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Immunology Letters 130 (2010) 19–25

Contents lists available at ScienceDirect

Immunology Letters

journa l homepage: www.e lsev ier .com/ locate /

urrent views

on channels in T lymphocytes: An update on facts, mechanisms and therapeuticargeting in autoimmune diseases

oltan Vargaa, Peter Hajdua, Gyorgy Panyia,b,∗

University of Debrecen, Department of Biophysics and Cell Biology, 98. Nagyerdei krt, Debrecen 4032, HungaryCell Biology and Signaling Research Group of the Hungarian Academy of Sciences, Department of Biophysics and Cell Biology, Research Center for Molecular Medicine,niversity of Debrecen, Debrecen 4032, Hungary

r t i c l e i n f o

rticle history:eceived 25 November 2009eceived in revised form 8 December 2009ccepted 10 December 2009

a b s t r a c t

During the last quarter of a century a large body of evidence was gathered about the involvement ofion channels in T lymphocyte activation. A series of remarkable findings promoted T cell ion channelsto become potential pharmaceutical targets in the therapy of autoimmune disorders. Numerous com-

vailable online 21 December 2009

eywords:on channelcell activation

mmunosuppression

prehensive reviews describe the types of ion channels found in the plasma membrane of T cells andtheir roles in signaling pathways leading to activation, the changes in the expression of these channelsbrought upon by differentiation to various T cell subsets, the formation and possible functions of signalingmolecular clusters that include ion channels in the immunological synapse, the discovery and refinementof structurally different ion channel blockers and the successful in vivo application of such compoundsto suppress hypersensitivity reactions and autoimmune processes. In this review we wish to provide a

opics

mmunological synapse concise update on these t

. Ion channels in T cells

The first ion channel described in T lymphocytes was theoltage-gated Kv1.3 potassium channel, which has a fundamentalole in the activation pathway and presently also seems to be theost important target to control T cell activation [1,2]. Kv1.3 chan-

els are activated by membrane depolarization over −60 mV andheir open probability steeply increases with more positive mem-rane potentials. The opening of Kv1.3 channels at a depolarizedoltage exceeding their activation threshold creates a hyperpolar-zing K+ efflux resulting in a negative feedback loop. Due to the veryigh electrical resistance of the lymphocyte membrane, the open-

ng of a few of these channels is sufficient to regulate the membraneotential.

The other plasma membrane potassium channel of T cells ishe Ca2+ activated KCa3.1 (formerly called IKCa1, for intermediateonductance Ca2+-activated K+ channel), which is activated when

he free intracellular Ca2+ concentration rises above about 200 nM3]. Thus, at resting Ca2+ levels these channels are silent and arectivated only during Ca2+ signaling, when they counteract theepolarizing effect of Ca2+ influx on the membrane potential by

∗ Corresponding author at: Department of Biophysics and Cell Biology, Universityf Debrecen, Medical and Health Science Center, 98. Nagyerdei krt, Debrecen 4012,ungary. Tel.: +36 52 412 623; fax: +36 52 412 623.

E-mail address: panyi@dote.hu (G. Panyi).

165-2478/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.imlet.2009.12.015

from recent years, highlighting the most notable developments.© 2009 Elsevier B.V. All rights reserved.

allowing K+ efflux. Ca2+ sensing is accomplished by calmodulinmolecules bound to the C terminus of KCa3.1 subunits [4] (Fig. 1).

Besides these two potassium channels several other channeltypes have been demonstrated or suggested to be expressed byT cells. These include the Ca2+ release-activated Ca2+ (CRAC) chan-nel, the swelling-activated Cl− channel, various members of the TRPchannel family, two-pore K+ channels and even voltage-gated Na+

and Ca2+ channels (reviewed in [5]). The function of some of thesechannels is questionable, but the existence and involvement of theCRAC channel in T cell activation has been known for a long time[6,7]. Despite this, its molecular identity and the coupling betweenendoplasmic reticulum (ER) emptying and activation of the chan-nel remained a mystery up until a few years ago. As it turned out,two proteins, named Orai1 and STIM1 are necessary to produceCRAC current. Orai1 is the pore forming subunit of the channel,while STIM1 is the sensor of Ca2+ depletion in the ER lumen [8–11].STIM1 is found in the ER membrane with an EF-hand in the ERlumen, while Orai1 is located in the plasma membrane mostly asa dimer [12]. Upon store emptying STIM1 senses the drop in Ca2+

concentration and relocalizes to puncta closely associated with theplasma membrane. This translocation brings Orai1 dimers togetherto form tetrameric active CRAC channels that allow the influx of

2+

Ca .The concerted action of the two K+ channels and the CRAC

channel achieves a precisely controlled and long lasting Ca2+ sig-nal that is required for the execution of the activation pathway.CRAC channels provide the entry route for Ca2+ while tightly

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egulated K+ efflux maintains a permissive membrane potential forhe prolonged Ca2+ influx.

