceedings of the National Academy of Sciences 90: 6295-6299, 1993. Page 49 of 117 Copenhagen, sep. 2013 Page 50 of 117 Copenhagen, sep. 2013 4 Paper I 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 Page 51 of 117 Copenhagen, sep. 2013 Page 52 of 117 Am J Physiol Cell Physiol 299: C844–C853, 2010. First published July 14, 2010; doi:10.1152/ajpcell.00024.2010. Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation Signe Skyum Kirkegaard,2 Ian Henry Lambert,1 Steen Gammeltoft,2 and Else Kay Hoffmann1 1Section for Cell and Developmental Biology, Department of Biology, University of Copenhagen, Copenhagen; and 2Department of Clinical Biochemistry, Glostrup Hospital, Glostrup, Denmark Submitted 22 January 2010; accepted in final form 6 July 2010 Kirkegaard SS, Lambert IH, Gammeltoft S, Hoffmann EK. Ac- molytes (e.g., taurine) are important in the RVD response (16), tivation of the TASK-2 channel after cell swelling is dependent on and it has been estimated that ϳ70% of the loss of osmolytes Downloaded from tyrosine phosphorylation. Am J Physiol Cell Physiol 299: C844–C853, in Ehrlich ascites tumor cells (EATC) during RVD is due to the 2010. First published July 14, 2010; doi:10.1152/ajpcell.00024.2010.— ϩ ϩ loss of KCl (14). Various K channels have been shown to be The swelling-activated K currents (IK,vol) in Ehrlich ascites tumor implicated in RVD depending on cell type; i.e., 1)Ca2ϩ- cells (EATC) has been reported to be through the two-pore domain ϩ (K ), TWIK-related acid-sensitive Kϩ channel 2 (TASK-2). The activated small conductance K (SK) channels (24, 42), inter- 2p mediate conductance Kϩ (IK) channels (3, 7, 24, 25, 46), and regulatory volume decrease (RVD), following hypotonic exposure in ϩ Big conductance K (BK) channels (10, 23); 2) stretch-acti- http://ajpcell.physiology.org/ EATC, is rate limited by IK,vol indicating that inhibition of RVD ϩ reflects inhibition of TASK-2. We find that in EATC the tyrosine kinase vated K channels; 3) voltage-dependent channels; 4) the ϩ inhibitor genistein inhibits RVD by 90%, and that the tyrosine phos- MinK channel; and 5) the two-pore domain K channel (4, phatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) [mpV(pic)] 31–35, 37). The volume-sensitive ion channels in EATC are shifted the volume set point for inactivation of the channel to a lower cell the volume-regulated anion channel (VRAC) (38) and the ϩ volume. Swelling-activated K efflux was impaired by genistein and TWIK-related acid-sensitive Kϩ channel-2 (TASK-2) (22, 33, the Src kinase family inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl) 41), which belongs to the family of two-pore domain channels pyrazolo[3,4-d]pyrimidine (PP2) and enhanced by the tyrosine phos- (K2p). TASK-2 is sensitive to clofilium (35), independent of phatase inhibitor mpV(pic). With the use of the TASK-2 inhibitor intracellular Ca2ϩ (35, 41), and highly sensitive to external pH clofilium, it is demonstrated that mpV(pic) increased the volume- ϩ sensitive part of the Kϩ efflux 1.3 times. To exclude Kϩ efflux via a (21). Besides being the swelling-activated K channel in Ϫ Ϫ EATC, TASK-2 has also been found to be involved in volume at Copenhagen University Library on July 10, 2013 KCl cotransporter, cellular Cl was substituted with NO3 . Also under these conditions Kϩ efflux was completely blocked by genistein. Thus regulation in kidney cells (4) and in murine spermatozoa (1). tyrosine kinases seem to be involved in the activation of the volume- Moreover, TASK-2 has been associated with apoptotic volume sensitive Kϩ channel, whereas tyrosine phosphatases appears to be decrease in EATC cells following cisplatin exposure (40). For involved in inactivation of the channel. Overexpressing TASK-2 in further details about volume-sensitive Kϩ channels see Refs. human embryonic kidney (HEK)-293 cells increased the RVD rate 19 and 44. and reduced the volume set point. TASK-2 has tyrosine sites, and Tyrosine kinases are important under several physiological precipitation of TASK-2 together with Western blotting and antibod- conditions such as cell proliferation, migration, differentiation, ies against phosphotyrosines revealed a cell swelling-induced, time- metabolism, and immune system function, but also a high dependent tyrosine phosphorylation of the channel. Even though we found an inhibiting effect of PP2 on RVD, neither Src nor the focal number of oncogenes are tyrosine kinases (for a review see adhesion kinase (FAK) seem to be involved. Inhibitors of the epider- Ref. 6). There are several evidences for the role of tyrosine mal growth factor receptor tyrosine kinases had no effect on RVD, kinases in RVD (8). The nonreceptor tyrosine kinases Src and whereas the Janus kinase (JAK) inhibitor cucurbitacin inhibited the focal adhesion kinase (FAK) has previously been reported to RVD by 40%. It is suggested that the cytokine receptor-coupled be activated during hypotonic stimulation in various cell types; JAK/STAT pathway is upstream of the swelling-induced phosphory- i.e., phosphorylation of an activation site in FAK have previ- lation and activation of TASK-2 in EATC. ously been reported in, for example, chicken retina cells (9), cell volume regulation; Janus kinase; volume-sensitive channels human umbilical vein endothelial cells (15), and in cerebellar granule neurons (30), whereas hypotonic activation of Src has been reported in cerebellar granule neurons (30), rat hepato- THE ABILITY of a cell to volume regulate after volume pertur- cytes (45), and in rat hepatoma cells (2). There is however no bations is fundamental for its homeostasis. When exposed to an clear evidence linking the activation of tyrosine kinases to ϩ increase in the internal or a decrease in the external osmolyte activation of swelling-activated K currents (IKvol). In EATC concentration, most mammalian cells swell and subsequently preliminary results showed that the relatively unspecific ty- compensate for this swelling by a release of osmolytes through rosine kinase inhibitor genistein (371 M) strongly inhibited different membrane transporters followed by a concomitant RVD, and the tyrosine phosphatase inhibitor monoperoxo(pi- efflux of water, a process termed regulatory volume decrease colinato)-oxo-vanadate(V) [mpV(pic)] (10 M) significantly (RVD) (19). Especially swelling-activated Kϩ and ClϪ chan- decreased the volume set point after RVD (17). Thus tyrosine nels as well as volume-sensitive transporters of organic os- phosphorylation appears to play a role in RVD in EATC, possibly as a part of the volume-sensing mechanism. The objective of this study was thus to examine the involvement of Address for reprint requests and other correspondence: E. K. Hoffmann, Section of Cell and Developmental Biology, Dept. of Biology, The August tyrosine kinases/phosphatases in the swelling-induced activa- Krogh Bldg, Univ. of Copenhagen, 13, Universitetsparken, DK-2100, Copen- tion/deactivation of TASK-2 in EATC. We specifically test hagen, Denmark (e-mail: [email protected]). whether overexpression of TASK-2 improves the RVD re- C844 0363-6143/10 Copyright © 2010 the American Physiological Society http://www.ajpcell.org TYROSINE PHOSPHORYLATION AND VOLUME-ACTIVATED CHANNELS C845 sponse and whether TASK-2 is directly tyrosine phosphory- 86Rb Efflux-Estimation of Kϩ Efflux lated following hyposmotic exposure. ϩ 86Rb is regarded as a tracer for K , and 86Rb efflux experiments were performed as previously described (20). Briefly, EATC were EXPERIMENTAL PROCEDURES equilibrated for 30 min with 86Rbϩ in standard NaCl-Ringer (2 ϫ 104 Cell Cultures: EATC, EATCC, and Human Embryonic Kidney Bq/ml), washed, and resuspended at a cytocrit of 2% in standard Cells-293 isotonic (300 mosM) or hypotonic Ringer (150 mosM), both contain- ing 0.01% bovine serum albumin (BSA) and 30 M bumetanide. In the present investigation we have used EATC maintained under Bumetanide, added to avoid 86Rbϩ reuptake via NKCC1, was present two separate conditions: 1) EATC maintained in first generation for the last 5 min of the preincubation and during the efflux experi- hybrids of female NMRI mice by weekly intraperitoneal transplanta- ment. [3H]Inulin was added for estimation of extracellular space. tion of 1.5 ϫ 107 cells per mouse. Six to seven days after transplan- 86Rbϩ efflux from the cells was measured as gain in the extracellular 86 ϩ tation the cells were harvested and transferred to isotonic NaCl-Ringer Rb activity (cpm/ml medium) within the initial 10 min after Downloaded from containing 2.5 U/ml heparin and resuspended at cytocrit 4%. 2) EATC transfer to hypotonic and isotonic Ringer by serially isolating cell-free cultures (EATCC) were obtained by transferring EATC to RPMI 1640 efflux medium by centrifugation (15,000 g, 30 s) through a silicone oil medium supplemented with 10% serum and 100 U/ml penicillin- phase [300 l 5:1 (wt/wt) AR200,200 and DC 200/20]. Separate streptomycin and grown at 37°C, 5% CO2. EATCC were maintained double samples were taken at time 1 min for estimation of cellular 5 by transfer of 8.5 ϫ 10 cells to 10 ml fresh RPMI 1640 medium content (mol/sample) and cell dry weight (g/sample) after correction every 3–4 days, and only passages 6–28 were used for experiments. for 86Rb and Kϩ trapped in the extracellular space using the [3H]inu- 3 86 The EATCC were introduced because we wanted to avoid the use of lin as a marker (20, 27). H and Rb activities were measured in a http://ajpcell.physiology.org/ mice for tumor cell growth. This was supported by The Danish Society liquid scintillation spectrometer (Packard 2000CA Tri-Carp Liquid for the Protection of Laboratory Animals. Propagation of ascites cells was Scintillation Analyzer) in channels ranging from 2.0 to 18.6 kV and approved by “Dyreforsøgstilsynet” 2007/561-1313. 3) Human embryonic 19 to 1,500 kV, respectively, using ULTIMA GOLD (Packard) as kidney cells (HEK-293) were grown in Dulbecco’s modified Eagle’s scintillation fluid. Kϩ content was determined by atomic absorption medium (DMEM with GlutaMAX-l, D-glucose, and sodium pyruvate, flame photometry (model 2380, Perkin Elmer atomic absorptions Invitrogen) with 10% fetal bovine serum and 100 U/ml penicillin- ϩ spectrophotometer) using samples and K standards diluted 100-fold streptomycin. Cells were subcultured every 3–4 days. ϩ with 1 mM CsCl. 86Rb activity (cpm/g cell dry wt), estimated from the extracellular 86Rb activity (cpm/ml medium) and the cell dry Solutions and Materials weight (g/ml medium), was plotted versus time, and the rate constant for 86Rbϩ efflux (minϪ1) was determined by fitting the efflux curve For EATC the standard isotonic NaCl-Ringer solution (300 mosM) 86 ϩ at Copenhagen University Library on July 10, 2013 contained (in mM) 143 NaCl, 5 KCl, 1 MgSO ,1Na HPO , 1 CaCl , between 1 and 10 min to an exponential function (18). The Rb 4 2 4 2 ϫ Ϫ1 3.3 MOPS, 3.3 N-tris-(hydroxymethyl) methyl-3-aminoethane sul- efflux (cpm/ml cell water min ) was calculated as the product of fonic acid (TES), and 5 HEPES, pH 7.4. Hypotonic Ringer (150 the rate constant and the cellular concentration (cpm/ml cell water). ϩ ϫ Ϫ1 mosM) was obtained from the isotonic solution by dilution with The K efflux ( mol/ml cell water min ) was calculated from the 86 ϩ buffered water (water containing 3.3 mM MOPS, 3.3 mM TES, and 5 Rb efflux by division with the specific activity (cpm/ml cell Ϫ Ϫ water·mol KϩϪ1·ml cell waterϪ1) and converted to mol/g dry wt ϫ mM HEPES, pH 7.4). The NO3 Ringer is a Ringer where all Cl is Ϫ minϪ1 by multiplication with the water content (ml cell water/g cell substituted with NO3 . For HEK-293 cells the isotonic NaCl-Ringer (300 mosM) contained (in mM) 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, dry wt). and 10 HEPES; pH was adjusted to 7.4. Hypotonic Ringer (150 mosM) was obtained as described above. Unless otherwise stated, Gel Electrophoresis and Western Blotting reagents were analytical grade and obtained from Sigma (St. Louis, 3 86 MO). [ H]Inulin and Rb were from Amersham Bioscience. EATCC were lysed in 95°C lysis buffer (10 mM Tris·HCl, pH 7.4, 1% SDS, and 20 mM EDTA) with protease inhibitors (Roche Applied Cell Volume Measurements Science) and phosphatase inhibitors added. The lysates were subse- quently homogenized, and the supernatant was collected by centrifu- Cell volume was determined using two different methods. Absolute gation. HEK-293 cells transfected with TASK-2 were lysed in 95°C cell volume of EATC/EATCC was estimated by electronic cell sizing lysis buffer (5 mM Tris·HCl, pH 7.4, 0.5% SDS, and 10 mM EDTA). by Coulter counter measurements (tube orifice 100 m, Coulter Protein concentrations were determined using Bio-Rad DC protein Multisizer 3, Coulter, Luton, UK). Relative cell volume of the mono- assay. SDS-polyacrylamide gel electrophoresis were performed layer HEK-293 cells was estimated by large angle light scattering measurements (RatioMaster model C44–2000, Photon Technology using a 10% gel (Invitrogen) and transferred to nitrocellulose International). membranes. The membranes were dyed with Ponceau Red (Sigma) Coulter counter measurements. Cell density was 90,000 cells/ml and washed in TBST (10 mM Tris·HCl, pH 7.5, 120 mM NaCl, and equivalent to a cytocrit at 0.008%. Cell volume was determined as the 0.1% Tween 20) before being blocked in a 5% nonfat dry milk and median of the cell volume distribution curves after calibration with TBST solution. Membranes were incubated with primary antibody latex beads (14.89 M). The Ringer solutions for the coulter counter at following concentrations: TASK-2 (Alomone) 1:500, nonphos- experiments were filtered (Millipore, 0.45 M) before use. Volume phorylated (np) FAK Y397 (Biosource) 1:200, phosphorylated (p) recovery was estimated as (Vmax Ϫ V3–4min)/(Vmax Ϫ Viso), where FAK Y397 (Biosource) 1:200, np-Src Y416 (Cell Signaling) 1:250, Vmax,V3–4min, and Viso are the maximal cell volume, cell volume at p-Src Y416 (Cell Signaling) 1:250, np-Src Y527 (Cell Signaling) time 3 or 4 min and cell volume under isotonic conditions, respectively. 1:250, p-Src Y527 (Cell Signaling) 1:250. Membranes were Light scatter measurements. Wild-type or TASK-2 transfected washed in TBST before introduction to secondary antibody HEK-293 cells were grown on coated (poly-L-lysine) coverslips until (Sigma) 1:5,000. The protein bands were visualized using 5-bro- ϳ80% confluence. Light scattering results were obtained using the mo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (BCIP/ monochromator settings: excitation 589 nm and emission 595 nm. For NBT Kirkegaard and Perry Lab) and scanned. Protein band density further description of the light scatter method see Ref. 39. was estimated using UN-SCAN-IT software. AJP-Cell Physiol • VOL 299 • OCTOBER 2010 • www.ajpcell.org C846 TYROSINE PHOSPHORYLATION AND VOLUME-ACTIVATED CHANNELS Transient Transfection RESULTS A TASK-2 containing vector (pcDNA3.1) was kindly given to us Effect of Tyrosine Kinase Inhibitors on RVD and on by Dr. Sepúlveda (Chile). The TASK-2 channel was transferred to Swelling-Activated Kϩ Efflux two other vectors. The first, pMT2-HA vector using PCR at the following conditions: denaturation at 98°C for 30 s followed by 25 In the present investigation we used EATC grown in cell cycles with the initial temperature at 98°C for 10 s, annealing at 65°C culture (EATCC) or in the abdominal cavity of mice (EATC) for 30 s extension at 72°C for 40 s, and a end extension at 72°C for to investigate the effect of the tyrosine kinase inhibitor 7 min. The restrictions sites Xho1 and kpn1 were used together with genistein on the RVD and the swelling-induced Kϩ loss. From the following primers: 5=-ATGGTGGACCGGGGT-3= (sense) and Fig. 1, A and B, it is seen that genistein is an effective inhibitor 5=-TCACGTGCCCCTGGG-3= (antisense). PCR product was purified of RVD in EATC grown both in culture and in the abdominal and resolved by agarose gel electrophoresis. DNA bands were cut out of the gel, purified, and subsequently cloned into the pMT2-HA cavity. Genistein was dissolved in DMSO, which can be vector. For all purification steps products from Qiagen was used. potentially toxic to the cells, but experiments showed that Downloaded from Sequencing was performed by Eurofins MWG Operon (Germany). the concentrations used (0.34%) did not affect the ability of the Second, pcDNA3.1/myc-His vector (Invitrogen) using PCR and the cells to perform RVD (data not shown). With the use of the restrictions sites BamH1 and Xho1. The following primers were recovery level after 3 min (EATC) or 4 min (EATCC), it is designed: 5=-AGTGTGGACCGGGGTCCTTTAC-3= (sense) and 5=- estimated that genistein reduced the RVD from 73% to 22% in CGTGCCCCTGGGGTTATCTGC-3= (antisense). PCR conditions EATC (Fig. 1C) and from 61% to 10% in EATCC (Fig. 1D). were the following: denaturation at 98°C for 5 min followed by 25 It is noted that the swelling response to hyposmotic media is cycles with the initial temperature at 98°C for 1 min, annealing at much faster (in seconds) in the EATCs than that in the http://ajpcell.physiology.org/ 62°C for 30 s and extension at 72°C for 40 s, and at the enda7min EATCCs, which probably reflects a difference in the number extension at 72°C. The rest of the procedure was identical to the one described above. of aquaporins. This has not been tested. To investigate whether genistein directly affects the swelling-activated Kϩ Light scatter. HEK-293 cells for transfection were grown on coated ϩ (poly-L-lysine) coverslips to ϳ50–80% confluence and transfected efflux, we measured the K efflux in EATC in hypotonic with a TASK-2 containing vector (pMT2-HA). For transfection we medium in the presence and absence of genistein. 