HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplemental Movie All Versions of this Article: 292/4/C1431    most recent 00376.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Nicolaou, S. A. Articles by Conforti, L. Search for Related Content PubMed PubMed Citation Articles by Nicolaou, S. A. Articles by Conforti, L.

MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

The Ca²⁺-activated K⁺ channel KCa3.1 compartmentalizes in the immunological synapse of human T lymphocytes

¹Department of Internal Medicine and ³Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio; and ²Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania

Submitted 10 July 2006 ; accepted in final form 29 November 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
T cell receptor engagement results in the reorganization of intracellular and membrane proteins at the T cell-antigen presenting cell interface forming the immunological synapse (IS), an event required for Ca²⁺ influx. KCa3.1 channels modulate Ca²⁺ signaling in activated T cells by regulating the membrane potential. Nothing is known regarding KCa3.1 membrane distribution during T cell activation. Herein, we determined whether KCa3.1 translocates to the IS in human T cells using YFP-tagged KCa3.1 channels. These channels showed electrophysiological and pharmacological properties identical to wild-type channels. IS formation was induced by either anti-CD3/CD28 antibody-coated beads for fixed microscopy experiments or Epstein-Barr virus-infected B cells for fixed and live cell microscopy. In fixed microscopy experiments, T cells were also immunolabeled for F-actin or CD3, which served as IS formation markers. The distribution of KCa3.1 was determined with confocal and fluorescence microscopy. We found that, upon T cell activation, KCa3.1 channels localize with F-actin and CD3 to the IS but remain evenly distributed on the cell membrane when no stimulus is provided. Detailed imaging experiments indicated that KCa3.1 channels are recruited in the IS shortly after antigen presentation and are maintained there for at least 15–30 min. Interestingly, pretreatment of activated T cells with the specific KCa3.1 blocker TRAM-34 blocked Ca²⁺ influx, but channel redistribution to the IS was not prevented. These results indicate that KCa3.1 channels are a part of the signaling complex that forms at the IS upon antigen presentation.

T cell activation; ion channels; membrane distribution

The onset of T cell activation is marked by an increase in intracellular Ca²⁺ that occurs immediately upon TCR engagement by the APC/antigen. Moreover, increased intracellular Ca²⁺ levels must be sustained for a long time before IL-2 is produced and activation becomes antigen independent (22). A sustained intracellular Ca²⁺ concentration is thus necessary for T cell activation and gene expression (7, 18). Ca²⁺ signaling in human T lymphocytes is modulated via two K⁺ channels: the voltage-gated K⁺ channel (Kv1.3) and the Ca²⁺-activated K⁺ channel (KCa3.1). Kv1.3 channels regulate the membrane potential in resting T cells where they represent the dominant conductance (22). However, when naive and central memory T cells are exposed to an antigen and become activated, the expression of KCa3.1 channels is strongly enhanced compared with a modest increase in Kv1.3 channels, and KCa3.1 channels become the major regulators of membrane potential in these cells (11, 13). Via regulation of the membrane potential, these channels provide the driving force for Ca²⁺ entry because the efflux of K⁺ assists in maintaining the necessary electrochemical gradient (22).

Interestingly, although recent evidence suggests that Kv1.3 channels localize in the IS in T cells, nothing is known about the ability of the KCa3.1 to compartmentalize in the IS (21). In the present study, we investigated KCa3.1 channel distribution on the plasma membrane upon T cell activation. By utilizing electrophysiological methods and fluorescence microscopy, we demonstrate that KCa3.1 channels redistribute to the IS on TCR binding and become part of the IS signaling complex that facilitates T cell activation.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Cells and transfection. CD3⁺ and CD4⁺ lymphocytes were isolated from healthy donors by E-rosetting (StemCell Technology, Vancouver, Canada) and Ficoll-Paque density-gradient centrifugation (ICN Biomedicals, Aurora, OH) and maintained as previously described (23). Freshly isolated human T cells were preactivated with 4 µg/ml phytohemmaglutinin (PHA; Sigma-Aldrich, St. Louis, MO) and transfected 18–24 h later with YFP-KCa3.1 with the Amaxa Nucleofector technology (Amaxa Biosystems, Cologne, Germany) using 10 x 10⁶ cells, 5 µg DNA, and program T20 according to the manufacturer's instructions. For ratiometric Ca²⁺ imaging experiments, human T cells were activated with 4 µg/ml PHA for 48–72 h, allowing for sufficient expression of the native KCa3.1 channels (13). This became necessary because of a low transfection efficiency that did not allow us to use the transfected T cells (10%). Blood was obtained from either healthy volunteers or healthy blood bank donors (unutilized blood units from the Hoxworth Blood Bank Center). The blood collection process was approved by the Institutional Review Board of the University of Cincinnati. Epstein-Barr virus (EBV)-infected B cells (gift of A. H. Filipovich) were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. Human embryonic kidney (HEK-293) cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. HEK-293 cells were transfected with YFP-KCa3.1 using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions at a ratio of DNA to Lipofectamine of 1:5.