. Kv1.3 in autoimmunity: a potential therapeutic target

The sensitivity of the membrane potential and that of the Ca2+

ignal to blockers of the lymphocyte K+ channels immediatelyurned the attention to the application of these molecules for theuppression of immune responses in autoimmune conditions. Andeal immunosuppressant would specifically inhibit the prolifera-ion of T cells being responsible for autoimmune tissue damage butould leave protective immune reactions unharmed. The expres-

ion of Kv1.3 and KCa3.1 channels in naïve, unstimulated cellseemed to contradict the applicability of Kv1.3 or KCa3.1 inhibitorso achieve specific suppression of autoimmune reactions.

This apparent contradiction has been resolved in the last decadey the discovery of the subset specific expression of ion channels inand B lymphocytes [13]. Based on the cell surface marker expres-

ion, cytokine secretion and physiological function T cells can belassified into three major groups [14]. Naïve T cells that have notncountered previously an antigen bear CCR7+CD45RA+ functionalarker expression. Central memory T cells (TCM, CCR7+CD45RA−),hich cells mediate reactive memory, are probably arrested at

ntermediate stages of terminal differentiation to become effectoremory cells (TEM). These cells have little or no effector function,

ut readily proliferate and differentiate to effector cells in responseo antigenic stimulation. Protective memory is governed by effector

emory TEM cells (CCR7−CD45RA+/−). TEM cells display character-stic sets of chemokine receptors and adhesion molecules that areequired for homing to inflamed tissues where they exert imme-iate effector function. In several autoimmune diseases, includingultiple sclerosis (MS) [13], rheumatoid arthritis and type-I dia-

etes mellitus [15], chronically activated TEM cells are responsibleor tissue damage.

Several studies confirmed that these T cell subsets react totimulation with a characteristic change in the expression of K+

hannels [13,16]. Naïve, TCM and TEM cells of either CD4+ orD8+ phenotype express similar number (200–300) of Kv1.3 and

ewer than 30 IKCa1 channels per cell. Upon specific stimula-ion of naïve and TCM cells the number of Kv1.3 channels per cellncreases modestly, ∼1.5-fold, whereas they dramatically upreg-late KCa3.1 channels (500 channel/cell) and thus, acquire anv1.3lowKCa3.1high phenotype. On the contrary, activation of TEM

s accompanied by an increase in the number of Kv1.3 chan-els to ∼1500 per cell without any change in the KCa3.1 levels,hereby the channel phenotype of the activated TEM becomesv1.3highKCa3.1low. Consistent with the ion channel phenotype

he proliferation of Kv1.3highKCa3.1low TEM cells becomes Kv1.3-ependent, i.e. proliferation of these cells can be completely andersistently inhibited by selective Kv1.3 inhibitors (e.g. ShK, see

ater). Although the proliferation of naïve and TCM cells is ini-ially sensitive to Kv1.3 blockers repeated stimulation of the cellselieves the Kv1.3 block-mediated inhibition of proliferation as

consequence of KCa3.1 upregulation [17]. Thus, proliferationf re-activated naïve and TCM becomes sensitive to blockers ofhe KCa3.1 channel. Based on these observations targeting ofv1.3 with high affinity and selectivity inhibitors may result inpecific immunosuppression manifesting in the inhibition of TEM-ediated autoimmune reactions but leaving protective immunity

nharmed.