86Rb was ϩ used 1 g DNA/3 l FuGENE 6 Transfection Reagent (Roche used as a tracer for K . Figure 2, A and C, shows that Applied Science) according to the manufacturer’s instructions. Exper- genistein reduces the measured 86Rb efflux and the calcu- iments were performed ϳ24 h after transfection. lated Kϩ efflux down to about 30% upon hypotonic expo- Precipitation of TASK-2. HEK-293 cells for transfection were sure; i.e., the control cells had a mean Kϩ loss of 100 2 ϳ at Copenhagen University Library on July 10, 2013 grown in coated (poly-L-lysine) wells (9.6 cm /well) to 50–80% mol·g proteinϪ1·minϪ1, and the treated cells had a mean confluence and transfected with a TASK-2-containing vector loss of 33.3 mol·g proteinϪ1·minϪ1. To exclude contribu- (pcDNA3.1/myc-His). For transfection we used 1 g DNA/3 l tion to the Kϩ efflux by the KCl cotransporter, we substi- FuGENE 6 Transfection Reagent (Roche Applied Science) according Ϫ Ϫ ϳ tuted all internal and external Cl with NO by preincu- to the manufacturer’s instructions. Experiments were performed 36 Ϫ 3 bating the cells for 60 min in NO3 Ringer. From Fig. 2, B h after transfection. It should be noted that we tried to use the HA tag ϩ to immunoprecipitate the TASK-2 without success. and D, it is seen that the swelling-activated K efflux, now ϩ Western blot analysis. HEK-293 cells for transfection were grown representing only efflux via K channels, is completely inhib- Ϫ Ϫ ϩ to 50–80% confluence in six-well trays and transfected as described ited by genistein. Substitution of NO3 for Cl reduced the K above. efflux from 222 Ϯ 13 to 171 Ϯ 10 mol·mg dry wtϪ1·minϪ1 (n ϭ 11 for each series), indicating a Kϩ efflux via the KCl TASK-2 Phosphorylation cotransporter equal to 51 mol·mg dry wtϪ1·minϪ1; i.e., 23%. ϩ HEK-293 cells were grown in coated (poly-D-lysine) six-well trays This is quantitatively similar to the genistein-insensitive K to 50–80% confluence and transfected with a pcDNA3.1/myc-His flux seen in NaCl medium (Fig. 2C). vector (Invitrogen) containing TASK-2 as described above. The cells were either hypotonically (150 mosM) or isotonically (300 mosM) Effect of Tyrosine Phosphatase Inhibitor on RVD (controls) stimulated for 1, 5, or 10 min at 37°C and subsequently and Loss Of Kϩ lysed using a Radio Immuno Precipitation Assay (RIPA) buffer (1% NP-40, 1% sodium deoxycholate, 0,1% SDS, 150 mM NaCl, 5 mM As inhibition of tyrosine kinases hinders RVD (Fig. 1) and Tris·HCl, 2 mM EDTA, and 10 mM imidazole). The TASK-2 channel inhibits the activation of volume-sensitive Kϩ channels (Fig. was precipitated using Ni-NTA agarose (Qiagen), and the specific 2), we investigated whether inhibition of tyrosine phosphatases tyrosine phosphorylation was visualized through SDS-PAGE separa- could potentiate the RVD in EATC. Cell volume measure- tion and Western blotting (see above) using the phosphospecific ments were performed in the presence of the tyrosine phos- tyrosine antibody pY-100 (1:500, Cell Signaling) and a TASK-2- specific antibody (1:500) (Alomone). Visualization of TASK-2 bands phatase inhibitor mpV(pic) (10 M). From Fig. 3A it is seen was performed using BCIP/NBT (Kirkegaard and Perry), whereas the that in control cells a new steady-state cell volume was ob- pY100 band was visualized using Lumigen solutions A and B (GE tained after 3 min exposure to hypotonic medium (150 mosM), Healthcare) and chemiluminiscens. Protein band density was esti- which was about 20% larger than the original cell volume. In mated using UN-SCAN-IT software. the presence of the tyrosine phosphatase inhibitor mpV(pic), the volume recovery at time 4 min improved and amounted to Statistically Analysis 90% compared with the 70% in control cells (Fig. 3B). These ϩ The values are given as means Ϯ SE. Statistically significance was findings were further substantiated through K efflux measure- analyzed by the use of Student’s t-test or ANOVA and shown as one ments. When treated with mpV(pic), the mean swelling-acti- ϩ asterisk when significant at a 5% significance level and two asterisks vated K efflux in EATC was increased to 150% compared when significant at a 1% significance level. with nontreated cells (Fig. 3C). Adding clofilium, a known AJP-Cell Physiol • VOL 299 • OCTOBER 2010 • www.ajpcell.org TYROSINE PHOSPHORYLATION AND VOLUME-ACTIVATED CHANNELS C847 Fig. 1. The general tyrosine kinase inhibitor genistein inhibits volume recovery in Ehrlich ascites tumor cells (EATC) and EATC cultures (EATCC). A and B: EATC and EATCC were placed in either isotonic Ringer (300 mosM) or hypotonic Ringer (150 mosM), and the absolute volume was observed as a function of time. Downloaded from EATC and EATCC were preincubated with the tyrosine kinase inhibitor genistein (371 M) for 45 min before they were transferred at time 0 to hypotonic medium also containing genistein (371 M). C and D: mean recovery after 3 min of EATC and EATCC in hypotonic medium was estimated from 3 independent sets of ex- periments and shown as means Ϯ SE. **Signif- http://ajpcell.physiology.org/ icantly reduced from control cells ability to regulate their volume. at Copenhagen University Library on July 10, 2013 TASK-2 blocker (35), the Kϩ efflux was inhibited to 27% of To test whether inhibition of tyrosine phosphatase could the Kϩ efflux value in nontreated control cells. In the presence prevent the effect of inhibition of tyrosine kinase, we of clofilium plus mpV(pic), the Kϩ efflux was reduced to the measured the RVD response in cells treated with genistein efflux value seen in the presence of clofilum alone. Thus a alone and genistein in combination with the phosphatase mpV(pic)-sensitive phosphatase seems to be involved in the inhibitor mpV(pic). From Fig. 3D it is seen that inhibition of inactivation of the volume-sensitive Kϩ channel (TASK-2) in the tyrosine phosphatase indeed prevented the effect of EATC. kinase inhibition. Fig. 2. Genistein inhibits swelling-induced, ClϪ-indepen- dent Kϩ efflux. EATC were incubated in isotonic NaCl Ringer (A and C) or isotonic NaNO3 Ringer (B and D). 86Rb, genistein (100 M) and [3H]inulin were added 60, 45, and 5 min before initiation of efflux estimation, respectively. Cells were subsequently washed once in isotonic Ringer (300 mosM, NaCl/NaNO3), and at time 0 resuspended in hypotonic Ringer (150 mosM, NaCl/ NaNO3) containing no genistein (control) or 100 M genistein. Twenty samples were drawn within the first 10 min and centrifuged through a silicone oil phase as indi- cated in Experimental Procedures. Double samples were used to estimate the cellular Kϩ and 86Rb content within 1 min following hypotonic exposure. 86Rbϩ activity (cpm/g cell dry wt) was plotted versus time (A: NaCl Ringer; B: NaNO3 Ringer), and the rate constant for 86Rbϩ efflux (minϪ1) was determined by fitting the efflux curve to an exponential function. The Kϩ efflux (mol·g dry wtϪ1·minϪ1), calculated by multiplying the 86Rb-rate constant (minϪ1) by the Kϩ content (mol/g dry wt), was estimated from 5 and 8 sets of experiments in NaCl Ringer (C) and NaNO3 Ringer (D), respectively and shown as mean values Ϯ SE. **Significantly reduced from control cells ability to regulate their volume (P Ͻ 0.01). AJP-Cell Physiol • VOL 299 • OCTOBER 2010 • www.ajpcell.org C848 TYROSINE PHOSPHORYLATION AND VOLUME-ACTIVATED CHANNELS Functional Effect of Overexpressing TASK-2 on RVD Swelling-Induced Tyrosine-Phosphorylation of TASK-2 To test the effect of TASK-2 overexpression on RVD, we To investigate whether TASK-2 is tyrosine-phosphorylated used HEK-293 cells, which according to Niemeyer and co- following hypotonic exposure, we transfected HEK-293 cells workers (33) show no swelling-activated Kϩ current. From with a TASK-2 containing pcDNA3.1/myc-His vector, stimu- Fig. 4A it is seen that overexpression of TASK-2 cloned from lated the cells hypotonically for 1, 5, or 10 min, and subse- EATC in HEK-293 was obtained after 24, 36, and 48 h. Using quently precipitated the TASK-2 channel using Ni-NTA aga- the light scattering technique, we found that TASK-2 overex- rose (Qiagen). Tyrosine phosphorylation was detected through pressing HEK-293 cells (Fig. 4C) had a faster volume recovery SDS-PAGE and Western blotting using a phospho-specific than the wild-type cells (Fig. 4B). It was estimated that tyrosine antibody (pY100). From Fig. 5 it is seen that TASK-2 TASK-2 overexpression resulted in a twofold faster volume expression was identical in isotonic- and hypotonic-treated recovery (Fig. 4D). It is noted that the TASK-2 overexpressing cells (Fig. 5A), whereas the phosphorylation of TASK-2 Downloaded from cells made an overshot in the RVD (Fig. 4C), which could be (pY100) increased within 1 min following hypotonic exposure taken to indicate that the cellular signaling system involved in and appeared to be maximal after 5 min (Fig. 5B). It is noted closing of the TASK-2 channel was not effective in the that ANOVA test on the total experimental data set indicated HEK-293 cells. This was not investigated further. It is also no difference of the pY100 band among 1, 5, and 10 min noted that the transfected cells showed a strong post-RVD isotonic medium. The relative specific activity for tyrosine- regulatory volume increase (RVI) response when reexposed to phosphorylated TASK-2 under hypotonic conditions (the ratio isotonic Ringer (Fig. 4C) in contrast to wild-type HEK-293 between pY100 band intensity and the TASK-2 band intensity http://ajpcell.physiology.org/ cells (Fig. 4B), indicating a far greater ion loss during the RVD given relative to the ratio in isotonic medium) was increased period compared with the wild-type cells. Thus expression of four times within the first minute (P ϭ 0.14), four times within TASK-2 contributed significantly to the net loss of Kϩ fol- 5 min (P ϭ 0.01), and three times after 10 min (P ϭ 0.03) lowed by ClϪ and water. hypotonic exposure. Hence, the TASK-2 channel was still at Copenhagen University Library on July 10, 2013 Fig. 3. Possible involvement of mpV(pic)-sensitive tyrosine phosphatases in regulatory volume decrease (RVD) and swelling-activated Kϩ efflux in EATC. EATC were preincubated with the tyrosine phosphatase inhibitor mpV(pic) (10 M) for 30 min before they were transferred to hypotonic medium also containing mpV(pic) (10 M). A: EATC were placed in either isotonic Ringer (300 mosM, closed circles) or hypotonic Ringer (150 mosM) without mpV(pic) (open circles) or with mpV(pic) (closed triangles), and the absolute cell volume was measured as a function of time. B: mean values for volume recovery of EATC after 4 min hypotonic exposure was calculated from 3 to 8 independent experiments and shown as means Ϯ SE. C:Kϩ efflux was estimated as indicated in Fig. 2. Cells were preincubated with 100 M clofilium and/or 10 M mpV(pic) for 30 min, and both inhibitors were present under the efflux experiment. Flux are given as mean values Ϯ SE and represent 12 control [mpV(pic)], 6 (clofilium), and 6 [clofilium ϩ mpV(pic)] independent experiments. In the case of clofilium the Kϩ efflux was calculated from 86Rb efflux within the initial 5 to 6 min after which the efflux suddenly increased by a yet undescribed pathway (data not shown). **Value is significantly different compared with control (P Ͻ 0.01). #Significantly reduced compared with mpV (pic) alone (P Ͻ 0.01). D: EATCC were exposed to the tyrosine kinases inhibitor genistein (371 M) in the absence or presence of the tyrosine phosphatase inhibitor mpV(pic) (10 M). The rate of RVD was estimated as the reduction in cell volume within the period 1 to 3 min following hypoosmotic exposure (150 mosM) using the Coulter counter. Genistein, when present (black and grey bars), was added to the cells 45 min before hypotonic exposure. mpV(pic) was added at the same time as genistein (light shaded bar) or 30 min (preincubation) before addition of genistein (dark shaded bar). All inhibitors were present during estimation of RVD. The number of experiments represents 6 (control), 4 (genistein), and 3 [genistein ϩ mpV(pic)]. *Value is significantly different compared with control. #Significantly increased compared with genistein alone. AJP-Cell Physiol • VOL 299 • OCTOBER 2010 • www.ajpcell.org TYROSINE PHOSPHORYLATION AND VOLUME-ACTIVATED CHANNELS C849 Fig. 4. TWIK-related acid-sensitive Kϩ channel-2 (TASK-2) overexpression improves volume recovery. A: HEK-293 cells transfected with TASK-2 contain- ing plasmid were harvested 24, 36, and 48 h after transfection. The success of the transfection was visu- alized through SDS-PAGE and Western blot analysis using antibody against TASK-2. B and C: wild type HEK-293 cells (control, B) and cells transfected with TASK-2 for 24 h (C) were grown on coated coverslips until ϳ80% confluence, and cell volume was mea- sured using the light scatter technique. When stable Downloaded from measurements in isotonic Ringer were seen, the me- dium was shifted to a hypotonic Ringer for 10–15 min. Volume measurements were carried out every 1–2 s. After this period of time the Ringer was shifted back to the isotonic Ringer and measurements contin- ued for another 10–15 min. D: volume recovery fol- lowing 4 min hypotonic exposure was estimated in 5 (wild type) to 6 (TASK-2) independent experiments http://ajpcell.physiology.org/ and given as mean values Ϯ SE. *Significantly in- creased compared with control cells (P Ͻ 0.05). tyrosine phosphorylated 10 min after hypotonic exposure, i.e., hypotonic stimulation (Fig. 7B). Thus an increase in the Src at that time the Kϩ efflux was still high (Fig. 2, A and B). and FAK kinase activities does not seem to be involved in ϩ activation of volume-sensitive channels K , in EATCC, and at Copenhagen University Library on July 10, 2013 Putative Tyrosine Kinases Involved in Swelling-Induced hence RVD. Activation of TASK-2 JAK2 and receptor-coupled tyrosine kinases. To substantiate Src and FAK. Various tyrosine kinase inhibitors were used the functional role of tyrosine kinases, we investigated the in an attempt to narrow down the potential tyrosine kinase(s) effect of inhibition of JAK2 [cucurbitacin I (5)], the insulin- involved in channel activation. As seen in Fig. 6A the volume- like growth factor I receptor [picropodophyllotoxin (13)], and activated Kϩ efflux was reduced with 25% in the presence of the epidermal growth factor receptor [GW583340, dihydro- the Src kinase inhibitor PP2 compared with controls with no chloride (11)] on the volume recovery after cell swelling. From inhibitor present. Hypotonic medium with and without PP2 Fig. 8 it is seen that cucurbitacin I reduced the recovery level present indicates a small but significant inhibition of the after 4 min by 40%, whereas the other inhibitors had no swelling-activated Kϩ efflux in EATC. The RVD, estimated as significant effect. This indicates that the JAK/STAT3 pathway the initial reduction in cell volume (10Ϫ15 l/min), in EATCC could be involved in the tyrosine phosphorylation of TASK-2 was significantly reduced to 35% (Fig. 6B), indicating an and hence in the RVD response. eventual role of Src in the RVD response. To pursue a role of Src in activation of the swelling-induced Kϩ channels and DISCUSSION RVD we used Western blot analysis with phospho-specific Tyrosine Kinases are Involved in the Initiation antibodies against 1) Src Y416, which is in the kinase domain of RVD and is phosphorylated during activation, and 2) Src Y527, which has to be dephosphorylated