Molecular biology. YFP was appended onto the COOH terminus of KCa3.1 via the addition of SalI and BamHI sites to the NH₂ and COOH terminus, respectively, in a single-step PCR reaction, resulting in the removal of the stop codon in KCa3.1. This PCR product was subcloned in-frame into the pEYFP-N1 vector (Clontech, Mountain View, CA). The fidelity of this construct was confirmed by sequencing (ABI PRISM 377 automated sequencer, University of Pittsburgh) and subsequent sequence alignment (NCBI BLAST) with KCa3.1 (GenBank accession number AF022150).

Measurement of intracellular Ca²⁺. Resting and PHA-preactivated T cells were plated on poly-L-lysine-coated coverslips and loaded with 1 µM fura 2-AM (Molecular Probes, Eugene, OR) for 35 min at room temperature (22–24°C) in RPMI 1640 and rinsed with 0.5 mM Ca²⁺-Ringer solution containing (in mM) 155 NaCl, 4.5 KCl, 2.5 MgCl₂, 10 HEPES, 10 glucose, and 0.5 CaCl₂, pH 7.4. Cells were stored at 37°C in the dark for up to 2 h before use. All cell-imaging experiments were performed on a Cyt-Im2 Ca²⁺ imaging system (Intracellular Imaging, Cincinnati, OH), using a ratiometric method as previously described (14, 23). Cells were imaged on a Nikon-inverted epifluorescence microscope equipped with a heated microscopy chamber, a x20 objective, and a xenon arc lamp, which was used for the alternative excitation of fura 2 at 340 and 380 nm. Emitted light passed through a 535 WB35 emission filter, and intensity values were averaged over either 10 s (experiments with CD3⁺ T cells) or 0.5- to 0.7-s intervals (experiments with CD4⁺ T cells) for analysis. For activation with EBV-infected B cells, B cells were prepulsed for 2 h at 37°C with 7 µg/ml staphylococcal enterotoxin B (SEB; Sigma-Aldrich), centrifuged, and resuspended in 1 ml of 0.5 mM Ca²⁺-Ringer solution and stored at 37°C. For experiments with the KCa3.1 blocker TRAM-34, cells were preincubated with 1 µM TRAM-34 (in 0.5 mM Ringer solution) for 15 min before recording. Fura 2-loaded T cells were recorded while bathed in 0.5 mM Ca²⁺-Ringer solution for 2 min before addition of SEB-pulsed B cells. The cells were then allowed to interact for 15 min before 1–2 µM ionomycin was added as a positive control. Visual inspection showed formation of stable APC-T cell conjugates in the bath. Initial experiments were performed in CD3⁺ T cells; however, because SEB only activates CD4⁺ T cells, subsequent experiments were performed in CD4⁺ T cells to increase the proportion of responding T cells. Cells that had an increase in 340 nm-to-380 nm ratio 0.1 ratio units were regarded as cells responding to antigen presentation. This value was well above two standard deviations of the average background noise: 0.023 ± 0.024 ratio units as determined in three separate experiments from 440 cells (110 cells per experiment) that showed no apparent response. In addition, to obtain the average of the 340 nm-to-380 nm ratio, the cells that exhibited a Ca²⁺ response were synchronized to reflect initiation of Ca²⁺ influx.

T cell activation. Transfected T cells were stimulated with 4.5-µm polystyrene beads coated with anti-CD3/CD28 antibodies (Dynal Biotech, Lake Success, NY) as previously described (30). Transfected T lymphocytes were mixed with the beads at a ratio of 1:1.5 and centrifuged for 5 min at 100 g. The cells were then incubated at 37°C for 30 min, resuspended, and plated onto poly-L-lysine (Sigma-Aldrich)-coated coverslips and allowed to attach for 3–5 min. Activation experiments were also performed with EBV-infected B cells as APCs. EBV-infected B cells were pulsed with SEB (7 µg/ml for 2 h) and loaded with 1 µM DDAO Far Red Cell Tracker (Molecular Probes) for 20 min. T and B cells were then mixed at a ratio of 1:1.5, spun briefly at 1,100 rpm, and incubated at 37°C for 1–30 min. Finally, the cells were plated onto poly-L-lysine-coated coverslips.

Immunocytochemistry. Immunolabeling was carried out, in part, as previously described (4). Cells attached onto poly-L-lysine-coated coverslips were washed with PBS and fixed with 4% paraformaldehyde for 20 min. To double label with CD3, the cells were blocked using 10% FBS, permeabilized with 0.2% Triton X-100, incubated for 1 h with goat anti-CD3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), washed, and incubated with donkey anti-goat Alexa fluor 546 fluorescent secondary antibody (Molecular Probes) for 1 h. To stain for F-actin, Alexa fluor 546 phalloidin (Molecular Probes) was added for 20 min. Finally, the cells were washed and mounted onto glass slides. Samples were visualized by confocal microscopy (Axioscope, Carl Zeiss Microimaging) using a x63 oil objective lens. The fluorescent probes were excited with the use of an argon ion laser and a HeNe laser. Data were obtained with the "Multi Track" option of the microscope to exclude cross talk between detection channels.