The presence of Kv1.3highKCa3.1low TEM has been confirmed for

everal human autoimmune diseases, such as multiple sclerosisMS), type 1 diabetes mellitus (T1DM) and rheuamtoid arthritisRA). Human T cell clones generated by repeated antigenic chal-enge of peripheral blood lymphocytes of MS patients become

tters 130 (2010) 19–25

Kv1.3highKCa3.11low TEM only if they were repeatedly stimulatedwith the proper autoantigen (myelin basic protein, MBP) but not ifthe antigenic challenge was with irrelevant antigens such as insulinor ovalbumin [13]. A latter study identified Kv1.3high TEM cells inpost-mortem brain slices of MS patients [18]. These cells werefound in the parenchimal infiltrate of MS plaques. Autoantigen-specific T cells (GAD65 and insulin) were recently isolated from theperipheral blood of T1DM patients either by MHC class II tetramersorting or by the generation of short-term antigen-specific T cellclones [15]. The Kv1.3 expression of these autoantigen-specific TEMcells was significantly higher than that of T cells isolated from T1DMpatients but specific for MBP. One patient in that study sufferedfrom both T1DM and MS and the T cell clones specific for GAD65,insulin and MBP all had Kv1.3high channel phenotype correspond-ing to the presence of TEMs specific for both of these antigens. Incase of RA T cells isolated form the synovial fluid are predominantlyCCR7− TEM cells having Kv1.3high channel phenotype [15]. The accu-mulation of these cells in the autoimmune-based arthritis (RA) wasspecific, the synovial fluid form non-autoimmune arthritis patients(osteoarthritis) contained naïve and TCM cells with Kv1.3low phe-notype. These results clearly indicate a correlation between theautoantigen-specific TEM phenotype and the Kv1.3 expression ofthe cells.

Given that autoantigen-specific TEM cells are Kv1.3high andthat their proliferation is persistently inhibited by selective Kv1.3inhibitors several excellent in vivo animal experiments revealedthe applicability of Kv1.3 inhibitors in the prevention or treatmentof various experimental autoimmune reactions mimicking humandiseases. A common characteristic of these animal experiments isthat they were done in rats where the ion channel expression ofnaïve, TCM and TEM cells is similar to that of the human counter-parts [13,16]. Murine T cells, on the contrary, express several typesof voltage-gated K+ channels in addition to Kv1.3. This, combinedwith the high KCa3.1 channel expression makes murine models ofautoimmune diseases unsuitable for evaluating a Kv1.3 inhibitor-based pharmacological approach.

In line with the above adoptive transfer of experimental autoim-mune encephalomyelitis (AT-EAE), an accepted animal model formultiple sclerosis in Lewis rats, could be prevented or the symp-toms of the disease could be greatly ameliorated using selectiveKv1.3 blockers alone or in combination with a KCa3.1 inhibitor,respectively [16]. Similarly, the symptoms of EAE in Lewis ratswere significantly reduced by inhibiting Kv1.3 function, which wasattributed to the great reduction in the proliferative response ofMBP specific T cells along with a decrease in the production of IL-2,TNF and in Ca2+ influx [19]. The use of high affinity and specificityKv1.3 inhibitors in in vivo treatment and prevention experimentsturned out to be very safe, the amount of the toxin (e.g. ShK(L5), seebelow) needed for therapeutic use does not cause cardiac toxicityassessed by continuous EKG monitoring and does not alter clinicalchemistry and hematological parameters after a 2-week therapy[20]. Several parameters characterizing the pharmacokinetics ofKv1.3 inhibitors were determined and improved for increased sta-bility (e.g. ShK192, see below) [21].

Kv1.3 inhibitors also ameliorate disease in rat models of RAand T1DM. In the pristane-induced MHC class II-restricted chronicarthritis model (model of RA) Kv1.3 inhibitor-treated (ShK(L5)-amide treatment) rats had significantly fewer affected joints duringthe 21 days of the treatment than the control group of animals[15]. A prevention trial in MHC class II-restricted DP-BB/W rats,a standard model for T1DM, showed that the development ofexperimental autoimmune diabetes (EAD) in the Kv1.3 specific

inhibitor-treated (PAP-1 treatment, see below) group was reducedto ∼50% of the control group. The prevention of EAD in PAP-1-treated rats is likely to be the result of specific immunosuppressionmanifesting in a decreased intraislet T cell and macrophage infiltra-

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ion and reduced � cell destruction compared with vehicle-treatedontrols [15].

In a rat model of allergic contact dermatitis (ACD) the infil-rating cells are CD8+ Kv1.3high TEM cells [22]. A PAP-1-meditatednhibition of the Kv1.3 activity potently suppressed oxazolone-nduced inflammation by inhibiting the infiltration of CD8+ T cellsnd reducing the production of the inflammatory cytokines IFN-, IL-2, and IL-17. Interestingly, topical application of PAP-1 wass effective as intraperitoneal or oral administration, which openshe possibility of developing a drug for the topical treatment ofnflammatory skin diseases such as psoriasis.