to activate the kinase. From In volume measurements on EATC and EATCC using the Fig. 6, C (left) and D, it is seen that 1 and 5 min of hypotonic Coulter counter, it was seen that the control cells did not reach stimulation elicited a significant decrease in the phosphoryla- the perfect osmometer value, which is due to the fact that the tion of Src Y416, reflecting a decreased Src activity after cells activate their volume-sensitive ion channels already after osmotic cell swelling. Looking at the inactivating site Y527, no no more than a 5% swelling. A RVD response will thus hinder change in the phosphorylation levels was observed over a the cells in reaching the perfect osmometer value. On the 10-min time period (Fig. 6, C, right, and E). The eventual contrary EATCC treated with genistein did in fact reach the effect of cell swelling on FAK was likewise investigated perfect osmometer value, indicating that their ability to volume using Western blot analysis and a phospho-specific FAK regulate was completely inhibited. Even after 4 min there was Y397 antibody. From Fig. 7, A (Western blot) and B no sign of a RVD response. The inhibition of RVD was evident (quantification), it is seen that initially (1 min after stimu- and similar in both cell lines. These results may be explained lation), there was no change in the phosphorylation of FAK, by the inhibition of either one or both of the volume-activated which would be expected if the kinase was activated during ion channels, which in EATC and EATCC are VRAC (ICl,vol) cell swelling. On the contrary there was a significant de- and TASK-2 (IK,vol) (see INTRODUCTION) upon hypotonic swell- crease in the phosphorylation levels at 5 and 10 min after ing. The Kϩ permeability increases much less when compared AJP-Cell Physiol • VOL 299 • OCTOBER 2010 • www.ajpcell.org C850 TYROSINE PHOSPHORYLATION AND VOLUME-ACTIVATED CHANNELS Tyrosine Phosphatases are Involved in the Inactivation of RVD When using the tyrosine phosphatase inhibitor mpV(pic), the set point for RVD in EATC (defined as the volume where the RVD response ends) was significantly lowered compared with control cells. The treated cells seemed to have lost more ions than the control cells, which could indicate that the Kϩ channel (being the inhibiting factor in RVD) was prevented from closing due to the impairment of tyrosine phosphatases. These findings were substantiated by Kϩ efflux measurements, in which it was found that inhibition of tyrosine phosphatases Downloaded from with mpV(pic) resulted in a significantly increased swelling- activated Kϩ efflux. Taken together these results strongly indicate that tyrosine phosphatases are involved in the closing of the volume-activated Kϩ channel TASK-2 in EATC. In a couple of experiments it was not possible to see an effect in EATCC treated with mpV(pic). We have no explanation on this apparent difference between EATC and EATCC. http://ajpcell.physiology.org/ Fig. 5. Tyrosine phosphorylation of the TASK-2 channel following hypotonic Overexpression of TASK-2 Accelerates the RVD exposure. TASK-2 expression and tyrosine phosphorylation of TASK-2 in ϩ HEK-293 cells transfected with a TASK-2 containing pcDNA3.1/myc-His As mentioned earlier the K conductance is rate limiting vector was measured under either isotonic or hypotonic conditions (1, 5, and during RVD, and the insertion of more channels would in 10 min). The TASK-2 channel was precipitated using Ni-NTA agarose theory increase the ability of the cells to volume regulate. In (Qiagen) and TASK-2 expression together with TASK-2 tyrosine phosphory- lation (pY100) visualized through SDS-PAGE and Western blotting (A). accordance with this theory, the TASK-2-transfected cells ANOVA test indicated no difference between isotonic pY100 values at 1, 5, showed a significantly increased RVD response, both in regard and 10 min. The relative specific activity for tyrosine phosphorylated TASK-2 to end point and velocity. This reflects a greater ion loss during under hypotonic conditions was estimated as the ratio between pY100 band RVD in cells transfected with TASK-2 channels than in wild- intensity and the TASK-2 band intensity given relative to the ratio in isotonic at Copenhagen University Library on July 10, 2013 medium (B). Values are given as means from 8, 9, and 6 sets of experiments type HEK-293 cells. Therefore when reintroduced to isotonic taken at time 1, 5, and 10 min, respectively, ϮSE. *Significantly increased Ringer, the transfected cells responded with a strong RVI like compared with isotonic conditions (t-test, P Ͻ 0.05). #Significantly increased response, a so-called post-RVD RVI response. When returning compared with isotonic conditions (ANOVA, P Ͻ 0.05). the cells to the isotonic Ringer, this now appeared hypertonic due to the excessive loss of ions during RVD, causing the cells to shrink beneath their original set point. The increased post- with the ClϪ permeability during cell swelling, which makes RVD RVI response in the cells overexpressing TASK-2 was the Kϩ permeability the limiting factor for the RVD rate (27). taken to indicate a greater loss of ions during RVD in trans- Inhibition of RVD will thus often reflect an inhibition of the fected cells than in the wild type. We thus have a model system swelling-activated Kϩ channel. However, it should be noted in which the RVD rate can be used to evaluate effects on the that an almost complete inhibition of VRAC would lead to the TASK-2 channel expressed in this system. The clear effect of same results. Results from efflux measurements comply with genistein on this RVD response is thus a clear evidence for the volume measurements, and it was seen that the swelling- tyrosin kinases being involved in activation of TASK-2 after activation of Kϩ efflux in EATC was strongly inhibited when cell swelling. Further experiments using mass spectrometry cells were treated with the tyrosine kinase inhibitor genistein. analysis will hopefully give us a more detailed picture of the This was true also when all intracellular ClϪ was substituted activation pattern of the channel. Ϫ ϩ ϩ with NO3 to assure that all measured K efflux was via a K channel and not involving the KCl cotransporter. It is thus clear Swelling-Induced Tyrosine Phosphorylation of TASK-2 that one or more tyrosine kinases are responsible for swelling- ϩ Several tyrosine kinases have been demonstrated to be activation of the K channels, which in EATC and EATCC are activated following cell swelling, and tyrosine kinase inhibitors known to be TASK-2 channels. It has previously been shown have been shown to block VRAC as well as the volume- that TASK-1 background current is inhibited by genestein and sensitive efflux pathway for organic osmolytes (19, 28, 29, 36). that an inhibitor of protein tyrosine phosphatase reduces this With respect to tyrosine kinases and swelling-activated Kϩ effect (12). In the present investigation it is demonstrated that channels, it has been shown that Src modulates the activity of, inhibition of tyrosine phosphatases prevents the inhibitory for example, the swelling-activated Kv channels (1.3 and 1.5) effect of the tyrosine kinase inhibitor genistein on RVD. A and the BK channels (19). In the present investigation we find similar phenomenon has been demonstrated on the volume- that swelling of HEK-293 cells overexpressing TASK-2 chan- sensitive release of organic osmolytes (26). We assume that it nels resulted in a time-dependent phosphorylation of the chan- is a balance between tyrosine kinase and tyrosine phospha- nel, which was evident within the period 1 to 10 min after tase activities that determines the degree of phosphorylation hypotonic treatment. Patch-clamp experiments performed on of TASK-2 and hence its activation following osmotic cell cell-attached patches with EATC have indicated that the vol- swelling. ume-sensitive Kϩ channel is activated within 65 s after hypo- AJP-Cell Physiol • VOL 299 • OCTOBER 2010 • www.ajpcell.org TYROSINE PHOSPHORYLATION AND VOLUME-ACTIVATED CHANNELS C851 Fig. 6. Possible involvement of the tyrosine Src kinase in RVD and swelling-activated Kϩ efflux in EATC. EATC in suspension were preincubated with the Src kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyra- zolo[3,4-d]pyrimidine (PP2, 5 M) for 30 min before they were transferred to hypotonic Ringer also containing PP2. A:Kϩ efflux was estimated as described in Fig. 2. B: volume recovery in EATCC was estimated as de- scribed in Fig. 1. Experiments were also per- formed with the use of 10 M PP2 but with Downloaded from no significantly difference compared with the use of 5 M (data not shown). Volume re- covery is shown as the water loss (flow/min) obtained by linear regression on cell volumes measured within the first 3 min after maximal cell swelling in hypotonic Ringer. Values are calculated from 3 independent experiments and shown as means Ϯ SE. C: SDS-page and West- http://ajpcell.physiology.org/ ern blot analysis performed on EATCC lysates from cells exposed to isotonic or hypotonic Ringer for 1, 5, or 10 min. The Src phosphor- ylation was visualized using specific antibodies against the activating site: SrcY416 (C, left) and the inactivating site: SrcY527 (C, right). D and E: hypotonic values for the phosphor- ylation of SrcY416 (D) and SrcY527 (E) are given relative to the isotonic values and the bars represent the mean values of 3 indepen- dent experiments Ϯ SE. at Copenhagen University Library on July 10, 2013 tonic exposure (7). Whole cell patch-clamp experiments in cause the cells do not volume regulate. The rate constant, EATC cells revealed that the volume-sensitive Kϩ channels calculated from our efflux data shown in Fig. 2B, was high and were detectable within 200 s after shift in solution (35). Whole unaltered in the time period 1 to 10 min after hypotonic cell patch-clamp experiments cannot be used to evaluate how exposure, indicating that the Kϩ channel was activated within long the channels remain open after hypotonic challenge be- the whole time frame. This is in accordance with the tyrosine phosphorylation pattern for TASK-2. Although it might not be possible to compare time scales for TASK-2 activation in Fig. 8. The Janus kinase inhibitor cucurbitacin I (CC1) inhibits volume recovery in EATCC. EATCC were placed in either isotonic Ringer (300 mosM) or hypotonic Ringer (150 mosM), and the absolute volume was Fig. 7. Possible phosphorylation of the tyrosine kinase focal adhesion kinase observed as a function of time. Cells were preincubated for 4 h with the JAK2 (FAK) after cell swelling. SDS-page and Western blot analysis were performed inhibitor CC1 (10 M), the insulin-like growth factor I receptor inhibitor on EATCC lysates made of cells exposed to isotonic or hypotonic Ringer for picropodophyllotoxin (PPPT, 1 M), and the epidermal growth factor receptor 1, 5, or 10 min. A: FAK phosphorylation (pFAK) was visualized using specific inhibitor dihydrochloride (DC, 1 M), and the inhibitors were present during antibodies against the site Y397. B: bar graphs represent the mean values of 6 volume estimations. Mean recovery after 4 min in hypotonic medium was independent experiments Ϯ SE. All calculations of phosphorylation are shown estimated from 3 independent sets of experiments and shown as means Ϯ SE. compared with their respective isotonic controls. *Significantly reduced from control cells ability to regulate their volume. AJP-Cell Physiol • VOL 299 • OCTOBER 2010 • www.ajpcell.org C852 TYROSINE PHOSPHORYLATION AND VOLUME-ACTIVATED CHANNELS EATC cells with time scales for tyrosine phosphorylation of REFERENCES TASK-2 in HEK-293 cells, we suggest that direct tyrosine 1. Barfield JP, Yeung CH, Cooper TG. The effects of putative Kϩ channel phosphorylation is involved in swelling-induced activation of blockers on volume regulation of murine spermatozoa. Biol Reprod 72: TASK-2. We cannot exclude that tyrosine phosphorylation is 1275–1281, 2005. also involved upstream in the signaling sequence from cell 2. Barfod ET, Moore AL, Melnick RF, Lidofsky SD. Src regulates distinct ϩ pathways for cell volume control through Vav and phospholipase swelling to activation of the K channel. Cgamma. J Biol Chem 280: 25548–25557, 2005. 3. Barrière H, Rubera I, Belfodil R, Tauc M, Tonnerieux N, Poujeol C, Tyrosine Kinases Involved in RVD Barhanin J, Poujeol P. Swelling-activated chloride and potassium con- ductance in primary cultures of mouse proximal tubules. Implication of As genistein is a known broad-spectrum tyrosine kinase KCNE1 Protein. J Membr Biol 193: 153–170, 2003. inhibitor, the results give no information to which tyrosine 4. Barriere H, Belfodil R, Rubera I, Tauc M, Lesage F, Poujeol C, Guy N, Barhanin J, Poujeol P. Role of TASK2 Potassium channels regarding kinase(s) could be involved. Experiments using the more volume regulation in primary cultures of mouse proximal tubules. J Gen Downloaded from specific Src kinase inhibitor PP2 showed a significant inhibi- Physiol 122: 177–190, 2003. tion of the volume-sensitive Kϩ efflux in EATC (Fig. 6A)as 5. Blaskovich MA, Sun J, Cantor A, Turkson J, Jove R, Sebti SM. well as of the concomitant RVD response (Fig. 6B) when Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/signal transducer and activator of transcription 3 signaling pathway inhibitor with introducing the cells to a hypotonic solution together with the potent antitumor activity against human and murine cancer cells in mice. application of PP2. As these experiments are performed in the Cancer Res 63: 1270–1279, 2003. presence of intracellular ClϪ, it might however be possible that 6. Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature 411: this small inhibition of the swelling-activated Kϩ efflux could 355–365, 2001. http://ajpcell.physiology.org/ 7. Christensen O, Hoffmann EK. Cell swelling activates Kϩ and ClϪ represent an inhibition of the swelling-activated KCl cotrans- channels as well as nonselective, stretch-activated cation channels in porter. We had bumetanide present in these experiments, but Ehrlich ascites tumor cells. J Membr Biol 129: 13–36, 1992. bumetanide, which is an excellent inhibitor of NKCC1, is not 8. Cohen DM. SRC family kinases in cell volume regulation. Am J Physiol very efficient toward KCl cotransport. Even though PP2 (a Src Cell Physiol 288: C483–C493, 2005. kinase family inhibitor) showed a small, but significant, inhi- 9. de la Paz L, Lezama R, Torres-Marquez E, Pasantes-Morales H. ϩ Tyrosine kinases and amino acid efflux under hyposmotic and ischaemic bition of the RVD and the K efflux, neither the Src kinase nor conditions in the chicken retina. Pflügers Arch 445: 87–96, 2002. FAK seemed to be activated during 10 min of hypotonic 10. Fernández-Fernández JM, Nobles M, Currid A, Vázquez E, Valverde stimulation in EATCC when analyzed by Western blotting. As MA. Maxi Kϩ channel mediates regulatory volume decrease response in a human bronchial epithelial cell line. Am J Physiol Cell Physiol 283: described in the INTRODUCTION, swelling-induced activation of C1705–C1714, 2002. at Copenhagen University Library on July 10, 2013 these kinases has been reported in different cell types, but they 11. Gaul MD, Guo Y, Affleck K, Cockerill GS, Gilmer TM, Griffin RJ, do not seem to play an important role in RVD in EATCC. With Guntrip S, Keith BR, Knight WB, Mullin RJ, Murray DM, Rusnak respect to tyrosine kinase activity coupled to membrane-bound DW, Smith K, Tadepalli S, Wood ER, Lackey K. Discovery and receptors, we found a significant inhibition of the RVD fol- biological evaluation of potent dual ErbB-2/EGFR tyrosine kinase inhib- itors: 6-thiazolylquinazolines. Bioorganic Med Chem Lett 13: 637–640, lowing addition of cucurbitacin I, an inhibitor of the JAK/ 2003. STAT pathway that is associated with cytokine signaling. 