Up-regulation of the IKCa1 Potassium Channel during T-cell Activation

We used whole cell recording to evaluate functional expression of the intermediate conductance Ca2+-activated K+ channel,IKCa1, in response to various mitogenic stimuli. One to two days following engagement of T-cell receptors to trigger both PKC- and Ca2+-dependent events, IKCa1expression increased from an average of 8 to 300–800 channels/cell. Selective stimulation of the PKC pathway resulted in equivalent up-regulation, whereas a calcium ionophore was relatively ineffective. Enhancement in IKCa1 mRNA levels paralleled the increased channel number. The genomic organization ofIKCa1, SKCa2, and SKCa3 were defined, and IKCa and SKCa genes were found to have a remarkably similar intron-exon structure. Mitogens enhancedIKCa1 promoter activity proportional to the increase inIKCa1 mRNA, suggesting that transcriptional mechanisms underlie channel up-regulation. Mutation of motifs for AP1 and Ikaros-2 in the promoter abolished this induction. Selective Kv1.3inhibitors ShK-Dap22, margatoxin, and correolide suppressed mitogenesis of resting T-cells but not preactivated T-cells with up-regulated IKCa1 channel expression. Selectively blockingIKCa1 channels with clotrimazole or TRAM-34 suppressed mitogenesis of preactivated lymphocytes, whereas resting T-cells were less sensitive. Thus, Kv1.3 channels are essential for activation of quiescent cells, but signaling through the PKC pathway enhances expression of IKCa1 channels that are required for continued proliferation.

pathways is capable of triggering different gene transcription events, and both are required for complete lymphocyte activation. A recent gene-chip survey of T-cells stimulated with a variety of mitogens, detected the induction of hundreds of genes (1). The rise in intracellular calcium ([Ca 2ϩ ] i ) activates calcineurin, a phosphatase that dephosphorylates the cytoplasmic transcription factor NFAT (nuclear factor of activated Tcells), enabling it to translocate to the nucleus and bind to NFAT-response elements of several genes, including the T-cell growth factor interleukin-2 (IL-2) (2, 3). The immunosuppressive drug, cyclosporin A (CsA), blocks this pathway by interacting with calcineurin and thereby suppresses activation (3,4). In a separate pathway, activation of PKC leads to phosphorylation of numerous substrates and results in assembly of Fos/Jun heterodimers that bind to activation protein-1 (AP1) elements on an overlapping set of genes via activation of the Ras and JNK (c-Jun N-terminal kinase) pathways. The functional significance of PKC, in particular, has been recently demonstrated (5,6). Cross-talk between these signaling pathways integrates the activation response. For example, the JNK pathway is co-activated by increases in cytoplasmic calcium (7). Sustained [Ca 2ϩ ] i signaling, mediated by calcium entry through calcium release-activated Ca 2ϩ (CRAC) channels, and PKC activation are both essential for complete activation.
Two potassium channels, the voltage-gated K ϩ channel Kv1.3 and the calcium-activated K ϩ channel IKCa1 (also known as KCNN4, IK1, hKCa4, and hSK4) (8 -10), modulate calcium influx through CRAC channels by regulating the membrane potential and hence the driving force for calcium entry (11). Freshly isolated resting human T-cells functionally express on average ϳ300 Kv1.3 channels (11-13) along with ϳ10 IKCa1 channels (14). During activation with phytohemagglutinin (PHA), expression of IKCa1 channels is strongly enhanced, while levels of Kv1.3 exhibit a modest enhancement (13,14). Changes in expression levels of K ϩ channels during activation have also been noted in murine T-cells (15) and in human and murine B cells (16,17). A recent gene chip survey (1) revealed a reduction in Kv1.3 mRNA levels in activated compared to resting T-cells, suggesting that post-transcriptional mechanisms contribute to the up-regulation of this channel.