The application of cyclosporine (CsA) in the long-term preven-ion of kidney transplant rejection is limited by its severe kidneyoxicity which emphasizes the need for novel immunosuppressivetrategies. A recent study addressed this issue by substituting aombination of Kv1.3 and KCa3.1 inhibitors for CsA following thenitial period of CsA application for 7 days [23]. Allografts wereransplanted from Fisher to Lewis rats and hystopathology showedhat the K+ channel inhibitor-based treatment is equally potent tohe CsA-treatment in the reduction of interstitial mononuclear cellnfiltration, the number of CD43+ T cells, CD8+ cytotoxic T cells andD68+ macrophages in the allografts.

Parallel to the discovery of the therapeutic applicability innimal experiments the pharmacological properties of Kv1.3nhibitors were also dramatically improved resulting in high affin-ty, high specificity and stable Kv1.3 inhibitors. These efforts areummarized in the next section.

. High affinity and specificity blockers of Kv1.3

The discovery that lymphocyte activation in vitro can be inhib-ted by agents that block their potassium channels is nearly as olds the discovery of the channels themselves [1,2]. The first in vivopplication of a high affinity Kv1.3 blocker with the purpose ofmmunosuppression came more than a decade later [24]. The find-ng that the activation of TEM cells involved in several autoimmuneiseases relies heavily on Kv1.3 channels while that of TCM andaïve cells does not, gave new momentum to the search for selec-ive Kv1.3 blockers [13]. During the last decade several compoundslocking Kv1.3 channels were shown to prevent the delayed typeypersensitivity reaction and improve the symptoms of variousodels of autoimmune diseases [15,16,19,25].The molecules shown to block Kv1.3 channels belong in two

arge classes: peptide toxins and small molecule blockers. Kv1.3-locking peptide toxins from scorpions, sea anemones or othernimals typically consist of 30–40 residues and have a molecu-ar weight of about 4 kDa. They carry a large positive net chargend consequently are impermeant through cell membranes. Pep-ide toxins utilize a “cork in the bottle” blocking strategy, coveringnd plugging the external end of the channel pore, thereby pre-enting the efflux of K+ ions. Their binding relies on a fairly largenteracting surface with the channel, where several correctly posi-ioned residue pairs contribute to the high affinity and selectivityf the binding [26].

In contrast, small molecule blockers typically have a moleculareight of less than 800 Da, are hydrophobic and thus easily per-eate through cell membranes. In the water-filled cavity located

t the middle of the channel they bind from the intracellular sideo the internal end of the selectivity filter. Due to their smaller sizehese molecules have fewer contact points with the channel than

eptide toxins, which generally results in lower affinity and poorerelectivity [27]. Furthermore, as opposed to the external mouth ofhe pore, which is practically isolated from gating-associated con-ormational changes, the internal end of the selectivity filter andhe cavity undergo significant structural rearrangements when the

tters 130 (2010) 19–25 21

channel shifts between gating states. As a result, external block bypeptide toxins is state-independent, while internal block by smallmolecules is state-dependent, usually preferring the inactivatedstate of Kv1.3 channels [28]. At the resting membrane potentialof T cells the majority, but not all, of Kv1.3 channels are in the inac-tivated state. A notable exception to the typical small moleculebehavior is the traditional K+ channel blocker tetraethylammo-nium, which is capable of blocking the channel from both sides,but is membrane-impermeant.

From a pharmaceutical aspect Kv1.3-blocking small moleculesare more desirable than peptides because of easier and less costlyproduction and more easily attainable oral bioavailability. Althoughless stressful delivery systems are being developed, the adminis-tration of peptide drugs usually requires injections and peptidesare also cleared from the body more quickly than small molecules.Moreover, peptides are more likely to trigger an immune reac-tion in the patient and may be less stable chemically [21]. On theother hand, due to their higher affinity and selectivity for Kv1.3,much lower concentrations of peptide toxins may be needed fora therapeutic effect, the possibility of unspecific binding to othermolecular structures (and therefore the possibility of side effects)is minimized, and the interaction with other drugs is also less likely.