Image quantification. To evaluate colocalization of proteins and their position within the IS, unprocessed images were analyzed with the linescan function of the MetaMorph program (Molecular Devices, Downingtown, PA). Briefly, a reference line was drawn along the T-B cell contact site. The software calculates the mean red and green fluorescence intensities along the reference line for four pixels of width and plots the measurements with respect to their position within the selected portion of the membrane. To determine the percentage of T-B cell conjugates that formed in the presence or absence of 1 µM TRAM-34, we counted the number of T-B cell conjugates/total number of T cells. Eight random fields were analyzed for each donor and each treatment condition.

Time-lapse microscopy. CD4⁺ T cells were transfected with YFP-tagged KCa3.1 channels and used for live microscopy experiments 6 h after transfection. EBV-infected B cells were prepulsed with SEB and loaded with 1 µM FarRed DDAO cell tracer (Molecular Probes). T cells were seeded into a heated microscopy chamber (37°C) on poly-L-lysine-coated coverslips. Next, B cells were added, and time-lapse images were recorded with a Plan-Apochromat x60 oil immersion objectives on a Nikon Microphot FXA inverted microscope coupled to an Orca-ER cooled camera (Axioscope, Zeiss Microimaging). Images were processed using the MetaMorph software.

Electrophysiology. Experiments were performed in the whole cell configuration using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) at room temperature (22°C). The external solution was (in mM): 140 NaCl, 4.5 KCl, 2 CaCl₂, 1 MgCl₂, and 10 HEPES, pH 7.4. The pipette solution was (in mM): 145 potassium aspartate, 8.5 CaCl₂, 10 EGTA, 2 MgCl₂, and 10 HEPES, pH 7.2, with an estimated free Ca²⁺ concentration of 1 µM (13). All solutions were 290–310 mosM. The cells were continuously perfused at a constant rate of 2 ml/min. Electrodes were pulled from TW150F-4 glass micropipettes (World Precision Instruments, Sarasota, FL) on a horizontal pipette puller (model P-97, Sutter Instrument) and had a resistance of 4–6 M. KCa3.1 current was measured in voltage-clamp mode and induced by ramp depolarization from –120 to +40 mV, 200 ms duration, every 10 s –80 mV holding potential. Data were corrected for a liquid junction potential of –10 mV (23). KCa3.1 slope conductance was measured between –100 and –60 mV. The digitized signals were stored and analyzed with pCLAMP 9 software (Axon Instruments).

Statistical analysis. All data are presented as means ± SE. Statistical analyses were performed with Student's t-test (paired or unpaired). P 0.05 was defined as significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Electrophysiological and pharmacological profile of the cloned YFP-KCa3.1 channel in HEK-293 cells matches the native KCa3.1 channel in human T cells. YFP-tagged KCa3.1 channels were expressed in HEK-293 cells, which lack endogenous KCa3.1 channels. Their pharmacological and electrophysiological properties were investigated and compared with native KCa3.1 channels previously described in the literature (13). KCa3.1 currents were recorded using the whole cell configuration and with a pipette solution of 1 µM Ca²⁺, which allows KCa3.1 activation (13). Ramp pulse depolarization induced K⁺ currents with a reversal potential of –79.0 ± 0.3 mV (n = 6) (Fig. 1A). This was indicative of a K⁺-selective current. Mock-transfected HEK-293 cells transfected with the empty YFP-vector displayed very small background K⁺ current (Fig. 1A). Overall, the KCa3.1 conductance was significantly higher in YFP-KCa3.1-transfected HEK-293 cells compared with mock-transfected cells (Fig. 1B). Furthermore, KCa3.1 current in YFP-KCa3.1-transfected cells was blocked by the specific KCa3.1 blocker TRAM-34 (kind gift of K. G. Chandy) (Fig. 1) (28). Collectively these electrophysiological and pharmacological studies confirm that transfection of HEK-293 cells with the YFP-KCa3.1 clone resulted in the expression of functional KCa3.1 channels, which are functionally identical to their native counterparts as their characteristics are in agreement with previous reports (13).

View larger version (11K): [in this window] [in a new window]   Fig. 1. Functional and pharmacological properties of recombinant YFP-tagged KCa3.1 channels. A: representative traces shown were obtained in HEK-293 cells transfected with YPF-KCa3.1 and YFP-vector (pEYFP-N1, control). YFP-KCa3.1 currents were blocked by 1 µM TRAM-34. Currents were induced by ramp depolarization from –120 to +40 mV (–80 mV holding potential). The theoretical K+ current was –88 mV. I, current; V, voltage. B: KCa3.1 conductance increased significantly in YPF-KCa3.1-transfected HEK-293 cells compared with mock-transfected cells (n = 6; #P = 0.003). The YFP-KCa3.1 currents decreased significantly after application of 1 µM TRAM-34 (n = 6; **P = 0.02).