12. Gierten J, Ficker E, Bloehs R, Sclömer K, Kathöfer S, Scholz E, Direct roles of cytokine receptors in volume sensing has been Zitron E, Kiesecker C, Bauer A, Becker R, Katus HA, Carle CA, reported (43), although the knowledge is yet rather limited but Thomas D. Regulation of two-pore-domain (K2P) potassium leak chan- interesting to pursue. Several reports points to a role for growth nels by the tyrosine kinase inhibitor genistein. Br J Pharmacol 154: 1680–1690, 2008. factor receptors in cell volume sensing (19). However, we see 13. Girnita A, All-Ericsson C, Economou MA, Aström K, Axelson M, no effect of the growth factor receptor tyrosine kinase inhibi- Seregard S, Larsson O, Girnita L. The insulin-like growth factor-I tors picropodophyllotoxin and dihydrochloride (GW583340) receptor inhibitor picropodophyllin causes tumor regression and attenuates on the cell volume recovery in the present investigation. From mechanisms involved in invasion of uveal melanoma cells. Acta Ophthal- mol Scand 86: 26–34, 2008. the present data a likely working hypothesis is that cell swell- 14. Hendil KB, Hoffmann EK. Cell volume regulation in Ehrlich ascites ing activates the cytokine receptor coupled JAK/STAT path- tumor cells. J Cell Physiol 84: 115–125, 1974. way, which is upstream of the swelling-induced phosphoryla- 15. Hirakawa M, Oike M, Karashima Y, Ito Y. Sequential activation of tion and activation of the TASK-2 in EATC. RhoA and FAK/paxillin leads to ATP release and actin reorganization in human endothelium. J Physiol 558: 479–488, 2004. 16. Hoffmann EK, Dunham PB. Membrane mechanisms and intracellular ACKNOWLEDGMENTS signalling in cell volume regulation. Int Rev Cytol 161: 173–262, 1995. 17. Hoffmann Else K. Intracellular signalling involved in volume regulatory The technical expertise by Birgit B. Jørgensen, Birthe J. Hansen, Birte decrease. Cell Physiol Biochem 10: 273–288, 2000. Kofoed, and Dorthe Nielsen is acknowledged. 18. Hoffmann EK, Simonsen LO, Sjøholm C. Membrane potential, chloride exchange, and chloride conductance in Ehrlich mouse ascites tumour cells. GRANTS J Physiol 296: 61–84, 1979. 19. Hoffmann EK, Lambert IH, Pedersen SF. Physiology of cell volume The present work was supported by The Danish Council for Independent regulation in vertebrates. Physiol Rev 89: 193–277, 2009. Research/Natural Sciences (Grants 272-07-0530, 272-08-0170, and 21-04- 20. Hoffmann EK. Leukotriene D4 (LTD4) activates charybdotoxin-sensitive 0535). The Danish Council for Independent Research/Medical Sciences (Grant and -insensitive Kϩ channels in Ehrlich ascites tumor cells. Pflügers Arch 271-08-0520 to I. H. Lambert). Lundbeck Foundation (J Nr R32-A3102 to E. 438: 263–268, 1999. K Hoffmann). The Danish Society for the Protection of Laboratory Animals (to 21. Hougaard C, Jørgensen F, Hoffmann EK. Modulation of the volume- I. H Lambert and E. K Hoffmann). sensitive Kϩ current in Ehrlich ascites tumour cells by pH. Pflügers Arch 442: 622–633, 2001. ϩ DISCLOSURES 22. Hougaard C, Niemeyer MI, Hoffmann EK, Sepúlveda FV. K currents activated by leukotriene D4 or osmotic swelling in Ehrlich ascites tumour No conflicts of interest, financial or otherwise, are declared by the authors. cells. Pflügers Arch 440: 283–294, 2000. AJP-Cell Physiol • VOL 299 • OCTOBER 2010 • www.ajpcell.org TYROSINE PHOSPHORYLATION AND VOLUME-ACTIVATED CHANNELS C853 23. Jakab M, Schmidt S, Grundbichler M, Paulmichl M, Hermann A, 35. Niemeyer MI, Hougaard C, Hoffmann EK, Jørgensen F, Stutzin A, Weiger T, Ritter M. Hypotonicity and ethanol modulate BK channel Sepúlveda FV. Characterisation of cell swelling-activated Kϩ selective activity and chloride currents in GH4/C1 pituitary tumour cells. Acta cunductance of ehrlich ascites tumour cells. J Physiol 524: 757–767, 2000. Physiologica 187: 51–59, 2006. 36. Pasantes-Morales H, Lezama RA, Ramos-Mandujano G. Tyrosine 24. Jørgensen NK, Pedersen SF, Rasmussen HB, Grunnet M, Klaerke kinases and osmolyte fluxes during hyposmotic swelling. Acta Physiol DA, Olesen SP. Cell swelling activates cloned Ca2ϩ-activated Kϩ chan- 187: 93–102, 2006. 37. Patel AJ, Honoré E, Maingret F, Lesage F, Fink M, Duprat F, nels: a role for the F-actin cytoskeleton. Biochim Biophys Acta 1615: ϩ 115–125, 2003. Lazdunski M. A mammalian two pore domain mechano-gated S-like K EMBO J 25. Khanna R, Chang MC, Joiner WJ, Kaczmarek LK, Schlichter LC. channel. 17: 4283–4290, 1998. 38. Pedersen SF, Prenen J, Droogmans G, Hoffmann EK, Nilius B. hSK4/hIK1, a calmodulin-binding KCa channel in human T lymphocytes. Separate Swelling- and Ca2ϩ-activated anion currents in Ehrlich ascites J Biol Chem 274: 14838–14849, 1999. tumor cells. J Membr Biol 163: 97–110, 1998. 26. Lambert IH. Reactive oxygen species regulate swelling-induced taurine 39. Pedersen SF, Beisner KH, Hougaard C, Willumsen BM, Lambert IH, efflux in NIH3T3 mouse fibroblasts. J Membr Biol 192: 19–32, 2003. Hoffmann EK. Rho family GTP binding proteins are involved in the 27. Lambert IH, Hoffmann EK, Jørgensen F. Membrane potential, anion regulatory volume decrease process in NIH3T3 mouse fibroblasts. J Downloaded from and cation conductances in Ehrlich ascites tumor cells. J Membr Biol 111: Physiol 541: 779–796, 2002. 113–131, 1989. 40. Poulsen KA, Andersen EC, Hansen CF, Klausen TK, Hougaard C, 28. Lambert IH. Regulation of the cellular content of the organic osmolyte Lambert IH, Hoffmann EK. Deregulation of apoptotic volume decrease taurine in mammalian cells. Neurochem Res 29: 27–63, 2004. and ionic movements in multidrug-resistant tumor cells: role of chloride 29. Lepple-Wienhues A, Szabò I, Wieland U, Heil L, Gulbins E, Lang F. channels. Am J Physiol Cell Physiol 298: C14–C25, 2010. Tyrosine kinases open lymphocyte chloride channels. Cell Physiol Bio- 41. Riquelme G, Sepúlveda FV, Jørgensen F, Pedersen S, Hoffmann EK. chem 10: 307–312, 2000. Swelling-activated potassium currents of Ehrlich ascites tumour cells. 30. Lezama R, Ortega A, Ordaz B, Pasantes-Morales H. Hyposmolarity- Biochim Biophys Acta 1371: 101–106, 1998. http://ajpcell.physiology.org/ induced ErbB4 phosphorylation and its influence on the non-receptor 42. Roman R, Feranchak AP, Troetsch M, Dunkelberg JC, Kilic G, tyrosine kinase network response in cultured cerebellar granule neurons. J Schlenker T, Schaack J, Fitz JG. Molecular characterization of volume- Neurochem 93: 1189–1198, 2005. sensitive SKCa channels in human liver cell lines. Am J Physiol Gastroi- 31. Maingret F, Fosset M, Lesage F, Lazdunski M, Honoré E. TRAAK Is ntest Liver Physiol 282: G116–G122, 2002. a mammalian neuronal mechano-gated Kϩ channel. J Biol Chem 274: 43. Rosette C, Karin M. Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors. 1381–1387, 1999. Science 15: 1194–1197, 1996. 32. Maingret F, Patel AJ, Lesage F, Lazdunski M, Honoré E. Mechano- or 44. Stutzin A, Hoffmann EK. Swelling-activated ion channels: functional acid stimulation, two interactive modes of activation of the TREK-1 regulation in cell-swelling, proliferation and apoptosis. Acta Physiol 187: potassium channel. J Biol Chem 274: 26691–26696, 1999. 27–42, 2006. 33. Niemeyer MI, Cid LP, Barros LF, Sepúlveda FV. Modulation of the 45. vom Dahl S, Schliess F, Reissmann R, Görg B, Weiergräber O, two-pore domain acid-sensitive Kϩ cChannel TASK-2 (KCNK5) by Kocalkova M, Dombrowski F, Häussinger D. Involvement of integrins at Copenhagen University Library on July 10, 2013 changes in cell volume. J Biol Chem 276: 43166–43174, 2001. in osmosensing and signaling toward autophagic proteolysis in rat liver. J ϩ 34. Niemeyer MI, Cid LP, Sepúlveda FV. K conductance activated during Biol Chem 278: 27088–27095, 2003. regulatory volume decrease. The channels in Ehrlich cells and their 46. Wang J, Morishima S, Okada Y. IK channels are involved in the possible molecular counterpart. Comp Biochem Physiol A Mol Integr regulatory volume decrease in human epithelial cells. Am J Physiol Cell Physiol 130: 565–575, 2001. Physiol 284: C77–C84, 2003. AJP-Cell Physiol • VOL 299 • OCTOBER 2010 • www.ajpcell.org Copenhagen, sep. 2013 5 Paper II KCNK5 is functionally down-regulated upon long-term hypotonicity in Ehrlich ascites tumor cells The paper has been submitted to “Cellular Physiology and Biochemistry” Status 09-30-2013: The paper has been found in “principle acceptable for publication”. Page 63 of 117 Copenhagen, sep. 2013 Page 64 of 117 KCNK5 is functionally down-regulated upon long-term hypotonicity in Ehrlich ascites tumor cells Signe Skyum Kirkegaard1, Tune Wulff1+2, Steen Gammeltoft3 and Else Kay Hoffmann1 1Section of Cell and Developmental Biology, Institute of Biology, University of Copenhagen, Denmark, 2National Food Institute, Technical University of Denmark, Kgs. Lyngby, Denmark, 3Molecular Biology, Sleep Research Unit, Diagnostic Department, Glostrup Hospital, Denmark Keywords: KCNK5, TASK-2, long-term volume regulation, RVD Running title: long-term hypotonicity and volume regulated channels Correspondence to: Prof. Else K Hoffmann Section for Cell and Developmental Biology Department of Biology, The August Krogh Building University of Copenhagen 13, Universitetsparken, DK-2100, Copenhagen Ø email: [email protected] 1 Abstract Background/aims: Regulatory volume decrease (RVD) in response to acute cell swelling is well described and KCNK5 (also known as TASK-2 or K2P5.1) 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. 2 Introduction Mammalian cells, with some exceptions, are susceptible to changes in intra- or extracellular amount of osmolytes due to the combination of aquaporins present in the membrane and an osmotic pressure across the membrane. An acute decrease in extracellular or increase in intracellular osmolarity respectively will in most cells result in water uptake and thus in cell swelling. The cell homeostasis is very vulnerable to volume changes and to counteract swelling the cell possess different regulatory mechanisms involving efflux of osmolytes through ion channels and membrane transporters – a response known as regulatory volume decrease (RVD). It is estimated that 70% of the osmolyte efflux in Ehrlich Ascites Tumor (EAT) cells during RVD is KCl efflux [3] and the remaining 30% is due to the efflux of organic osmolytes [4]. For a detailed description of the mechanisms of volume regulation see [1, 2]. 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 K2p5.1) [5-7], and the Volume Regulated Anion Channel (VRAC, ICl,vol) [8]. KCNK5 has besides in Ehrlich cells [5-7] 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. For a recent review on KCNK5 see [15]. Response to hypotonicity can be divided into an acute and a long-term phase. The acute phase which includes RVD happens within minutes of cell swelling. During long-time exposure to osmolarity changes, the cell uses other mechanisms to ensure a steady intracellular environment, such as an altered gene expression and an altered protein synthesis [16]. The role of KCNK5 in acute volume regulation is well described in different cell types (see above). It has previously been shown how the KCNK5 channel is tyrosine phosphorylated after acute cell swelling in an time dependent manner [17] and recently it has been shown how KCNK5 is inhibited by Gβγ subunits of heteromeric G protein and thereby how a G protein coupled mechanism also can regulate the KCNK5 channel [18]. Effects of long-term hypertonicity is well studied and is known to elicit an increase in gene transcription of a number of osmoregulatory genes, which will affect the uptake and synthesis of organic osmolytes (see [2, 19]). On the other hand only very limited work has been done on the effects of long-term hypotonicity on cells, thus a potential role for KCNK5 in long-term regulation has never been studied. Preliminary studies (referred in [2]) using micro array screening suggested that the expression of KCNK5 is 2.7 times down-regulated after 48 hours in a hypotonic medium correlating with preliminary measurements of the maximum swelling- 3 activated KCNK5 current. The purpose of this study was thus to examine the effect of long-term hypotonicity on mRNA-, protein- and functional levels of the KCNK5 channel. Experimental procedures Solutions and materials Hypotonic media (180mOsm) for long-term hypotonic stimulation was obtained by diluting growth media with buffered water, containing 5 mM HEPES, 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (p/s), pH 7.4. The solution was sterile filtered before use. Patch- clamp experiments: hypotonic NaCl Ringer’s solution (180mOsm) contained in mM: 71 Na+, 5 K+, 1 Mg2+, 1 Ca2+, 38 Cl-, 33 gluconate, 10 HEPES, pH 7.4. Isotonic Ringer’s solution (300mOsm) was generated by addition of 110 mM mannitol and contained only 5 mM HEPES. The pipette solution contained in mM: 2 Na+, 116 K+, 1.2 Mg2+, 42.4 Cl-, 62 gluconate, 10 HEPES, 30 mannitol, 10 EGTA, 2.5 ATP and 0.1 GTP. Coulter counter experiments: hypotonic Ringer’s solution + - + 2+ 2- - 2+ (180mOsm) contained in mM: 72 Na , 74.5 Cl , 2.5 K , 0.5 Mg , 0.5 SO4 , 0.5 HPO4 , 0.5 Ca , 3.3 MOPS, 3.3 TES and 5 HEPES, pH 7.4. Isotonic Ringer’s solution (300mOsm) was obtained by the addition of sucrose. Cell cultures ELA cells and EAT cells were cultured in RPMI1640 media supplemented with 10% FBS and 1% p/s o at 37 C and 5% CO2. EAT and ELA cells were maintained by transferring 1 ml cell suspension to 10 ml fresh RPMI1640 medium every 3-4 days. ELA cells were loosened from the surface by trypsination, 1.5 ml 0.25% trypsine/EDTA solution per T-75 culture flask. Only passages 6-30 were used for experiments. Patch-clamp measurements ELA cells were kept in isotonic (300mOsm) or hypotonic medium (180mOsm) for 24 or 48 hours and transferred to 25 mm cover-slips after which they were placed in a chamber mounted on an inverted microscope (Zeiss Axiovert 10, Carl Zeiss, Germany). Solution shifts were generated using gravity-fed and pump-suction mechanisms. The current was measured using standard whole-cell patch-clamp using a suitable amplifier (Axopatch 200B, Axon instruments, California, USA). A Digidata 1200 Interface board and pClamp7 software (Axon Instruments) were used to generate voltage-clamp command voltages, and to digitize data. In the cell attached configuration, prior to establishing the whole-cell configuration the fast capacity transients were eliminated. Following whole-cell formation, the series resistance and the whole-cell capacitance were compensated and annulled with mean values that did not change significantly during experiments [20]. Pipettes were made from Vitrex glass capillary tubing with an outside 4 diameter of 1.7 mm (Modulohm, Herlev, Denmark) using a Narishige PP-830 puller (Tokyo, Japan) and had resistances of ≈4 MΩ. All experiments were carried out at 37 °C. IK was measured as previously described in [5]. Briefly, the membrane potential was clamped in pulses of 500 ms at the equilibrium potential for Cl– which was 5 mv during isotonic conditions and 0 mv under hypotonic conditions and these reversal potentials have previously proven to be good estimates [5, 6, 21]. The voltage correction was due to a dilution of the intracellular ion compositions as a result of hypotonicity and the following cell swelling. The current increase was measured after 300 sec of hypotonic stimulation and taken relative to the cell membrane surface (pF). mRNA measurements ELA and EAT cells were stimulated with hypotonic media (180 mOsm) or isotonic media (300 mOsm) for 24 or 48 hours and lysed in RNA lysis buffer (Machery-Nagel, Düren, Germany). RNA was purified from lysates using NucleoSpin® RNA II (Macherey-Nagel, Düren, Germany) according to manufacturer’s instructions. Reverse transcriptase PCR with SuperScript II and oligo(dT)12-18 primer (Invitrogen, Life Technologies, Naerum, Denmark) was used to generate cDNA from the purified mRNA and performed on an Eppendorf Mastercycler. In a total of 10 µl ddH2O, dNTP mix (500 µM) and oligo(dT)12-18 primer (500 ng) was mixed on ice, 1 µg mRNA was added and incubated for 5 min at 65 °C. The solution was put on ice and briefly centrifuged. 4 µl 5 x first strand buffer, DTT (10 µM) and 1 µl ddH2O was subsequently added and the solution incubated for 2 min at 42 °C. 1 µl SuperScript II was added and incubated at 42 °C for 50 min followed by incubation at 70 °C for 15 min and ending at 4 °C. Real-time qPCR was performed in triplicates using the Stratagene MX4000 PCR system, Brilliant® II SYBR® Green QPCR Master Mix (Stratagene, Agilent Technologies) and the following primers: mKCNK5 forward: 5’-GTC AAG GCC ACT TGG TGA GG-3’ and mKCNK5 reverse: 5’-TGC TGG TGA AGG TGG ACT CA-3’ (KCNK5 GenBank accession number NM_021542) and ARP: mARP forward: 5’-CGA CCT GGA AGT CCA ACT AC-3’ mARP reverse: 5’-ACT TGC TGC ATC TGC TTG-3’ (GenBank accession number NM_007475), which was used as reference gene. 10 µl Master Mix, 0.4 µl forward and reverse primer (200 nM final concentration), 0.4 µl 500 x diluted ROX II reference dye (40 nM final concentration), 7.8 µl ddH2O and 1 µl cDNA was mixed and quantification was done using the following cycles: 95 ˚C 10 min and 95 ˚C 30 sec, 58 ˚C 1 min, 72 ˚C 30 sec x 40. Standard curves were done in order to measure primer efficiency which was adjusted for in calculations. Primers were selected using Primer3 software and purchased from MWG Eurofins (Germany). Quantification was carried out using the Pfaffl method [22]. 