When the structure of a natural lead peptide that blocks Kv1.3channels with high affinity is altered with the purpose of develop-ing it into a therapeutically usable drug, achieving high selectivityfor Kv1.3 over other channels in vitro is the primary step. If thetoxin blocks multiple channels then the comparison of sequencesand affinities, structure determination by X-ray crystallography orNMR, thermodynamic mutant cycle analysis and in silico dock-ing simulations can identify the interacting amino acid pairs inthe channel and the toxin that determine the toxin’s selectivityamong the various channels [29–32]. By introducing point muta-tions at these locations toxin selectivity can be shifted towardthe desired target channel [33,34]. However, for the peptide tobe a safe and effective drug all of the aspects mentioned abovemust be considered. The evolution of a native peptide into apotent, highly selective, chemically stable, non-toxic and non-immunogenic molecule is best exemplified by the sea anemonetoxin ShK from Stichodactyla helianthus. The native peptide potentlyblocks Kv1.3 channels along with Kv1.1, Kv1.4, Kv1.6 and Kv3.2channels and in an effort to improve its selectivity the lysineresidue that is highly conserved among K+ channel blocking tox-ins was replaced by the non-natural amino acid diaminopropionicacid [35–37]. Although the mutant toxin (ShK-Dap22) displayedimproved selectivity for Kv1.3, its efficacy to inhibit T cell activa-tion was later shown to be much lower than that of ShK, and italso turned out to block heterotetramers of Kv1.1 and 1.2 subunits[38]. Two other ShK mutants, ShK-F6CA containing a fluoresceinmoiety attached to its N-terminus, and ShK(L5) containing an l-phosphotyrosine at the same position were produced with greatlyimproved properties [20,39]. The latter blocks Kv1.3 channels andsuppresses the proliferation of TEM cells in picomolar concentra-tions and is at least 100-fold less effective on other related channels.This peptide was shown not to be cytotoxic or mutagenic on mam-malian cell lines, yet remained stable in the plasma of rats anddid not influence EKG records, blood chemistry or hematologicalparameters even during prolonged administration. ShK(L5) effec-tively prevented the delayed type hypersensitivity reaction andimproved the symptoms of acute adoptive experimental autoim-mune encephalomyelitis in rats. However, at acidic pH and elevatedtemperatures dephosphorylation of the tyrosine residue and oxi-

dation of a methionine residue was observed indicating instabilityof the molecule [21]. The peptide was also degraded by proteasesruling out the possibility of oral administration in that form. Toovercome stability problems the phosphotyrosine was exchangedfor a non-hydrolysable replacement group, the methionine for nor-

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eucine to avoid oxidation and the C-terminal carboxyl for an amide.he final peptide, named ShK192, kept the advantageous proper-ies of high affinity (Kd = 140 pM) and selectivity (>100-fold) withhe potential to be used for slow release formulation due to itsmproved stability.

In parallel with peptide development the search for potent smallolecule Kv1.3 blockers is also going on with high-throughput

creening and structure-based drug design. Many molecules haveeen identified that block Kv1.3 in the nanomolar range, but mostf them lack the necessary selectivity to be a drug candidate28,40–42]. The small molecule with the best properties to dates PAP-1, which blocks Kv1.3 with an IC50 = 2 nM, and is at least3-fold selective over other channels [43]. PAP-1 can be orallydministered and was found non-toxic in vivo. It was shown touppress oxazolone-induced allergic contact dermatitis [22] and tonhibit the development and reduce the severity of experimentalutoimmune diabetes in rats [15].

Thus, in vivo experimental results indicate that the application ofompounds that block the Kv1.3 channel of T cells for the treatmentf certain autoimmune diseases is a viable and promising approach.long with their pros and cons both peptide and small moleculelockers may be developed into drugs that can represent valuablelternatives to general immunosuppression currently used for thereatment of autoimmune diseases.