View larger version (10K): [in this window] [in a new window]   Fig. 2. Expression of functional YFP-tagged KCa3.1 channels in human T cells. A, top: representative I-V relationships obtained from YPF-KCa3.1-transfected T cells and nontransfected (control) T cells. Control T cells have undergone the same transfection procedure as YFP-KCa3.1 channel-transfected T cells. Currents were obtained by ramp depolarization as described in Fig. 1. Inset: representative images of a control (top) and transfected (bottom) cell are shown. Both cells were from the same donor and in the same image. Scale bar = 5 µm. A, bottom: there was a significant increase in KCa3.1 conductance in YPF-KCa3.1-transfected T cells compared with nontransfected T cells, which corresponds to an increase in the number of functional KCa3.1 channels/cell. Data are the average of 5 cells from 2–4 donors. *P = 0.02. B: KCa3.1 currents decreased significantly after application of 1 µM TRAM-34. Currents, elicited by ramp depolarization as described in Fig. 1, were recorded continuously before (–TRAM-34) and during exposure to the drug (+TRAM-34).

View larger version (51K): [in this window] [in a new window]   Fig. 3. KCa3.1 channels and F-actin localize at the T cell-bead contact point. YFP-KCa3.1-transfected T cells were activated with anti-CD3/CD28 antibody-coated beads for 30 min, fixed, permeabilized, and stained with phalloidin Alexa fluor 546 (to visualize F-actin). T cells not conjugated with beads display uniform distribution of KCa3.1 and F-actin around the membrane (top), whereas KCa3.1 and F-actin localize at the T cell-bead contact interface on conjugation (bottom). Scale bar = 5 µm. The three-dimensional T-bead interface reconstruction (xz projection) is shown under the corresponding two-dimensional (2D) image. The reconstructed portion of the T-B cell complex is indicated by a box in the 2D merged image. Scale bar for the xz projection = 2 µm for both x- and z-axes. DIC, differential interference contrast.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Staphylococcal enterotoxin B (SEB)-pulsed B cell interaction with T cells induces a KCa3.1-dependent increase in cytoplasmic Ca2+ in activated T cells. A: human T cells were loaded with the ratiometric dye fura 2 and stimulated with SEB-pulsed B cells for 15 min. Each panel shows a representative trace of cytoplasmic Ca2+ in individual cells. 340/380, Ratio of 340 nm to 380 nm. Arrows indicate point of introduction of the B cells into the bath. T cells coming in contact with a B cell displayed differential responses, including a sustained increase of intracellular Ca2+, a transient response, and an oscillatory response. These data are representative of a total of 35 activated T cells. Unstimulated cells (cells not contacting a B cell) showed little or no response. Yet they responded to ionomycin (I; 1–2 µM). Scale bars correspond to 200 s. Experiments were performed at 29°C. B: resting and activated CD4+ T cells were or were not pretreated with 1 µM TRAM-34 before interacting with B cells. The numbers of T-B cell conjugates showing a significant increase in intracellular Ca2+ were normalized for the total number of T cells. Resting T cells showed no significant difference between untreated and pretreated cells (n = 4, >70 cells/experiment, and n = 4, >50 cells/experiment, from 3 donors; P = 0.3). However, activated T cells showed a significant decrease in the number of cells that responded when treated with the blocker (untreated: n = 4, >40 cells/experiment; treated: n = 5 >40 cells/experiment, from 2 donors; P = 0.002). C: average increase in intracellular Ca2+ in control and TRAM-34-treated cells that responded to antigen stimulation (same experiment as B). Average cytoplasmic Ca2+ levels for control (118 cells from 4 separate experiments) and cells treated with TRAM-34 (47 cells from 5 separate experiments) were obtained by alignment of the traces so that the times of onset of the Ca2+ response corresponded. Experiments in B and C were performed at 34.7 ± 0.2°C (n = 16). D: number of T-B cell conjugates that form in control (–TRAM-34) and TRAM-34 (1 µM)-pretreated activated T cells was determined in fixed micrographs and reported as percentage of total T cells counted. P = 0.2.

Redistribution of KCa3.1 channels at the IS. Confocal microscopy experiments were performed to study the KCa3.1 channel distribution upon contact with APCs. The YFP-KCa3.1 channels were expressed in human T cells, and the T cells were prepared for immunocytochemistry experiments 6 h after transfection. It is known that the TCR and associated molecules redistribute to the T cell-APC contact interface on T cell activation (9). CD3 is part of the TCR complex that localizes in the center of the mature IS; thus it can be used as a marker of IS formation (8). Our data indicate that KCa3.1 channels redistributed at the T cell-APC interface and colocalized extensively with CD3 on conjugation (Fig. 5, B and C), but both remained evenly distributed on the membrane when the T-APC were not conjugated (Fig. 5A) or in the absence of SEB (Fig. 5B, top). Notably, KCa3.1 channels acquired a central localization within the contact interface early upon IS formation, and this localization is maintained for at least 30 min of conjugation. This localization within the IS and the colocalization with CD3 is clearly visible in the xz projection of the T-B cell interface, and it was further confirmed by the position of the peak fluorescence intensities in the linescan graphs (Fig. 5). Interestingly, despite pretreatment with 1 µM TRAM-34, a concentration already demonstrated to block channel current (Figs. 1 and 2) and the Ca²⁺ response (Fig. 4, B and C), KCa3.1 channels were still recruited in the IS, and they also maintained the same central location within the IS (Fig. 5C, bottom). Overall, KCa3.1 blockade did not seem to affect IS formation because CD3 was still recruited, and its localization within the IS, together with that of the channel, remained unchanged. Our data therefore suggest that functional KCa3.1 channels are not required for IS formation and maintenance or for channel membrane trafficking.