5 SDS-PAGE and western blotting ELA and EAT cells were stimulated with isotonic or hypotonic media for 24 or 48 h and subsequently lysed in 95°C lysis buffer (10 mM Tris-HCl pH 7.4, 1% SDS and 20 mM EDTA) with protease inhibitors (Roche Applied Science) and phosphatase inhibitors added. SDS-PAGE and western blotting were performed as previously described [17]. Briefly, we used a SDS-PAGE- western blotting system form Invitrogen (Lfe technologies) and with the following antibodies; KCNK5 (Alomone Lab., Israel) 1:250 and β-actin (Sigma-Aldrich) 1:1000. Since we use two different antibodies on every blot, membranes were cut around 50kDa letting us visualize both KCNK5 and β-actin (65 and 42 kDa respectively) using BCIP/NBT. Protein bands were quantified using UN-SCAN-IT software. Cell membrane protein labeling and purification EAT and ELA cells were grown in 40% hypotonic medium (180mOsm, standard medium diluted with buffered water, see above) or isotonic medium (300mOsm, standard medium) for 48h before cell membrane purification. Cell membrane proteins were biotinylated and purified using Pierce Cell Surface Protein Isolation Kit (Thermo Fisher Scientific) according to manufactures instructions, with two exceptions; after lysis and before purification a sample was drawn from the lysate and protein concentration measured. These samples were used as a control of total KCNK5 protein content under the two circumstances. SDS-PAGE and western blotting was used to analyze the amount of KCNK5 in whole cell lysate as well as purified samples (see above). Cell volume measurements Absolute cell volume was measured by electronic cell sizing using the Coulter Multisizer ll (Coulter, Luton, UK) with a tube orifice of 100 μm. For each experiment 2.5x106 cells were used and cell volume was determined as the median of the cell volume curves after calibration with latex beads (15 µm). Cells were either kept in isotonic (300mOsm) medium or in a 40% hypotonic medium for 24 or 48h. Prior to cell volume measurements cells were centrifuged and resuspended in standard medium (isotonic) were they were kept for 30 min in order to acclimatize before experiencing a hypotonic chock. The duration in standard medium allowed the cells to adapt to the changed environment without influencing protein synthesis and gene transcription. It should be mentioned that the cells shrink when transferred from the hypotonic into the isotonic medium and thus perform a RVI process. As KCNK5 channels are inhibited in shrunken cells [7, 18] we have included the 30 min isotonic acclimatization period and have controlled that there is no significant difference between the cell volumes in the three experimental groups. The mean isotonic, 24h hypotonic pre-treatment and 48h hypotonic pre- treatment cell volumes where 803.7±61.5 µm3, 829.7±58 µm3 and 783.7±46.9 µm3 respectively, with no significant difference between the control and pre-treated cells. 6 All Ringer’s solutions were micro-filtered (Millipore, 0.45 µM) before use. Volume recovery was estimated as (Vmax-V4min)/(Vmax-Viso), where Vmax, V4min and Viso are the maximal cell volume, cell volume at time 4 min and cell volume under isotonic conditions, respectively. Statistics Student’s T-test and one-way ANOVA was used to test for statistical significance. 95% and 99% levels of significance were shown by one star or two stars respectively. Results Patch-clamp studies revealed a decreased maximum swelling-activated current in ELA cells upon 48h of hypotonicity Fig. 1 shows the maximum swelling-activated K+ current through the volume sensitive K+ channel and it is seen that after 24h there is a decreased (not significant) K+ current. This is supported by experiments measuring K+ efflux after 24h hypotonic incubation using 86Rb+ as a tracer. We found that the rate constant for the swelling-activated K+ efflux in stimulated cells was lower than in control cells. At 12 minutes after cell swelling the rate constant was 0.0134 in stimulated cells compared to 0.0154 in the control cells (data not shown), thus supporting the results obtained by patch-clamp. After 48h of hypotonicity the maximum swelling-activated current through KCNK5 was significantly decreased from 28.7 ± 7.2 pA/pF to 7.0 ± 2.1 pA/pF corresponding to a decrease of 75.6%. Long-term exposure to hypotonicity (24 and 48 h) decreased RVD in EAT cells In addition we studied the effect of 24 and 48 hours hypotonicity (180mOsm) on RVD in EAT cells (Fig. 2) and found that after both 24h and 48h RVD was significantly decreased both when looking at % recovery after 4 min (fig. 2A+B) and at the initial rate of RVD (fig. 2A+C). 24h of hypotonic stimulation resulted in a decreased RVD when re-exposing the cells to a hypotonic shock, with a recovery after 4 minutes of 19.3 ± 1.33% compared with 41.5 ± 2.51% seen in the control cells (fig. 2B), corresponding to a 53% decrease. When looking at the initial rate of RVD, 24h of hypotonicity resulted in a relative decrease in the rate of RVD to 46.5±3.79% compared to untreated control cells (fig. 2D). After 48h the initial rate of RVD was further decreased to 30.8±3.69% compared to untreated control cells equal to a 69% RVD inhibition (fig. 2D). The % recovery after 4 min was decreased to 12±1.51% compared to 41.5±2.51% of the control cells corresponding to a 71% decrease after 48h of hypotonicity. There was no significantly change in mRNA levels in EAT and ELA cells upon long-term hypotonicity To further elucidate the nature of the decreased physiological function of the KCNK5 channel we investigated the expression of KCNK5 on mRNA and protein levels. 7 Real-Time qPCR was performed on treated (48h of hypotonicity) and untreated (control) EAT and ELA cells and fig. 3 shows the mRNA expression data for ELA (Fig. 3A) and EAT (Fig. 3B) cells upon long-term hypotonicity. It is seen that even though there is a 10.6% decrease in mRNA levels in 48h hypotonically treated ELA cells compared to the untreated control cells the decrease is not significant. In the EAT cells an apparent increase in KCNK5 mRNA was detected, though the difference was also not significant. KCNK5 protein levels were significantly decreased in EAT and ELA cells upon 48h of hypotonically exposure Western blot analysis on EAT and ELA cells revealed a significant decrease in KCNK5 protein expression levels after 48h, but not after 24h of hypotonically stimulation. Fig. 4A shows how the mean KCNK5 protein level in EAT cells is approximately the same in both control cells and after 24h of hypotonicity, but is significantly decreased to 70±4.4% after 48h of treatment. The same analysis in ELA cells likewise revealed a significant difference between protein amount in control and treated cells (fig. 4B). After 48h of hypotonicity a ≈30% decrease to 70.9±12.2% was seen, while an apparent increase (not significant) after 24h was observed. The results were confirmed in a single experiment by measuring cell surface KCNK5 protein amount using membrane protein biotinylation and showing how 48h of hypotonic treatment decreases the amount of KCNK5 protein inserted into the membrane by approximately 50%, thus confirming and not very different from the decrease seen in total KCNK5 protein (see fig 4C). Discussion Since the KCNK5 channel is a very important player in the RVD response in Ehrlich cells, we investigated a possible effect of long-term hypotonicity on RVD and on expression of KCNK5. The RVD measurements were performed on the EAT cell line using a Coulter Counter. EAT cells were chosen for these volume measurements since the most accurate volume measurements are obtained on cells in suspension. A significant impairment of RVD was found both after 24 and 48 hours of long-term exposure to the hypotonic medium. The inhibited RVD performance in EAT cells could reflect a decreased function of either KCNK5 (K+) or of VRAC (Cl-). However since the KCNK5 channel is the rate limiting factor for RVD in these cells [14] and since patch-clamp measurements showed a decreased maximum swelling-activated current through KCNK5 upon long-term hypotonic stimulation, the results were taken to indicate a down-regulation of the KCNK5 channels or reduced ability of the KCNK5 channel during RVD. The physiological impairment of RVD and of the maximum swelling-activated K+ current through KCNK5 channels could be due to regulation on a number of levels such as I) a down-regulation of KCNK5 protein synthesis II) KCNK5 mRNA transcription III) post-translational modifications 8 resulting in an inhibited channel function IV) an unknown regulatory mechanism “turning off” the channel V) an internalization of membrane embedded channels or a combination of the above. We found that there was no significant difference between the mRNA amount of KCNK5 in control cells compared to hypotonic stimulated cells which were true in both EAT and ELA cells. Since there was no significant difference in the mRNA expression pattern between control and stimulated cells we conclude that the physiological changes most likely are not due to regulation on the transcriptional level. It might be argued that cells with down-regulated KCNK5 expression could have died before 48h and this is why no decrease in mRNA level is seen. However, the fact that we do see a clear down-regulation at the protein level argues against the possibility. Although there was no effect of long-term stimulation on gene transcription levels, we found significant lower levels of KCNK5 protein after 48h of hypotonicity in both cell lines, thus indicating a higher degree of KCNK5 protein degradation or decreased synthesis of the KCNK5 protein. Our results were further substantiated in one experiment, where it was shown how 48h of long-term hypotonicity resulted in a decrease in KCNK5 protein present in the plasma membrane, thus supporting the theory that long-term hypotonicity decreases KCNK5 protein expression and may in addition cause an altered KCNK5 sorting to the membrane. As the decrease in membrane bound KCNK5 was not very different from the total decrease in KCNK5 protein, we have not looked further into this. At least three factors could influence this change in KCNK5 protein expression level: the lowered ion strength, the decrease in K+ concentration and the decreased Cl- concentration all generated by the response to the hypotonic medium. We speculate if e.g. the decreased K+ concentration could prompt the cells to down-regulate the K+ leak-channel KCNK5 in order to govern the smaller amount of K+ more strictly. The lowered Cl- concentration could result in the same mechanisms, but since we are dealing with a significant decrease in KCNK5 protein expression, we suggest that the physiological changes are predominantly due to alterations in potassium rather that in chloride level. To our knowledge not much work has been published on long-term effects of hypotonicity on protein synthesis but it should be noted that it has been shown that protein synthesis is up- regulated in hepatocytes of air-breathing walking catfish upon hypotonicity (≈ 2 hours) [23]. Since we are dealing with a down-regulation of the KCNK5 protein this is thus not likely to be caused by a general effect on the protein synthesis but rather by a specific effect on the KCNK5 protein synthesis or breakdown. 9 There is a time discrepancy between the physiological data and the protein expression data obtained in EAT cells, since there is a significant impairment of RVD in EAT cells already after 24h of hypotonicity but the down-regulation of KCNK5 protein amount is first detectable after 48h of hypotonicity. It is likely that this is caused by a regulatory inhibition of the channel followed by a later decrease in protein synthesis. This has not been further investigated. Taken together our results show that the clear physiological changes of the KCNK5 channel in Erhlich cells upon long-term hypotonic stimulation is predominantly due to a decreased protein synthesis. We cannot rule out other regulatory mechanisms as contributors to the down- regulation of the maximum swelling-activated KCNK5 current seen after long-tem hypotonicity. We have previously shown how protein tyrosine kinases are vital in KCNK5 channel opening and that tyrosine phosphatases are important for the closing of the channel upon a swelling- mediated activation of the channel [17], and it has furthermore been suggested that KCNK5 activation can be modulated by G protein Gβγ subunits [18]. Thus an altered tyrosine kinase/phosphatase profile or changes in the G protein coupled mechanism could potentially be involved in the functional inhibition of KCNK5. The technical skills of Dorthe Nielsen and Birthe Juul Hansen (University of Copenhagen) is gratefully acknowledged and appreciated. This work was supported by The Danish Council for Independent Research/Natural Sciences (grants 09–064182 and 10–085373) and The Lundbeck Foundation, Denmark (J Nr R32-A3102). Conflict of interest The authors declare no conflict of interest. 10 References 1 Lang F, Busch GL, Ritter M, Völkl H, Waldegger S, Gulbins E, Häussinger D: Functional Significance of Cell Volume Regulatory Mechanisms. Physiol Rev 1998;78:247-306. 2 Hoffmann EK, Lambert IH, Pedersen SF: Physiology of Cell Volume Regulation in Vertebrates. Physiol Rev 2009;89:193-277. 3 Hendil KB, Hoffmann EK: Cell volume regulation in Ehrlich ascites tumor cells. J Cell Physiology 1974;84:115-125. 4 Hoffmann Else K, Hendil Klavs B: The role of amino acids and taurine in isoosmotic intracellular regulation in Ehrlich ascites mouse tumour cells. J Comp Physiol 1976;108:279-286. 5 Riquelme G, Sepúlveda FV, Jørgensen F, Pedersen S, Hoffmann EK: Swelling-activated potassium currents of Ehrlich ascites tumour cells. Biochim Biophys Acta 1998;1371:101-106. 6 Hougaard C, Niemeyer MI, Hoffmann EK, Sepúlveda FV: K+ currents activated by leukotriene D4 or osmotic swelling in Ehrlich ascites tumour cells. Pflügers Archiv European Journal of Physiology 2000;440:283-294. 7 Niemeyer MI, Cid LP, Barros LF, Sepúlveda FV: Modulation of the Two-pore Domain Acid-sensitive K+ Channel TASK-2 (KCNK5) by Changes in Cell Volume. J Biol Chem 2001;276:43166-43174. 8 Pedersen SF, Prenen J, Droogmans G, Hoffmann EK, Nilius B: Separate Swelling- and Ca2+-activated Anion Currents in Ehrlich Ascites Tumor Cells. J Membr Biol 1998;163:97-110. 9 Barriere H, Belfodil R, Rubera I, Tauc M, Lesage F, Poujeol C, Guy N, Barhanin J, Poujeol P: Role of TASK2 Potassium Channels Regarding Volume Regulation in Primary Cultures of Mouse Proximal Tubules. J Gen Physiol 2003;122:177-190. 10 Bobak N, Bittner S, Andronic J, Hartmann S, ühlpfordt F, Schneider-Hohendorf T, Wolf K, Schmelter C, Göbel K, Meuth P, Zimmermann H, Döring F, Wischmeyer E, Budde T, Wiendl H, Meuth SG, Sukhorukov VL: Volume regulation of murine T lymphocytes relies on voltage-dependent and two- pore domain potassium channels. Biochim Biophys Acta 2011;1808:2036-2044. 11 Andronic J, Bobak N, Bittner S, Ehling P, Kleinschnitz C, Herrmann AM, Zimmermann H, Sauer M, Wiendl H, Budde T, Meuth SG, Sukhorukov VL: Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes. Biochimica et Biophysica Acta (BBA) - Biomembranes 2013;1828:699-707. 12 Barfield JP, Yeung CH, Cooper TG: The Effects of Putative K+ Channel Blockers on Volume Regulation of Murine Spermatozoa. Biology of Reproduction 2005;72:1275-1281. 13 Skatchkov SN, Eaton MJ, Shuba YM, Kucheryavykh YV, Derst C, Veh RW, Wurm A, Iandiev I, Pannicke T, Bringmann A, Reichenbach A: Tandem-pore domain potassium channels are functionally expressed in retinal (Müller) glial cells. Glia 2006;53:266-276. 14 Lambert IH, Hoffmann EK, Jørgensen F: Membrane potential, anion and cation conductances in Ehrlich ascites tumor cells. J Membr Biol 1989;111:113-131. 11 + 15 Cid LP, Roa-Rojas HA, Niemeyer MI, González W, Araki M, Araki K, Sepúlveda FV: TASK-2: a K2P K channel with complex regulation and diverse physiological functions. Frontiers in Physiology 2013;4:1- 9. 16 Cohen DM: SRC family kinases in cell volume regulation. Am J Physiol Cell Physiol 2005;288:C483- C493. 17 Kirkegaard SS, Lambert IH, Gammeltoft S, Hoffmann EK: Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation. American Journal of Physiology - Cell Physiology 2010;299:C844-C853. 18 Añazco C, Peña-Münzenmayer G, Araya C, Cid LP, Sepúlveda F, Niemeyer M: G protein modulation of K2P potassium channel TASK-2. Pflugers Arch - Eur J Physiol 2013;1-12. 19 Burg MB, Kwon ED, Kültz D: Regulation of gene expression by hypertonicity. Annual Review of Physiology 1997;59:437-455. 