. Ion channels are recruited into the immunologicalynapse

Several years ago our group showed for the first time that Kv1.3hannels, which are essential participants of T cell activation, wereo-localized with CD3 molecules in Jurkat T cells and were able toedistribute to the contact area between the cytotoxic T cell andhe target cell [44,45]. Since this first discovery of the accumula-ion of Kv1.3 channels in the IS many other studies have reportedhe recruitment of other ion channels of T lymphocytes into the IS,hich also play important roles in Ca2+ signaling and subsequent

ctivation: both KCa3.1 and the Orai 1-STIM1 functional complexCRAC channels) are localized in the IS [46–48]. In addition, variousransmembrane proteins such as TCR/CD3, CD28, CD4, and intra-ellular molecules, e.g. PKC-�, F-actin, p56lck, etc. are also recruitedo the synapse [49,50]. At present there is no experimental proof,hich would adequately clarify the role of the ion channel redis-

ribution – Kv1.3, KCa3.1 and CRAC – into the IS. Despite the lack of

irect evidence on the physiological relevance of ion channel accu-ulation a great deal of data suggests that these channels are able

o manipulate the process of Ag recognition and subsequent sig-aling processes, thereby, they are also likely to be modified uponhis redistribution. In the following paragraphs we detail facts/our

ig. 1. Kv1.3 and KCa3.1 current in human T cells. (A) Kv1.3 current in a human T cell. Af −120 mV to +50 mV. The Kv1.3 current evoked by this stimulus is characterized by aell. A whole-cell patch-clamped human T cell was stimulated with a voltage-ramp prooltage-independent K+ current. The experimental conditions were discussed elsewhere

tters 130 (2010) 19–25

hypothesis related to the possible consequences and role of therearrangement.

4.1. Kv1.3 channels in the IS

Several lines of evidence support that Kv1.3 serves as anactive participant of the signaling complex recruited into the IS.It was described that both kinases and phosphatases can regulatethe activity of Kv1.3 channels in human peripheral lymphocytes[51,52]: the window current is altered upon activation of thesemolecules. Furthermore, costimulation of CD3 and CD28 receptorsin Jurkat cells suppresses the current of Kv1.3 channels, which ismediated by CD45 phosphatase activation. Modification of Kv1.3channels by other tyrosine kinases like EGFR/insulin receptor wasalso reported in heterologous expression systems [53,54]. p56lckand PKC were shown to bind to the channel protein via differ-ent adaptor proteins, which also accumulate in the immunologicalsynapse [55,56]. hDlg (human homologue of the Drosophila discslarge tumor suppressor protein or SAP97) couples p56lck to theC terminus of Kv1.3 upon binding to the recognition site for PDZdomain channel, hence allowing phosphorylation of tyrosine sidechains [55]. Another member of the PDZ domain recognition adap-tor protein family, PSD95 or SAP90 was shown to be a crucialelement in clustering and interaction of Kv1.3 with a tyrosinekinase (insulin receptor) [57]. hDlg as well as PSD95 can also bindto some cytoskeletal components in T cells, hence this couplingmay facilitate or guide the redistribution of Kv1.3 channels to the IS[58,59]. Under pathological conditions such as SLE (systemic lupuserythematosus) but not RA (rheumatoid arthritis) Nicolaou et al.determined that diffusion rate of the Kv1.3 channel into the IS aswell as out of it is significantly higher as compared to healthy T cells[47]. However, a well-defined molecular mechanism, which couldaccount for this facilitated diffusion of Kv1.3 in SLE lymphocytes,is still unknown. However, it clearly emphasizes that residence ofKv1.3 channel in the IS is necessary for its regulation, since the var-ious postsynthetic modulatory molecules arrive at the synapse atdifferent times.

One of the auxiliary subunits of Kv1.3 channels, Kv�2 or Kv�1.2,which binds to a conservative site on the N-terminus is a linkbetween the channel and the ZIP proteins [56]. Alternatively, Kv�’scan also modify the biophysical properties of Kv1.3 channels andthe expression level of various Kv� subunits may change uponactivation and depends on the mode of activation [60]. Via ZIP

adaptors two kinases, PKC and p56lck can couple to the channel.In two recent papers a possible role of p56lck in Kv1.3 regulationwas described: it can mediate inhibition of Kv1.3 channels underhypoxic conditions, since p56lck silencing in Jurkat T cells or admin-istration of p56lck antagonists prevented the suppression of Kv1.3

whole-cell patch-clamped human T cell was depolarized from a holding potentialrapid activation and a slow inactivation phase. (B) KCa3.1 current in a human T

tocol, the cytosolic free Ca2+ concentration was 1 �M. The voltage-ramp evokes a[30].