View larger version (70K): [in this window] [in a new window]   Fig. 5. KCa3.1 channel redistribution in the immunological synapse. KCa3.1 channel-transfected human T cells were incubated with Epstein-Barr virus (EBV)-infected B cells that had been exposed to medium with or without SEB at 37°C. B cells were labeled with cell trace FarRed DDAO (pseudocolored blue). T cells, pretreated or not pretreated with TRAM-34 (1 µM) for 15 min, were mixed with B cells and incubated for 1–30 min at 37°C, plated on coverslips, fixed, permeabilized, and stained with anti-CD3 antibody. A: representative images of T cells that did not form a stable conjugate with SEB-infected B cells; B: T cells conjugated with B cells in the absence (top) or presence (bottom) of SEB after 1 min. C: T cells not treated (top) or treated (bottom) with TRAM-34 after 30-min conjugation with SEB-pulsed B cells. Scale bar = 5 µm. The three-dimensional T-B cell interface reconstructions are shown under the corresponding 2D images. The area used for the reconstruction is marked in the 2D merged images by a box. The line scan analyses of KCa3.1 (green) and CD3 (red) fluorescent intensity (FI) over the T-B cell contact area are shown (right, adjacent to the corresponding micrographs). These images are representative of the results obtained from 5 donors for control experiments and 2 donors for pretreatment experiments. Scale bar for the xz projection = 2 µm for both x- and z-axes.

To more precisely define the kinetics of KCa3.1 channel distribution in the IS, we used time-lapse microscopy to directly image live KCa3.1 channel translocation in T cells as it develops upon encounter with APCs. Activation and IS formation of transfected T cells was induced by SEB-pulsed EBV-infected B cells. The results obtained in these studies confirmed the observations in the fixed microscopy studies, although a certain degree of variability in the kinetics of KCa3.1 channel translocation to the T-B cell interface was also observed. In the majority of conjugates imaged, the channels were recruited at the IS within 40 s to 2 min (5 of 7 conjugates) (Fig. 6 and supplemental movie S1) (please note that the online version of this manuscript contains supplemental data). Only in two conjugates was a longer time necessary for channel recruitment. Interestingly, in some experiments, we observed that the channels were recruited on contact with an APC but then readily relocalized to a second APC on contact (data not shown) (n = 3). It is not uncommon for T cells to undergo serial stimulation by APCs during the activation process (10). However, because of the multiple encounters and transient nature of this polarization, these experiments were not included in the overall analysis. In addition, we observed two distinct patterns of retention in the IS. Specifically, in 43% of the cells, the channels resided in the IS throughout the duration of the whole experiment for at least 14–30 min (n = 3), whereas in 57% of the conjugates the channels stayed in the synapse for a shorter time (7:04 ± 0:35 min, n = 4). Moreover, the channels appear to obtain a central localization in 86% of the conjugates studied and maintained a more peripheral distribution in only 14% (n = 7). These data further define the dynamics of KCa3.1 channel recruitment in the IS.

View larger version (60K): [in this window] [in a new window]   Fig. 6. KCa3.1 channel redistribution in live T cells. Human T cells transfected with YFP-KCa3.1 were seeded on a heated microscopy stage and imaged while interacting with SEB-pulsed EBV-infected B cells. Top rows: brightfield images. Bottom rows: corresponding images of KCa3.1 staining intensity using a pseudocolor scale. The location of the B cells is indicated by a white line around the cell membrane. Snapshot sequence corresponds to supplemental movie S1 (note that the online version of this manuscript contains supplemental data). We observed that at time 0:00, before the transfected T cell comes in contact with an antigen presenting cell (APC), KCa3.1 channels are uniformly expressed all along the cell membrane. After 0:42 min, an APC comes in contact with the T cell, and, on contact, the channels begin to accumulate at the immunological synapse that forms at the T cell-APC interface where they remain for over 25 min. At 28:42 min, the channels began to redistribute along the cell membrane, and they acquire a uniform distribution by 35:05 min. Scale bar = 5 µm.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