20 Hougaard C, Jørgensen F, Hoffmann EK: Modulation of the volume-sensitive K+ current in Ehrlich ascites tumour cells by pH. Pflügers Archiv European Journal of Physiology 2001;442:622-633. 21 Niemeyer MI, Hougaard C, Hoffmann EK, Jørgensen F, Stutzin A, Sepúlveda FV: Characterisation of cell swelling-activated K+ selective cunductance of ehrlich ascites tumour cells. J Physiol 2000;524:757- 767. 22 Michael W.Pfaffl: A new mathematical model for relative quantification in real-time RT–PCR. Nucleic acids Research 2001;29:2002-2007. 23 Biswas K, Jyrwa L, Häussinger D, Saha N: Influence of cell volume changes on protein synthesis in isolated hepatocytes of air-breathing walking catfish (Clarias-ábatrachus). Fish Physiol Biochem 2010;36:17-27. 12 Fig. 1 Activity of KCNK5 current in ELA cells upon long-term hypotonic stimulation Cells were kept in isotonic (300mOsm) or hypotonic medium (180mOsm) for 24 or 48 hours. The maximum swelling-activated K+ current was measured using standard whole-cell patch-clamp. The current increase was measured after 300 sec of hypotonic stimulation (n=3) and taken relative to the cell membrane surface (pF). Student’s T-test was used to test for statistical significance and one star (*) indicate a 95% significance level. Fig. 2 RVD in EAT cells A Coulter counter was used to measure the volume of isotonic control (300m) and long-term (24 or 48h) hypotonically (180 mOsm) treated EAT cells. A: representative figure showing relative RVD over time (sec) for untreated control cells and cells kept in hypotonic medium for 24 or 48 h. B: Mean volume recovery of n=5 experiments after 4 min was calculated as (Vmax-V4min)/(Vmax- Viso), where Vmax, V4min and Viso are the maximal cell volume, cell volume at time 4 min and cell volume under isotonic conditions, respectively. C: initial rate of RVD or the slope was calculated (n=5) using linear regression on the linear part of the RVD curve, from maximum volume to the end of linearity. One-way ANOVA was used to test for statistical significance and two stars (**) indicate a 99% significance level. Fig. 3 mRNA levels of KCNK5 in ELA and EAT cells ELA (A) (n=4) and EAT (B) (n=5) cells were stimulated with hypotonic (180 mOsm) or isotonic media (300 mOsm) for 24 or 48 hours and Real-Time qPCR was performed. Student’s T test or one-way ANOVA was used to test for statistical significance. Fig. 4 KCNK5 protein levels in Ehrlich ascites tumor and Ehrlich Lettré ascites cells. Cells were isotonic (300mOsm) or hypotonically (180mOSm) treated for 24 or 48 hours and SDS- PAGE and western blotting was performed on the lysates using antibodies against KCNK5 and β- actin. A: representative western blot and mean protein levels (n=3) in EAT cells. B: representative western blot and mean protein levels (n=9) in ELA cells. C: membrane purification (n=1) showing KCNK5 protein amount inserted into the membrane under control and 48h hypotonic conditions. Student’s T-test was used to determine statistical significance and one star (*) represents a statistical significance level of 95%. 13 14 15 16 17 Copenhagen, sep. 2013 Page 82 of 117 Copenhagen, sep. 2013 6 Paper III The potential role of KCNK5 in activated T cell physiology with specific focus on cell volume control Paper in preparation. Page 83 of 117 Copenhagen, sep. 2013 Page 84 of 117 The potential role of KCNK5 in activated T cell physiology with specific focus on cell volume control Signe S. Kirkegaard1, Pernille Dyhl Strøm1, Anker Jon Hansen2, Steen Gammeltoft3 and Else K. Hoffmann1 1Section for Cell and Developmental Biology, University of Copenhagen, Denmark. 2Novo Nordisk, Måløv, Denmark. 3Molecular Biology Sleep Research Unit, Diagnostic Department, Glostrup Hospital, Denmark Running title: KCNK5 and T cell activation Correspondence to: Else K Hoffmann, PhD Section for Cell and Developmental Biology Department of Biology, The August Krogh Building University of Copenhagen 13, Universitetsparken, DK-2100, Copenhagen Ø email: [email protected] 1 Summary The potential role of the two-pore domain potassium channel KCNK5 (also known as TASK-2 and K2P5.1) in activated T cell physiology has only recently been described and for years the main focus of potassium channels in T cell physiology have been on the voltage-gated K+ channel Kv1.3 and the Ca2+-activated KCa3.1. So far KCNK5 has been described to be up-regulated in multiple sclerosis patients and to be implicated in the volume regulatory mechanism regulatory volume decrease (RVD) in T cells. KCNK5 has furthermore been shown to be involved in RVD in Ehrlich cells, proximal tubules and spermatozoa. Here we further elucidate on the potential role of KCNK5 in activated T cell physiology, thus we investigated the time-dependent expression pattern of KCNK5 in activated T cells together with its role in RVD. We find that KCNK5 is indeed up-regulated in CD3/CD28 activated T cells both on mRNA and protein levels, but despite this strong up-regulation we find the RVD response to be inhibited in activated T cells compared to non-activated control cells. In addition we find that T cells in response to activation increase their volume and swell more than non-activated control cells when subjected to hypotonicity. We furthermore find the swelling-activated Cl- permeability in activated T cells to be strongly decreased and thus find the RVD inhibition predominantly to be due to the decreased Cl- permeability. Our results suggest that the up-regulation of KCNK5 in activated T cells does not play a volume regulatory role and we speculate that it might play a role in hyperpolarizing the cell membrane and thus increasing the Ca2+ influx. 2 Introduction The importance of ion channels in T cell activation and activated T cell function is established. Especially the role of the voltage-gated Kv1.3, the Ca2+-activated Kca3.1 and Ca2+ release- activated Ca2+ channel (CRAC) is well known. For reviews on ion channels and T cells see (8; 30; 34). Activation of the T cell when encountering an antigen requires a sustained rise in 2+ 2+ 2+ intracellular Ca concentration ([Ca ]i) which is generated by IP3 mediated Ca release from stores in ER. This in turn mediates the activation of CRAC channels allowing Ca2+ entry from the 2+ 2+ cytosol. This Ca influx and subsequent rise in [Ca ]i activates KCa1.3 and also causes a depolarization activating the voltage sensitive Kv1.3 K+ channel. Activation of Kv1.3 and KCa3.1 causes K+ efflux and hyperpolarization of the plasma membrane allowing further Ca2+ entry via 2+ the CRAC channels in the membrane. The sustained increase in [Ca ]i is essential for the transcription and expression of interleukin 2 (IL-2) which in turn is essential for keeping the T cells activated without the requirement of further antigen stimulation (see (23)). Other ion channels, however, have been implicated in T cell activation. The leak conductance channel (TREK-2) was described in WEHI-231 cells which is a murine immature B cell line (27; 37). Further studies have also revealed the presence of two-pore domain K+ channels in lymphocytes e.g. TASK-1 and TASK-3 (25) and of special interest to this study Bittner and co-workers suggested that the two-pore domain potassium channel KCNK5 (also known as TASK-2 or K2P5.1) has a role in T cell activation and in multiple sclerosis (5). KCNK5 has previously been shown to be volume sensitive and to be implicated in the volume regulatory process of regulatory volume decrease (RVD) in response to cell swelling in various cell types and tissues including Ehrlich Ascites Tumor (EAT) cells (19; 29), mouse proximal tubules (4), human and murine spermatozoa (2; 3) and murine T lymphocytes (6). Recently it was shown that two-pore domain potassium channels are also involved in volume regulation in human T cells (1). For a recent review on KCNK5 see (12). T cells like most other mammalian cells are subject to changing intracellular environment causing potential volume changes and subsequent regulation. T cells are in constant movement around the body and thereby subjected to extracellular osmotic changes e.g. in the kidney and as such, T cells are as most other mammalian cells capable of performing RVD in response to cell swelling (7; 10; 11; 14). As seen in all volume regulating cells the volume regulated anion channel (VRAC) is responsible for the swelling-activated Cl- current (8) whereas different K+ channels have been suggested to play a part in T cell volume regulation including the voltage-gated potassium channel Kv1.3 (9; 22), the Ca2+-activated KCa3.1 (33), and two-pore domain potassium channels (1; 6). 3 KCNK5 is up-regulated during T cell activation and is also involved in volume regulation (1; 5) but there is no description on the time dependency of the expression pattern of KCNK5 during T cell activation and its physiological consequences. This is the subject of the present paper and in addition we demonstrate a strong decrease in the swelling-activated Cl- permeability during the activation which inhibits the RVD process in the large activated T cells. Experimental procedures Solution and Materials Volume measurements: Hypotonic Ringer’s solution (180mOsm) contained (in mM): 71.5 NaCl, 2.5 KCl, 0.5 MgSO4, 0.5 Na2HPO4, 0.5 CaCl2, 3.3 MOPS, 3.3 TES and 5 HEPES, pH 7.4. Isotonic Ringer’s solution (300mOsm) was obtained by addition of sucrose. Cl- permeability: the hypotonic (150mOsm) low Na+ Ringer’s solution contained (in mM): 70 NMDGCl-, 0.8 NaCl, 5 KCl, 1 K2HPO4, 1 MgSO4, 1 CaCl2, 3.3 MOPS, 3.3 TES and 5 HEPES, pH 7.4. Gramicidin and Clofilium was purchased from Sigma and used in the concentrations 1µM and 100µM respectively Purification and maintenance of cells Human T cells were purified from buffy coats from healthy donors. Human buffy coats were obtained from the blood bank at Rigshospitalet, Copenhagen, Denmark. All procedures were performed at room temperature. The T cell population was purified from buffy coats using RosetteSep™ (Human T Cell Enrichment Cocktail, StemCell Tech.) accordingly to manufactures description. After purification any residual red blood cells was lysed using RBC lysis buffer (eBioscience). Purified T cells were kept in RPMI1640 GlutaMax medium supplemented with 30 o U/ml IL-2, 10% FBS and 1% P/S at 37 C and 5% CO2. RNA purification and Real-Time qPCR T cells were stimulated for 2, 4, 8, 12, 24, 48, 72 and 144 hours CD3/CD28 beads (Invitrogen) and RNA was purified from lysates using NucleoSpin® RNA II (Macherey-Nagel) according to manufacturer’s instructions. Reverse transcriptase PCR with SuperScript II (Invitrogen) and oligo(dT)12-18 primer (Invitrogen) was used to generate cDNA from the purified mRNA and performed on a Eppendorf Mastercycler as previously described (20). Real-Time 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’- 4 ACGTGCTTCTCTGCCTTGTT-3’. A total volume of 20 μl containing 1 μl of the cDNA, 200 nM of primers, and 10 μl 2× MasterMix was used. Despite testing numerous potential reference genes none of them lived up to standards and instead determination of total cDNA concentration was used to correct the data obtained. To determine cDNA content upon PCR reaction and to normalize samples Quant-iT™ OliGreen® ssDNA Assay Kit was used as described in (24). Standard curves were also done to measure primer efficiency which was corrected for in calculations. Primers were selected using Primer3 software and purchased from MWG Eurofins (Germany). Quantification was carried out using the Pfaffl method: (26) Western blotting T cells were stimulated for 2h, 4h, 8h, 12h, 24h, 48h, 72h or 144h days with CD3/CD28 beads and lysed in 95°C lysis buffer (10 mM Tris-HCl pH 7.4, 1% SDS, 20 mM EDTA) with protease inhibitors (Roche Applied Science) and phosphatase inhibitors added. SDS-PAGE and western blotting was performed as previously described (19). We used the following antibodies and concentrations: KCNK5 (TASK-2) (Alomone Lab., Israel) 1:100, β-actin (Sigma-Aldrich) 1:1000. Volume measurements by Coulter Counter Volume measurements: T cells were stimulated with CD3/CD28 beads for 2h, 4h, 8h, 12h, 24h, 48h, 72h or 144 before the absolute cell volume was measured by electronic cell sizing using the Coulter Multisizer ll (Coulter, Luton, UK) with a tube orifice of 80 μm. Before measurements calibration was performed using 15 µm latex beads. Approximately 2.5x106 cells were used per experiment and cell volume was determined in either isotonic or hypotonic Ringer’s solutions (all micro-filtered before use). Volume recovery was estimated in two different ways, by % recovery after 3 min as previously described (19) and by calculating the initial rate of RVD measured as the slope of the recovery part of the RVD curve. Cl- permeability: to determine if the Cl- permeability was decreased in activated T cells we used a method previously described in (17) with some minor alterations, thus we used clofilium as a blocker of potassium channel KCNK5 and a NMDG Na+ substitution Ringer’s solution (see above). Briefly, the principle of the experiments is as follows: the addition of clofilium blocks the KCNK5 channel allowing the cells to swell when subjected to hypotonicity but inhibiting K+ efflux and thus RVD. When adding gramicidin a high cation permeability is introduced making Cl- the limiting factor. This way makes it possible to indirect measure the Cl- permeability since a cell shrinkage will reflect the cells Cl- permeability which then can be measured in relative values. 5 Statistical methods Statistical significance was determined by Student’s T-test or one-way analysis of variance (ANOVA) where one star represent statistical significance at a 95% level, two stars at a 99% level and three stars at a >99% significance level. Results KCNK5 and KCa3.1 is highly up-regulated on mRNA level in CD3/CD28 activated T cells When activating the T cells for 2, 4, 8, 12, 24, 48, 72 and 144 hours with CD3/CD28 beads both KCa3.1 and KCNK5 were up-regulated while no significant change was seen in Kv1.3 (fig. 1A) mRNA expression level compared to un-stimulated controls using Real-Time qPCR. An initial down-regulation of KCa3.1 (fig. 1B) mRNA level was observed upon 2 and 4 h of stimulation followed by an increasing up-regulation after 8, 12 and 24 h reaching a stable level of a 13 fold up-regulation after 48 h and with statistic significant values after 72 and 144 h (p<0.05). KCNK5 (fig. 1C) showed the largest up-regulation of the three with a 115 fold peak-increase in mRNA level following 24 h of T cell activation when comparing with the non-activated T cells. No initial down-regulation was observed regarding KCNK5 thus a 4 fold increase was already seen after 2 h. Statistical significance was only seen at 24h (p<0.05) when using ANOVA test though the up- regulation seem clear. This could be due to the rather large standard deviations seen in the data. When using Student’s T test on the data-set comparing each test value to its respective control statistical significance is found at times 2, 4, 8 and 144h (p<0.05). KCNK5 is also highly up-regulated on protein level Figure 2 shows a biphasic KCNK5 protein expression pattern with an initial significant decrease (p<0.001) during the initial stimulation (2 and 4h) of the cells with CD3/CD28 beads followed by a still increasing protein expression after 8, 12, 24 and 48 h. The expression peaks upon 72 h of stimulation with a 277% increase compared to the quiescent control cells with a subsequent slight decrease to 209% at 144 h of activation both time-points showing significant more KCNK5 protein in activated T cells than in the controls (p<0.001 and p<0.05 respectively). The activation of the T cells resulted in fast proliferation of more than 300% measured 72 h after CD3/CD28 stimulation compared to non-stimulated control cells (seen in two experiments, data not shown). Activation causes isotonic cell swelling, decreased RVD ability and increased hypotonic swelling Since the KCNK5 channel is a known volume regulator we tested the activated T cells ability to perform regulatory volume decrease (RVD) upon hypotonicity. From the volume measurements 6 depicted in fig. 3 it is seen that the activated T cells when measured in isotonic Ringer’s solution are larger (fig. 3A+B) – as described many times before, but it is also seen that in hypotonic Ringer’s solution the activated cells swell more than the non-activated control cells (fig. 3C). After 8 h of CD3/CD28 stimulation T cells in isotonic Ringer’s solution begin increasing their volume and after 24 h