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Fig. 2. Ion channels and Kv1.3-associated proteins in the immunological synapse(IS). Kv1.3, KCa3.1 Orai 1 and STIM1 is depicted schematically in the IS between anantigen presenting cell (APC) and a T cell. hDlg binds to the PDZ domain in the CtKs

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erminus of Kv1.3, which is coupled to the p56lck. Kv�2 links the N terminus ofv1.3 channel to the ZIP1/2 protein, which can have several interacting partnersuch as p56lck, PKC, PPAR�, ras-GAP, p38MAPK, TRAF6.

urrent detected in hypoxia [61,62]. Moreover, ZIP/p62 can interactith several proteins, which induce gene transcription through var-

ous signaling pathways (NF-�B, PPAR�, p38MAPK); and are likelyo lead to T cell activation (see Fig. 2) [63–67]. The accumulationf Kv1.3 in the IS makes it possible to interact with the all proteinsinked to it. Recently, Toth et al. has demonstrated that rearrange-

ent of Kv1.3 channels into the IS modifies kinetic and equilibriumarameters of the activation and inactivation [68]. The slower acti-ation of Kv1.3 current may be an evidence for the redistributionf the channel into membrane domains with higher viscosity, theaster inactivation kinetics can be explained by dephosphorylationf the channel [69].

.2. Role of Ca2+ influx in the IS

Opening of CRAC channels, which is triggered by emptying of ERa2+ stores and formed upon STIM1-Orai 1 coupling, elevates thea2+ level in T cells. The Ca2+ concentration in the vicinity of theite of entry may go up to a concentration range, which in turn canwitch on KCa3.1 in a direct manner or Kv1.3 due to the local depo-arization. Moreover, the elements of the supramolecular complexn the IS can be Ca2+-dependent enzymes, which may become acti-ated, too. Due to the increased intracellular Ca2+ CRAC channelsnactivate, while the translocation of mitochondria to the synapseounteract this negative feedback and maintain the driving forceor the Ca2+ ions [70–72]. Interestingly, it has been reported latelyhat immobilization of Kv1.3 channels by crosslinking (accumula-ion in the IS is blocked) elevates Ca2+ response of Jurkat T cellshallenged by anti-CD3/anti-CD28 coated beads [73]. Nonetheless,he elevated intracellular Ca2+ level stabilizes and maintains therm contact between the T cell and the APC, but rearrangement ofhe CD3 molecules into the IS is not affected [74].

Finally we should mention that in their recent paper Cahalannd Chandy suggested that changes of the ion concentrations inhe synaptic cleft (mainly the elimination of Ca2+ ions and increasen the K+ level) between the T cell and the APC may result in theepolarization of the T cell membrane [5]. The putative conse-

uences of this are the following: (1) attenuated Ca2+ flux intohe T lymphocytes, (2) increased negative surface charge due tohe polar head of lipid molecules, which can shift the transmem-rane potential toward depolarization, (3) stronger interaction

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tters 130 (2010) 19–25 23

between the interacting membrane proteins of the APC and lym-phocyte, (4) activation of voltage activated Ca2+ channels of the Tcell, and (5) changes in the biophysical properties of Kv1.3 chan-nels. These scenarious are certainly viable, but presently are stillassumptions with no solid experimental support. In summary,ion channels are important elements in efficient transmembranesignaling upon antigen recognition. Besides being responsible formaintaining the appropriate ion milieu during T cell activation, theyare also remarkable players in the crowd of molecules in signal-ing clusters. Thus, their role may be twofold: they may accomplishrequired steps in the signaling cascade (Ca2+-dependent enzymeactivation) or they may serve as a collectors of various interactingmolecular partners (as for Kv1.3).

5. Concluding remarks

Compounds influencing ion channel activity in nerve or musclecells to achieve certain therapeutic goals have already establishedthemselves as powerful pharmaceutical tools and the potential forsimilar successful immunological application was recognized about25 years ago. We are not quite yet at the end of this road, butthe wealth of information collected during this period about theintriguing and diverse function of T cell ion channels in one of thecritical steps of cellular immunity, along with major developmentsin specific channel blockers, took us a long way down on it. The suc-cess of in vivo experiments for the prevention of undesired actionsof the immune system via T cell ion channels predicts the avail-ability of a potent alternative to conventional immunosuppression,hopefully, in the near future.

Acknowledgements

Supported in part by grants: OTKA K 60740 and NK 61412,and TÁMOP-4.2.2/08/1 19 to G.P. Zoltan Varga and Peter Hajdu areBolyai fellows.

References

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