Experiments were performed on human T cells expressing YFP-tagged KCa3.1 channels. This was made necessary by the fact that, to our knowledge, no specific anti-KCa3.1 antibodies are commercially available. The availability of a specific antibody would have allowed us to study the behavior of native KCa3.1 channels that exist at the appropriate conformational and phosphorylation state as well as in association with other cellular components. Furthermore, from a technical point of view, this would have excluded the limitation inherent in the low efficiency of transfection of primary T cells (i.e., low number of transfected cells available for the experiments). Recombinant channels instead carry the risk of overexpression, which we avoided as much as possible by using the cells as soon as expression was seen. In our experiments, the transfected T cells expressed on average a KCa3.1 conductance of 1.3 nS/µm². This is comparable to the level of expression of native KCa3.1 channels in human T lymphocytes reported after 2 days of activation with PHA (1.12 nS/µm²) (13). It is also possible that the YFP tag could have hindered other molecular interactions, although it did not compromise the ability of the channel to enter and exit the IS. Furthermore, a more cytoplasmic distribution has been observed in GFP-tagged channels compared with native K⁺ channels (17). Still, a YFP protein is extremely photostable, thus allowing detailed and prolonged live cell imaging experiments, minimizing the risk of photobleaching. Furthermore, these studies with recombinant channels set the stage for future structural-functional studies, that will allow the determination of the channel protein sequence or sequences necessary for its recruitment in the IS and thus the possible mechanisms driving this process.

Electrophysiological experiments indicated that the YFP-KCa3.1 channels displayed biophysical properties identical to their native counterparts and were inhibited by the specific KCa3.1 blocker TRAM-34 (13). This allowed us to express these channels in primary human T lymphocytes and perform localization experiments to demonstrate that KCa3.1 channels localize at the IS. Two methods were used to induce IS formation and T cell activation: CD3/CD28-coated beads and SEB-pulsed EBV-infected B cells. The former have been used as surrogate APCs, and they have been shown to induce reorganization of F-actin and accumulation of structural proteins at the bead-T cell contact area (30). We have also shown that CD3/CD28 beads were able to induce a productive activation in human T cells because they can elicit an increase in intracellular Ca²⁺ concentration on contact (23). However, stimulation of T cells with superantigen-loaded B cells more closely resembles the "in vivo" situation where the antigen is presented to the T cell by either B or dendritic cells (9). The association between T cells and these APCs involves adhesion and other costimulatory molecules not provided by the CD3/CD28 beads (9). We have confirmed that SEB-pulsed EBV-infected B cells can be used as effective APCs by monitoring the Ca²⁺ response induced in T cells upon binding (Fig. 4). These experiments were performed in human T cells preactivated by exposure to PHA for 72 h. This intervention was shown to induce expression of KCa3.1 channels in human T cells, and, in these cells, the Ca²⁺ response becomes dependent on these channels (13). Different patterns of Ca²⁺ response were elicited in individual T cells by exposure to the SEB-pulsed B cells. Similar heterogeneity to TCR stimulation was previously observed and described by use of soluble antigens and CD3/CD28 beads (15, 23, 25). This reflects the mixed T cell population that comprises T cells freshly isolated from the blood and includes T cells at different degrees of activation and development.

Overall, the results presented reveal that KCa3.1 channels moved into the IS immediately upon its formation, and they localized with F-actin and CD3. Furthermore, fixed microscopy experiments, representative snapshots of the process, revealed that KCa3.1 channels are rapidly recruited at the center of the IS where they reside for at least 30 min after stimulation. We observed that KCa3.1 channels colocalized with CD3 and were surrounded by F-actin. This is in agreement with F-actin forming a peripheral ring within the IS, whereas the TCR accumulates at the core of the IS (2, 5).

To further substantiate our data, we also performed time-lapse microscopy experiments. KCa3.1 channels are recruited early on in the IS in the majority of the cells imaged. Intriguingly, we also noted two patterns of recruitment: a sustained recruitment and a shorter lived one. This might reflect the variability in activation and differentiation state of our mixed T cell population, an observation also demonstrated by the variability of our Ca²⁺ response.

KCa3.1 localization in the IS could have important implications on the channel activity and overall T cell function. It is well known that, in activated T cells, KCa3.1 channels regulate the membrane potential and, as a consequence, Ca²⁺ influx as well (11, 13). In agreement with the literature, we observed that blockade of KCa3.1 channel activity inhibits the TCR-mediated Ca²⁺ response in these cells (Fig. 4). Notably, blocking the Ca²⁺ increase does not prevent the formation of tight T-B cell interfaces and accumulation of adhesion molecules at site of contact (29). Interestingly, we observed that, when KCa3.1 channels were blocked, neither formation of T-B cell conjugates nor KCa3.1 and CD3 transition to the IS was prevented. Thus our results suggest that KCa3.1 channel transition to the IS is not dependent on Ca²⁺ influx. Furthermore, it suggests that the functionality of the channel is not integral to its migration to the IS. Similarly, we have observed that Kv1.3 channels can also translocate to the IS when their activity is pharmacologically abrogated (data not shown).