the mean volume is significantly (p<0.001) larger than the control cells with a mean volume of 171.6 µm3 compared to 118.8 µm3of the control cells. A plateau is seen at times 72 and 144 h where the activated cells have significantly (p<0.001) increased their volume by 213% (fig. 3B). Figure 3A+C shows how both activated and non-activated cells swell in hypotonic Ringer’s solution but to a different degree. CD3/CD28 stimulated T cells have increased maximum swelling starting at 8 h of stimulation, with highest measured values after 72 h and a small decrease seen after 144 h. At 72 h their maximum swelling reaches a value of 309.3 µm3 compared to a gain of volume of 71.9 µm3 seen in control cells equivalent to a 4 times increase. Maximum swelling values 24, 72 and 144 h after stimulation are significantly larger than control values (p<0.001). Besides swelling more when hypotonically challenged, activated T cells have poorer RVD performance compared to control cells. As seen from fig. 3A both stimulated and non- stimulated T cells swell and perform RVD but when measuring % recovery after 3 min (19) and the initial rate of RVD (see experimental procedures) stimulated T cells show much less RVD capability than unstimulated control cells. As a control the RVD response in non-stimulated control cells was measured as a function of time, and it is seen that the recovery percentages of non-activated T cells are not significantly different from each other at the measured time-points (average recovery of 36% after 3 min). From fig. 3D it is seen how the relative recovery of activated T cells decreases beginning after 8 h of stimulation and with significant decrease at 12, 24, 72 and 144 h. After 72 h of CD3/CD28 stimulation the cells have the lowest ability to perform RVD with a recovery of 13.6% compared to 35.7% (data not shown) of the un-stimulated control cells and equivalent to a 60% decrease (fig. 3D). After 144 h the cells seem to regain a small fraction of RVD performance now with an absolute mean recovery of 15.5% or 53% relative to the control. When measuring RVD by the initial rate of shrinkage the results are similar – activated T cells have a lesser RVD performance than control cells (fig. 3E). It is seen that the initial rate of RVD is relatively faster in all controls when comparing them to the activated T cells though the values are only significantly lower for 8, 12, 24, 72 and 144 h. 8 h of CD3CD28 stimulation results in a 26% decreased RVD rate of (p<0.05) followed by a further decrease after 12 and 24 h to 56% and 53% respectively (p<0.001). Stimulation for 72 and 144 h results in a slightly faster RVD with rates of 67% (p<0.01) and 63% (p<0.001) compared to that of the control cells. 7 The Cl- permeability of activated T cells are inhibited Even though KCNK5 is a well-known player in RVD, the considerable up-regulation of the channel in activated T cells does not seem to increase the cells ability to perform RVD on the contrary RVD is inhibited in CD3/CD28 activated cells. Since RVD is driven by the extrusion of KCl we speculated if the Cl- permeability of the activated cells could be decreased. To determine if a decreased Cl- permeability could be responsible for the inhibited RVD we used the method described in materials and methods and in (17). As described in (17) we use the rate of RVD after addition of gramicidin in a low Na+ media as a measure of the swelling-activated Cl- permeability. As seen in fig. 4 the addition of gramicidin to hypotonically swollen non-stimulated control cells result in a significant faster RVD (higher Cl- permeability) with a 34.3±1.02% recovery compared to -0.102±1.24% (statistically not different from 0) seen in the CD3/CD28 activated T cells. Thus there is a significant decrease (p<0.05) in the Cl- permeability in non- stimulated T cells when comparing them to their non-activated counterparts. Discussion In the current study we find that the activated T cells enlarge upon activation which is a well described feature in the activated and proliferating T cells (31; 36). We find that this enlargement occurs in a time-dependent manner which coincides with the massive up- regulation of KCNK5 protein. The strong up-regulation is in agreement with earlier findings (1; 5). Thus we here confirm these findings while adding a time profile. The time profile described here shows an initial (after 2 and 4 hours of CD3/CD28 stimulation) and significant decrease in KCNK5 protein expression in activated T cells which correlates with a minimal though not significant early shrinkage and decreased maximum swelling when comparing the activated T cells to non-activated control cells. CD3/CD28 T cell activation causes an initial loss of cell number which in two experiments was found to be of 56% and 41% after 2 and 4 hours of stimulation respectively. This could reflect that only the activated cells survive whereas the stimulated but non-activated cells undergo programmed cell death. The initial decrease in cell number is only seen in activated cells and is therefore not due to purification procedures or culturing conditions. The loss of cell number after 2 and 4 hours correlates with the decrease in KCNK5 protein and we speculate if this decrease could occur in order to protect the T cells from apoptosis. It should be noted that the initial cell loss does not influence the results obtained by western blot, since protein expression measurements are performed on the same amount of total protein at each time-point. It has been shown that the same channels involved in RVD are also active in apoptosis thus apoptotic volume decrease (AVD) is a vital signal in the cells commitment to apoptosis (21). We therefore speculate if the down-regulation of KCNK5 protein expression seen in the heterogenic T cell 8 population including both apoptotic and surviving/proliferating cells reflect an initial protection against apoptosis whereas the later up-regulation of KCNK5 protein is involved in the increased proliferation in the activated T cell CD3/CD28 activated T cells not only get larger when measured in isotonic Ringer’s solution they also swell significantly more when subjected to a hypotonic Ringer’ solution as if they have an inhibited KCl efflux. Despite the up-regulation of the potent volume regulator KCNK5 we also find the activated T cells to have an inhibited RVD performance upon cell swelling compared to the control cells. Since an up-regulation of KCNK5 protein expression with a decreased RVD might seem contradictory we speculate if the up-regulation occurs in order to facilitate proliferation. Proliferating cells are enlarged to keep daughter cells the same size as the parental cell a feature vital for successful proliferation, thus a strong RVD mechanism would be problematic for the activated T cell whose main objective is to increase in size and number to sufficient fight off a pathogenic intrusion. The KCNK5 up-regulation will help insure a hyperpolarization of the cell membrane and thus a sustained Ca2+ influx vital for the long-term activation of the T cells keeping them activated in an antigen-independent manner (13; 16; 18; 28; 35) this would facilitate optimal conditions for the T cell expansion. Andronic et al. find the RVD performance in stimulated and non-stimulated T cells to be the same, an observation contradicting our findings (1), but Andronic et al only measure the 20 min volume recovery and from their figure 2 we calculate the initial rate of volume recovery to be slower in stimulated cells compred to non-stimulated cell as found in the present study. With respect to maximum swelling we and Deutch and Lee (15) find the stimulated cells to swell more than non-stimulated control cells an observation contradicting what was found by Andronic et al (1). We do not have an explanation for these apparent differences. Since the RVD in activated T cells was found to be inhibited despite the strong up-regulation of KCNK5 protein we speculated whether the swelling-mediated Cl- permeability in activated T cells could be inhibited thereby setting a limit for the cells RVD capacity. We tested this and found that the Cl- permeability through the volume sensitive anion channel VRAC indeed was inhibited in the activated T cells compared to control cells. This means that regardless of the increase in KCNK5 protein quantity the cells will not perform RVD as long as there is a functional down- regulation of VRAC since the KCl efflux during RVD is an electroneutrale process. It should be noted that our results are in conflict with those published by Deutsch and Lee in 1988 (15) where it is shown how phytohemagglutinin activated human peripheral blood lymphocytes (PBL) respond to gramicidin in a set-up similar to ours by decreasing cell volume, and it is thus speculated that K+ is the rate limiting factor of RVD in activated T cells. A major difference seems to be that Deutsch and Lee are not adding gramicidin until 30+ minutes after cell swelling whereas we add it immediately after cell swelling when the channels are maximally stimulated. 9 We have previously shown in Ehrlich cells that the volume activated channels close before 15 min after swelling activation (17) and Sarkadi and co-workers show that it is also true in human lymphocytes (32). Another difference between our experiments and those presented by Deutsch and Lee is the fact that they used PBL’s which contained T cells, B cells and monocytes whereas we use purified T cells. In summary we suggest that the KCNK5 up-regulation detected in CD3/CD28 activated human T cells helps ensure a hyperpolarization of the membrane thus favoring a sustained Ca2+ influx and T cell activation. We further suggest that to keep the cells from loosing volume thereby supporting a strong proliferation the Cl- channel VRAC is functionally down-regulated. Acknowledgements The technical skills of Dorthe Nielsen (University of Copenhagen) and Pia Birn (Novo Nordisk) is gratefully acknowledged and appreciated. This work was supported by The Danish Council for Independent Research/Natural Sciences (grants 09–064182 and 10–085373) and The Lundbeck Foundation, Denmark (J Nr R32-A3102). Conflicts of interest The authors declare that they have no conflict of interest. 10 Reference List 1. Andronic J, Bobak N, Bittner S, Ehling P, Kleinschnitz C, Herrmann AM, Zimmermann H, Sauer M, Wiendl H, Budde T, Meuth SG and Sukhorukov VL. Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes. Biochimica et Biophysica Acta (BBA) - Biomembranes 1828: 699-707, 2013. 2. Barfield JP, Yeung CH and Cooper TG. Characterization of potassium channels involved in volume regulation of human spermatozoa. Molecular Human Reproduction 11: 891-897, 2005. 3. Barfield JP, Yeung CH and Cooper TG. The Effects of Putative K+ Channel Blockers on Volume Regulation of Murine Spermatozoa. Biology of Reproduction 72: 1275-1281, 2005. 4. Barriere H, Belfodil R, Rubera I, Tauc M, Lesage F, Poujeol C, Guy N, Barhanin J and Poujeol P. Role of TASK2 Potassium Channels Regarding Volume Regulation in Primary Cultures of Mouse Proximal Tubules. J Gen Physiol 122: 177-190, 2003. 5. Bittner S, Bobak N, Herrmann AM, Göbel K, Meuth P, Höhn KG, Stenner MP, Budde T, Wiendl H and Meuth SG. Upregulation of K2P5.1 potassium channels in multiple sclerosis. Ann Neurol 68: 58- 69, 2010. 6. Bobak N, Bittner S, Andronic J, Hartmann S, ühlpfordt F, Schneider-Hohendorf T, Wolf K, Schmelter C, Göbel K, Meuth P, Zimmermann H, Döring F, Wischmeyer E, Budde T, Wiendl H, Meuth SG and Sukhorukov VL. Volume regulation of murine T lymphocytes relies on voltage- dependent and two-pore domain potassium channels. Biochim Biophys Acta 1808: 2036-2044, 2011. 7. Bui AH and Wiley JS. Cation fluxes and volume regulation by human lymphocytes. J Cell Physiol 108: 47-54, 1981. 8. Cahalan MD and Chandy KG. The functional network of ion channels in T lymphocytes. Immunological Reviews 231: 59-87, 2009. 9. Cahalan MD and Lewis RS. Role of potassium and chloride channels in volume regulation by T lymphocytes. Soc Gen Physiol Ser 43: 281-301, 1988. 10. Cheung RK, Grinstein S, Dosch H-M and Gelfand EW. Volume regulation by human lymphocytes: characterization of the ionic basis for regulatory volume decrease. J Cell Physiol 112: 189-196, 1982. 11. Cheung RK, Grinstein S and Gelfand EW. Volume regulation by human lymphocytes. Identification of differences between the two major lymphocyte subpopulations. J Clin Invest 70: 632-638, 1982. 12. Cid LP, Roa-Rojas HA, Niemeyer MI, González W, Araki M, Araki K and Sepúlveda FV. TASK-2: a K2P K+ channel with complex regulation and diverse physiological functions. Frontiers in Physiology 4: 1- 9, 2013. 13. Crabtree GR. Contingent Genetic Regulatory Events in T Lymphocyte Activation. Science 243: 355- 361, 1989. 11 14. Deutsch C, Slater L and Goldstein P. Volume regulation of human peripheral blood lymphocytes and stimulated proliferation of volume-adapted cells. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 721: 262-267, 1982. 15. Deutsch C and Lee SC. Cell volume regulation in lymphocytes. Renal Physiol Biochem 11: 260-276, 1988. 16. Goldsmith MA and Weiss A. Early signal transduction by the antigen receptor without commitment to T cell activation. Science 240: 1029-1031, 1988. 17. Hoffmann E, Lambert I and Ole Simonsen L. Separate, Ca2+-activated K+ and Cl- transport pathways in Ehrlich ascites tumor cells. J Membrain Biol 91: 227-244, 1986. 18. Imboden JB, Weiss A and Stobo JD. The antigen receptor on a human T cell line initiates activation by increasing cytoplasmic free calcium. The Journal of Immunology 134: 663-665, 1985. 19. Kirkegaard SS, Lambert IH, Gammeltoft S and Hoffmann EK. Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation. American Journal of Physiology - Cell Physiology 299: C844-C853, 2010. 20. Kirkegaard, Signe Skyum, Wulff, Tune, Gammeltoft, Steen, and Hoffmann, Else K. KCNK5 is functionally down-regulated upon long-term hypotonicity in Ehrlich ascited tumor cells. 2013. Ref Type: Unpublished Work 21. Lang F, Ritter M, Gamper N, Huber S, Fillon S, Tanneur V, Lepple-Wienhues A, Szabo I and Bulbins E. Cell Volume in the Regulation of Cell Proliferation and Apoptotic Cell Death. Cellular Physiology and Biochemistry 10: 417-428, 2000. 22. Lee SC, Price M, Prystowsky MB and Deutsch C. Volume response of quiescent and interleukin 2- stimulated T-lymphocytes to hypotonicity. American Journal of Physiology - Cell Physiology 254: C286-C296, 1988. 23. Lewis RS. CALCIUM SIGNALING MECHANISMS IN T LYMPHOCYTES. Annu Rev Immunol 19: 497-521, 2001. 24. Lundby C, Nordsborg N, Kusuhara K, Kristensen K, Neufer P and Pilegaard H. Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies. European Journal of Applied Physiology 95: 351-360, 2005. 25. Meuth SG, Bittner S, Meuth P, Simon OJ, Budde T and Wiendl H. TWIK-related Acid-sensitive K+ Channel 1 (TASK1) and TASK3 Critically Influence T Lymphocyte Effector Functions. J Biol Chem 283: 14559-14570, 2008. 26. Michael W.Pfaffl. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic acids Research 29: 2002-2007, 2001. 27. Nam JH, Woo JE, Uhm DY and Kim SJ. Membrane-delimited Regulation of Novel Background K+ Channels by MgATP in Murine Immature B Cells. J Biol Chem 279: 20643-20654, 2004. 28. Negulescu PA, Shastri N and Cahalan MD. Intracellular calcium dependence of gene expression in single T lymphocytes. Proceedings of the National Academy of Sciences 91: 2873-2877, 1994. 12 29. Niemeyer MI, Cid LP, Barros LF and Sepúlveda FV. Modulation of the Two-pore Domain Acid- sensitive K+ Channel TASK-2 (KCNK5) by Changes in Cell Volume. J Biol Chem 276: 43166-43174, 2001. 30. Panyi G, Varga Z and Gáspár R. Ion channels and lymphocyte activation. Immunology Letters 92: 55- 66, 2004. 31. Petrzilka GE and Schroeder HE. Activation of human T-lymphocytes. Cell Tissue Res 201: 101-127, 1979. 32. Sarkadi B, Mack E and Rothstein A. Ionic events during the volume response of human peripheral blood lymphocytes to hypotonic media. II. Volume- and time-dependent activation and inactivation of ion transport pathways. J Gen Physiol 83: 513-527, 1984. 33. Schlichter LC and Sakellaropoulos G. Intracellular Ca2+ Signaling Induced by Osmotic Shock in Human T Lymphocytes. Experimental Cell Research 215: 211-222, 1994. 34. Varga Z, Hajdu P and Panyi G. Ion channels in T lymphocytes: An update on facts, mechanisms and therapeutic targeting in autoimmune diseases. Immunology Letters 130: 19-25, 2010. 35. Weiss A, Imboden J, Shoback D and Stobo J. Role of T3 surface molecules in human T-cell activation: T3-dependent activation results in an increase in cytoplasmic free calcium. Proceedings of the National Academy of Sciences 81: 4169-4173, 1984. 36. Zaffran Y, Destaing O, Roux As, Ory Sp, Nheu T, Jurdic P, Rabourdin-Combe C and Astier AL. CD46/CD3 Costimulation Induces Morphological Changes of Human T Cells and Activation of Vav, Rac, and Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase. The Journal of Immunology 167: 6780-6785, 2001. 37. Zheng H, Nam JH, Pang B, Shin DH, Kim JS, Chun YS, Park JW, Bang H, Kim WK, Earm YE and Kim SJ. Identification of the large-conductance background K+ channel in mouse B cells as TREK-2. American Journal of Physiology - Cell Physiology 297: C188-C197, 2009. 13 Fig. 1: mRNA levels of KCNK5, KCa3.1 and Kv1.3 determined by Real-Time qPCR mRNA levels were determined using Real-Time qPCR and primers against KCa3.1 (A), Kv1.3 (B) and KCNK5 (C). ANOVA test was used to test for statistical significance on 5-8 independent experiments (n=2 for 48h). One star (*) indicates a 95% significance level. Fig. 2: KCNK5 protein expression T cells were purified from human buffy coats and stimulated with CD3/CD28 activation beads for 2h, 4h, 8h, 12h, 24h, 48h, 72h or 144h. SDS-PAGE and western blotting was performed using antibodies against KCNK5 and β-actin. Protein bands were visualized and pixel density calculated. ANOVA was used to determine statistical significance on 3-10 independent experiments and one star (*) represents a statistical significance level of 95% whereas (***) indicate a significance level >99%. No significance was seen after 2 and 4 h using the statistical test ANOVA but when using Student’s T-test both times showed significance here represented by (###) indicating a level of significance of >99%. Fig. 3: RVD in CD3/CD28 stimulated T cells and non-activated controls T cells were purified from human buffy coats and stimulated with CD3/CD28 activation beads for 2, 4, 8, 12, 24, 72 or 144 hours before absolute volume was measured using a coulter counter. Stimulated T cells and non-stimulated controls were measured in either isotonic (300mOsm) or hypotonic (160mOsm) Ringer’s solution. A: representative figure showing RVD over time (min) for untreated control cells and CD3/CD28 stimulated T cells. B: Initial mean volume measured in isotonic Ringer’s solution C: Mean maximal swelling seen in hypotonic Ringer’s solution. D: Mean volume recovery after 3 min was calculated as (Vmax-V3min)/(Vmax-Viso), where Vmax, V3min and Viso are the maximal cell volume, cell volume at time 3 min and cell volume under isotonic conditions, respectively. E: initial rate of RVD (the slope) was calculated using linear regression on the linear part of the RVD curve, from maximum volume to the end of linearity. ANOVA test was used to test for statistical significance on 5-7 independent experiments and one star (*) represent a 95% significance level whereas two (**) and three stars (***) indicate significance levels of 99% and >99% respectively. Fig. 4: Cl- permeability in RVD in CD3/CD28 activated and non-activated T cells T cells were purified from human buffy coats and stimulated with CD3/CD28 beads for 72 hours. Activated T cells and non-activated control cells were treated with 100µM clofilium for 30 min to block the potassium channels. The cells were swollen in hypotonic low Na+ NMDG Ringer’s solution and their volume was measured using a coulter counter. After one min. 1 µM gramicidin was added to facilitate cation flux thereby letting the Cl- flux being the limiting factor. Student’s T-test was used to test for statistical significance on 3 independent experiments and two stars (**) indicate a significance level of 99%. 14 15 16 17 *** 18 Copenhagen, sep. 2013 7 Final thesis discussion, future work and perspectives Throughout my time as a PhD student I have worked with the two-pore domain potassium channel KCNK5 in various cell systems and with different focus points in mind. My contribution to the field has mainly been on the channels role in cell volume control, and I have thus worked with its short time activation, effect of long-term anisotonicity and the potential role of KCNK5 in activated T cell physiology. In this section I will focus on the perspectives of my work presented in the three papers included in the thesis. Each of the papers holds their own discussion section and I will thus refer to those, but at the same time try to look a bit further into the perspectives and future work to be done. Protein tyrosine phosphorylation of KCNK5 upon acute cell swelling In Paper I we show how over-expression of KCNK5 channel protein enhances RVD in human embryonic kidney (HEK)-293 cells which confirms earlier findings (104), indicating that the K+ efflux upon swelling is rate-limiting for the RVD response in this set up (Paper I, fig. 4). We furthermore show how protein tyrosine kinases (PTK’s) play an important role in the opening of the channel, since inhibiting PTK’s resulted in an inhibited RVD response in response to hypotonic cell swelling (Paper I, fig. 1) and how protein tyrosine phosphatases are important in the closing of the channel (Paper I, fig. 3). We test whether the Src kinase (Paper I, fig. 6) or the focal adhesion kinase (FAK) (Paper I, fig. 7) could be the PTK’s involved, but our results suggest that neither of them are likely candidates. On the contrary we find the Janus kinase (JAK) could be a potential candidate (Paper I, fig. 8). PTK phosphorylation could be involved at different steps in pathway, from cell swelling to KCNK5 channel (called TASK-2 in the paper, see section 1.3) activation and K+ efflux (53), and we found that cell swelling resulted in a direct tyrosine phosphorylation of KCNK5 itself with a time course following the time course of the RVD response (Paper I, fig. 5). We have not tested the potential role of PTK’s in other steps of the pathway and thus cannot rule out the possibility that tyrosine kinases and tyrosine phosphatases could also be involved somewhere else in the pathway from cell swelling to KCNK5 activation – a question that could be interesting to look further into. Our findings opens up for a lot of new questions, as to which tyrosine phosphorylation site (or sites) are phosphorylated upon swelling-activation of the channel and which kinase (or kinases) are involved? When submitting the KCNK5 sequence (GenBank accession number NM_021542) to the NetPhos 2.0 server predicting tyrosine phosphorylation sites, 5 possible sites are identified (fig. 12). Page 103 of 117 Copenhagen, sep. 2013 NetPhos 2.0 Server - prediction results Technical University of Denmark 502 Sequence MVDRGPLLTSAIIFYLAIGAAIFEVLEEPHWKEAKKNYYTQKLHLLKEFPCLSQEGLDKILQVVSDAADQGVAITGNQTF 80 NNWNWPNAMIFAATVITTIGYGNVAPKTPAGRLFCVFYGLFGVPLCLTWISALGKFFGGRAKRLGQFLTRRGVSLRKAQI 160 TCTAIFIVWGVLVHLVIPPFVFMVTEEWNYIEGLYYSFITISTIGFGDFVAGVNPSANYHALYRYFVELWIYLGLAWLSL 240 FVNWKVSMFVEVHKAIKKRRRRRKESFESSPHSRKALQMAGSTASKDVNIFSFLSKKEETYNDLIKQIGKKAMKTSGGGE 320 RVPGPGHGLGPQGDRLPTIPASLAPLVVYSKNRVPSLEEVSQTLKNKGHVSRPLGEEAGAQAPKDSYQTSEVFINQLDRI 400 SEEGEPWEALDYHPLIFQNANITFENEETGLSDEETSKSSVEDNLTSKEQPEQGPMAEAPLSSTGEFPSSDESTFTSTES 480 ELSVPYEQLMNEYNKADNPRGT 560 ...... Y...... 80 ...... Y...... 160 ...... Y...... 240 ...... Y...... 320 ...... Y...... 400 ...... 480 ...... 560 Phosphorylation sites predicted: Tyr: 5 Tyrosine predictions Name Pos Context Score Pred ______v______Sequence 15 AIIFYLAIG 0.019 . Sequence 38 AKKNYYTQK 0.061 . Sequence 39 KKNYYTQKL 0.789 *Y* Sequence 101 TTIGYGNVA 0.740 *Y* Sequence 118 FCVFYGLFG 0.076 . Sequence 190 EEWNYIEGL 0.917 *Y* Sequence 195 IEGLYYSFI 0.248 . Sequence 196 EGLYYSFIT 0.418 . Sequence 219 PSANYHALY 0.124 . Sequence 223 YHALYRYFV 0.014 . Sequence 225 ALYRYFVEL 0.007 . Sequence 232 ELWIYLGLA 0.075 . Sequence 301 KEETYNDLI 0.965 *Y* Sequence 349 PLVVYSKNR 0.042 . Sequence 387 PKDSYQTSE 0.958 *Y* Sequence 412 EALDYHPLI 0.210 . Sequence 486 LSVPYEQLM 0.104 . Sequence 493 LMNEYNKAD 0.194 . ______^______ Figure 12: NetPhos tyrosine phosphorylation prediction NetPhos server 2.0 was used to predict tyrosine phosphorylation sites on KCNK5 (mus musculus GenBank accession number NM_021542). 5 possible tyrosine phosphorylation sites were predicted by the software. http://www.cbs.dtu.dk/services/NetPhos/ Future experiments using e.g. mass spectrometry could help answer the questions of which site/sites are phosphorylated upon swelling-induced activation of the channel and which kinases are involved. Answering those questions would be really beneficial for the further and full story of PTK’s in the swelling-activation of KCNK5. Page 104 of 117 Copenhagen, sep. 2013 As described in section 1.3.3 there are various other gating and activation mechanisms involved in the swelling-induced activation of KCNK5 and we cannot exclude the possibilities of other gating mechanisms being involved. Long-term hypotonic stimuli and its consequences for KCNK5 Paper II deals with the long-term effects of hypotonicity on KCNK5 channel expression and physiology. In patch-clamp (Paper II, fig. 1) and Coulter counter experiments (Paper II, fig. 2) we find that there is a decreased maximum current through the channel and at the same time, we see an inhibited RVD response after cell swelling, when subjecting the cells to long-term hypotonicity. The RVD results could also reflect an inhibition of VRAC or perhaps a combination of decreased KCNK5 and VRAC activities. Since the molecular identity of VRAC remains unknown, it would not be possible to measure mRNA or protein expression, but the volume activated Cl- current through VRAC is measurable and it would be interesting to see what potential effect long-term hypotonicity would have on VRAC. It should though be noted that K+ efflux have been shown to be rate-limiting of RVD in Ehrlich cells, thus it is very likely that the physiological changes seen is due to K+ channel regulation. Since we speculated that the physiological impairment was likely to be caused by changes related to K+ efflux, we studied the KCNK5 expression pattern both on mRNA and protein levels. We found a down-regulation of the KCNK5 channel protein (Paper II, fig. 4) suggesting that the functional inhibition of the K+ current and the RVD response is indeed likely to be due to lowered amount of KCNK5 protein. Not much work has to my knowledge been done on the long-term effects of hypotonicity, thus we haven’t got much to relate our findings to, nor to help explain the exact mechanisms behind, which again leaves some questions un-answered. Future experiments could, and should, aim at explaining the mechanisms behind the functionally down-regulation of the KCNK5 channel. We suggest that the physiological impairment is the effect of a decreased KCNK5 synthesis or increased KCNK5 protein degradation, but we cannot exclude the possibility of a contribution from one or more still un-known regulatory mechanisms. Such mechanisms could e.g. involve direct or indirect regulation of the channel by one or more of the mechanisms described in section 1.3.3., a decreased protein insertion into the membrane by an altered protein sorting on its way to the membrane, internalization of KCNK5 proteins already embedded in the membrane or post-translational modifications. Since our results show a lowered KCNK5 protein expression and since we (in one experiment) show an approximately equal decrease in KCNK5 inserted into the membrane we do favor the idea that an altered KCNK5 protein expression is the main reason for the changes observed rather than a decreased recruitment to the membrane. Page 105 of 117 Copenhagen, sep. 2013 We find that there is some discrepancy between the physiological data and the protein expression in which a physiological effect is already seen after 24 hours of stimulation whereas the inhibited protein expression is first detectable upon 48 h of hypotonicity, thus suggesting that something else causes initial impairment of the channel. We therefore speculate that the cell uses other regulatory mechanisms (see section 1.3.3 and above) initially in the response to the acute environmental change until an altered KCNK5 protein expression can be effective. As to the question of why the cell down-regulates the KCNK5 protein and thus introduce an impaired physiological function in response to long-term hypotonicity different factors could be involved. As the long-term hypotonic stimulated cell experiences a loss of cellular ionic strength and a decrease in cellular KCl concentration it is very likely that the cell down-regulates potassium channels to protect the cell potassium homeostasis. As described in section 1.2 K+ channels and thus potassium ions are involved in numerous physiological mechanisms e.g. setting the membrane potential, proliferation secretion of hormone and transmitters, thus the cellular potassium concentration is important for many cellular functions. Furthermore a decrease in intracellular K+ is seen in apoptosis (see section 1.4.2.1) thus a functional down- regulation of the channel in a low K+ media could help protect the cell. KCNK5 in activated T cell physiology We got the opportunity to work together with Novo Nordic on the project of KCNK5 in T cell activation and thus the previously cell systems studied were changed from a murine cell line to primary human T cells and the focus point changed to T cell activation. I have not regretted taking a directional shift since the work on T cell physiology proved to extremely exciting and the chance to work together with an industrial firm to be very educational. I can only regret that limited time means that there is still work to be done before this story is complete. While changing cell type and host species we still worked on KCNK5 and its activation, involvement in volume regulation and expression pattern. A potential role for KCNK5 and other two-pore domain K+ channels in lymphocyte biology is by some regarded as questionable (112; 146), but recent publications from different groups have made the presence and physiological function of two-pore domain K+ channels including KCNK5 in lymphocytes more than likely (3; 10; 11; 35; 98; 100). Paper III ads to the increasing evidence for a role for KCNK5 in activated T cell physiology, thus we find the channel to be massive up-regulated both on mRNA and protein levels upon T cells activation (Paper III, fig 1C and fig. 2). The roles of the two potassium channels Kv1.3 and KCa3.1 in T cell activation has long been known and is undoubtedly of great importance in T cell activation and physiology, thus we also looked at the mRNA expression of Kv1.3 and KCa3.1. We found Kv1.3 mRNA to be expressed in T cells but with no change in expression level upon T cell activation. KCa3.1 mRNA is on the other hand, like KCNK5 mRNA, Page 106 of 117 Copenhagen, sep. 2013 was also up-regulated in activated T cells. Kv1.3 is believed to be of most importance in regulating membrane potential in quiescent T cells, whereas KCa3.1 is up-regulated upon activation, though some variation in expression profile is seen in different T cell sub-populations (see (16)). Since we find that KCNK5 is so heavily up-regulated with no resulting increase in RVD (Paper III, fig. 3) we suggest that KCNK5 holds another function in activated T cells namely as a important player in hyperpolarizing the plasma membrane upon activation and thus facilitating a sustained Ca2+ influx (see section 1.5.3.) in co-operation with Kv1.3 and KCa3.1 and possible other K+ channels. Since the RVD response in activated T cells is inhibited even though KCNK5 is up- regulated we also investigated the volume sensitive Cl- permeability after T cell activation in order to see if a down-regulation of the volume sensitive Cl- current could account for the decreased RVD performance seen in activated T cells, and we did find this to be the case (Paper III, fig. 4). In future experiments it would be interesting to perform patch-clamp measurements of the Cl- current upon T cell activation, to confirm our indirect findings of a decreased permeability. Furthermore membrane potential and proliferation measurements on KCNK5 knock-down T cells would help elucidate the potential importance of KCNK5 in activated T cell function. All in all new knowledge has come from the work presented here though it has also raised several new questions as research often does and only time can tell if and how these questions will be answered. Page 107 of 117 Copenhagen, sep. 2013 8 Appendix Co-authorship statement for papers I, II and III can be found on the following pages. Page 108 of 117