The functional consequences of KCa3.1 channel translocation in the IS might reflect on the Ca²⁺ response that is triggered on antigen presentation. It is generally accepted that, following TCR engagement, the magnitude and pattern of the Ca²⁺ signaling are in part regulated by the activity of Kv1.3 and KCa3.1 channels (26). It has already been shown that Kv1.3 channels translocate to the IS in human T cells, and in the present study we show that KCa3.1 channels also move to the IS on T cell activation (21). Moreover, it is commonly believed that formation of the IS occurs to provide close proximity between various elements of the T cell activation machinery and thus more efficient signaling among them (9). Interestingly, signaling molecules such as protein kinases A (PKA) and C (PKC), which are known regulators of KCa3.1 channel function, have been shown to accumulate in the IS on T cell activation as well (1, 6, 12, 19, 24, 31). Investigation of the reorganization of PKC- during IS formation reveals that this PKC isoform sustains a central localization in the IS supramolecular activation complex (16). Furthermore, PKA also moves into the IS 30 min after activation, and it partially colocalizes with the TCR-CD3 complex to facilitate the termination of the activation process (31). Thus the spatial and temporal distributions of PKC- and PKA allow for access to the KCa3.1 channels and as such could provide a regulatory mechanism affecting the channel's activity. As a result, it is quite possible that recruitment of KCa3.1 channels could lead to differential regulation of these channels. Consequently, modulation of KCa3.1 channel activity will determine the magnitude and duration of the Ca²⁺ response triggered by antigen presentation as it contributes to the driving force for Ca²⁺ influx.

In view of this, we propose that the functional relevance of KCa3.1 channel translocation to the IS could be to facilitate the better regulation of the channel by signaling molecules recruited at this site during T cell activation with the ultimate goal to shape the Ca²⁺ response, which is integral for differential gene expression (7).

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Cancer Institute Grant CA-95286 to L. Conforti, American Heart Association-Ohio Valley Affiliate predoctoral fellowship 0615213B to S. A. Nicolaou, and National Institutes of Health Grants DK-54941 and HL-083060 to D. C. Devor.

	ACKNOWLEDGMENTS

 
We thank M. K. Ragupathy for technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. Conforti, Dept. of Internal Medicine, 231 Albert Sabin Way, Univ. of Cincinnati, Cincinnati, OH 45267-0585 (e-mail: laura.conforti{at}uc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
1. Bi K, Tanaka Y, Coudronniere N, Sugie K, Hong S, van Stipdonk MJ, Altman A. Antigen-induced translocation of PKC- to membrane rafts is required for T cell activation. Nat Immun 2: 556–563, 2001.[CrossRef][ISI]

2. Bunnell SC, Kapoor V, Trible RP, Zhang W, Samelson LE. Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT. Immunity 14: 315–329, 2001.[CrossRef][ISI][Medline]

3. Cannon JL, Burkhardt JK. The regulation of actin remodeling during T-cell-APC conjugate formation. Immunol Rev 186: 90–99, 2002.[CrossRef][ISI][Medline]

4. Conforti L, Bodi I, Nisbet JW, Millhorn DE. O2-sensitive K+ channels: role of the Kv1.2-subunit in mediating the hypoxic response. J Physiol 524: 783–793, 2000.[Abstract/Free Full Text]

5. Das V, Nal B, Roumier A, Meas-Yedid V, Zimmer C, Olivo-Marin JC, Roux P, Ferrier P, Dautry-Varsat A, Alcover A. Membrane-cytoskeleton interactions during the formation of the immunological synapse and subsequent T-cell activation. Immunol Rev 189: 123–135, 2002.[CrossRef][ISI][Medline]

6. Del Carlo B, Pellegrini M, Pellegrino M. Modulation of Ca²⁺-activated K⁺ channels of human erythrocytes by endogenous protein kinase C. Biochim Biophys Acta 1612: 107–116, 2003.[Medline]

7. Dolmetsch RE, Xu K, Lewis RS. Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392: 933–936, 1998.[CrossRef][Medline]

8. Ehrlich LI, Ebert PJ, Krummel MF, Weiss A, Davis MM. Dynamics of p56lck translocation to the T cell immunological synapse following agonist and antagonist stimulation. Immunity 17: 809–822, 2002.[CrossRef][ISI][Medline]

9. Friedl P, den Boer AT, Gunzer M. Tuning immune responses: diversity and adaptation of the immunological synapse. Nat Rev Immunol 5: 532–545, 2005.[CrossRef][ISI][Medline]

10. Friedl P, Gunzer M. Interaction of T cells with APCs: the serial encounter model. Trends Immunol 22: 187–191, 2001.[CrossRef][ISI][Medline]

11. George Chandy K, Wulff H, Beeton C, Pennington M, Gutman GA, Cahalan MD. K⁺ channels as targets for specific immunomodulation. Trends Pharmacol Sci 25: 280–289, 2004.[CrossRef][Medline]

12. Gerlach AC, Gangopadhyay NN, Devor DC. Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1. J Biol Chem 275: 585–598, 2000.[Abstract/Free Full Text]

13. Ghanshani S, Wulff H, Miller MJ, Rohm H, Neben A, Gutman GA, Cahalan MD, Chandy KG. Up-regulation of the IKCa1 potassium channel during T-cell activation. Molecular mechanism and functional consequences. J Biol Chem 275: 37137–37149, 2000.[Abstract/Free Full Text]

14. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract/Free Full Text]

15. Hess SD, Oortgiesen M, Cahalan MD. Calcium oscillations in human T and natural killer cells depend upon membrane potential and calcium influx. J Immunol 150: 2620–2633, 1993.[Abstract]

16. Kupfer A, Kupfer H. Imaging immune cell interactions and functions: SMACs and the Immunological Synapse. Semin Immunol 15: 295–300, 2003.[CrossRef][ISI][Medline]

17. Kupper J. Functional expression of GFP-tagged Kv1.3 and Kv14 channels in HEK 293 cells. Eur J Neurosci 10: 3908–3912, 1998.[CrossRef][ISI][Medline]

18. Negulescu PA, Shastri N, Cahalan MD. Intracellular calcium dependence of gene expression in single T lymphocytes. Proc Natl Acad Sci USA 91: 2873–2877, 1994.[Abstract/Free Full Text]

19. Neylon CB, D'Souza T, Reinhart PH. Protein kinase A inhibits intermediate conductance Ca²⁺-activated K⁺ channels expressed in Xenopus oocytes. Pflügers Arch 448: 613–620, 2004.[CrossRef][ISI][Medline]

20. Panyi G, Bagdany M, Bodnar A, Vamosi G, Szentesi G, Jenei A, Matyus L, Varga S, Waldmann TA, Gaspar R, Damjanovich S. Colocalization and nonrandom distribution of Kv1.3 potassium channels and CD3 molecules in the plasma membrane of human T lymphocytes. Proc Natl Acad Sci USA 100: 2592–2597, 2003.[Abstract/Free Full Text]

21. Panyi G, Vamosi G, Bacso Z, Bagdany M, Bodnar A, Varga Z, Gaspar R, Matyus L, Damjanovich S. Kv1.3 potassium channels are localized in the immunological synapse formed between cytotoxic and target cells. Proc Natl Acad Sci USA 101: 1285–1290, 2004.[Abstract/Free Full Text]

22. Panyi G, Varga Z, Gaspar R. Ion channels and lymphocyte activation. Immunol Lett 92: 55–66, 2004.[CrossRef][ISI][Medline]

23. Robbins JR, Lee SM, Filipovich AH, Szigligeti P, Neumeier L, Petrovic M, Conforti L. Hypoxia modulates early events in T cell receptor-mediated activation in human T lymphocytes via Kv1.3 channels. J Physiol 564: 131–143, 2005.[Abstract/Free Full Text]

24. Skalhegg BS, Tasken K, Hansson V, Huitfeldt HS, Jahnsen T, Lea T. Location of cAMP-dependent protein kinase type I with the TCR-CD3 complex. Science 263: 84–87, 1994.[Abstract/Free Full Text]

25. Verheugen JA, Vijverberg HP. 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.[CrossRef][ISI][Medline]

26. Winslow MM, Crabtree Immunology GR. Decoding calcium signaling. Science 307: 56–57, 2005.[Abstract/Free Full Text]

27. Wulff H, Calabresi PA, Allie R, Yun S, Pennington M, Beeton C, Chandy KG. The voltage-gated Kv1.3 K⁺ channel in effector memory T cells as new target for MS. J Clin Invest 111: 1703–1713, 2003.[CrossRef][ISI][Medline]

28. Wulff H, Miller MJ, Hansel W, Grissmer S, Cahalan MD, Chandy KG. Design of a potent and selective inhibitor of the intermediate-conductance Ca²⁺-activated K⁺ channel, IKCa1: a potential immunosuppressant. Proc Natl Acad Sci USA 97: 8151–8156, 2000.[Abstract/Free Full Text]

29. Wulfing C, Sjaastad MD, Davis MM. Visualizing the dynamics of T cell activation: intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels. Proc Natl Acad Sci USA 95: 6302–6307, 1998.[Abstract/Free Full Text]

30. Xavier R, Rabizadeh S, Ishiguro K, Andre N, Ortiz JB, Wachtel H, Morris DG, Lopez-Ilasaca M, Shaw AC, Swat W, Seed B. Discs large (Dlg1) complexes in lymphocyte activation. J Cell Biol 166: 173–178, 2004.[Abstract/Free Full Text]

31. Zhou W, Vergara L, Konig R. T cell receptor induced intracellular redistribution of type I protein kinase A. Immunology 113: 453–459, 2004.[CrossRef][ISI][Medline]

This article has been cited by other articles:

				M. I. Lioudyno, J. A. Kozak, A. Penna, O. Safrina, S. L. Zhang, D. Sen, J. Roos, K. A. Stauderman, and M. D. Cahalan Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation PNAS, February 12, 2008; 105(6): 2011 - 2016. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Movie All Versions of this Article: 292/4/C1431    most recent 00376.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Nicolaou, S. A. Articles by Conforti, L. Search for Related Content PubMed PubMed Citation Articles by Nicolaou, S. A. Articles by Conforti, L.