In this study, we define the pathway leading to IKCa1 upregulation using phorbol myristate acetate (PMA) to trigger PKC selectively or ionomycin to stimulate the calcium-dependent cascade, and other mitogens (anti-CD3 Ab or PHA) that stimulate both pathways. By combining electrophysiological and molecular methods we show that stimulation of the PKC pathway alone is sufficient to enhance IKCa1 channel expression via transcriptional activation of the IKCa1 promoter. A reporter gene assay combined with mutational analysis defined the minimally active promoter region of the IKCa1 gene, demonstrating the importance of AP1, the PKC-dependent site of binding by Fos/Jun heterodimers. Using selective Kv1. 3 and IKCa1 inhibitors we demonstrate an important functional role of Kv1.3 channels in resting T-cells and IKCa1 channels in activated T-cells.
Genomic Organization of IKCa1-1.1 ϫ 10 6 plaques from a human EMBL3 genomic library (CLONTECH, Palo Alto, CA) were screened with a human IKCa1 (accession number AF033021) coding region probe to a final stringency of 1 ϫ SSC and 0.1% SDS at 65°C for 45 min. Five clones were isolated and two of these, KCNN4 -9 and KCNN4 -16 (which hybridized to both the 5Ј and 3Ј fragments of the probe) were further characterized. Precise location of the exon/intron boundaries was established by sequencing across the junctions in genomic DNA with primers derived from the cDNA sequence.
Northern Blot Analysis-Northern blots (CLONTECH) were hybridized to an IKCa1-specific probe in Expresshyb solution (CLONTECH), washed at a final stringency of 0.1 ϫ SSC, 0.1% SDS for 40 min at 55°C, and exposed to x-ray film at Ϫ80°C with an intensifying screen for 3-5 days. The IKCa1 probe corresponds to amino acid residues 380 -427 and includes ϳ490 bp of 3Ј noncoding sequence. Blots were stripped and re-probed with the control ␤-actin probe (CLONTECH). For Northern blot experiments on peripheral blood lymphocytes, poly(A) ϩ RNA was isolated from resting (2 ϫ 10 8 cells) and mitogen-activated human MNC (9 ϫ 10 7 cells) using the Ambion Pure mRNA Isolation kit (Ambion, Austin, TX). Cells were activated for 24 h with PHA (5 g/ml) or PMA (40 nM). A Northern blot containing 2 g of mRNA/lane was probed and washed as described. Northern blots were scanned and the intensity of bands determined by densitometry. The IKCa1 mRNA levels were normalized against the control probe, LEF (lymphoid enhancing factor).
Primer Extension-The Primer Extension System (Promega) was used to define the transcription initiation site. Briefly, an antisense primer (5Ј-ATGGGCTTTGTCACACACAATGG-3Ј) located 52 bases downstream of the 5Ј-end of the previously reported IKCa1 cDNA (accession number AF022797) was end-labeled using T4 polynucleotide kinase. In parallel reactions, 0.4 pmols labeled primer was annealed to either 20 g of human placental total RNA (Ambion) or 10 g of yeast tRNA at 68°C for 20 min and cooled for 10 min at room temperature. The annealed primer was next extended at 48°C for 30 min in the presence of AMV Primer Extension Buffer (50 mM Tris-HCl, pH 8.3, 50 mM KCl, 10 mM MgCl 2 , 10 mM dithiothreitol, 1 mM of each of four dNTPs, and 0.5 mM spermidine), 3 mM sodium pyrophosphate, and 20 units of AMV reverse transcriptase. Extension products were concentrated, and loaded onto a 6% polyacrylamide gel adjacent to a sequencing reaction of genomic DNA primed with the same oligonucleotide. In vitro transcribed kanamycin RNA and the control primer (Promega) produced an extension product that served as a positive control for the reaction.
Transfection of Human Peripheral Blood T Lymphocytes-In order to transfect primary T-cells, we stimulated them with a submitogenic dose of PHA (1 g/ml) which induces these cells to pass through a "window" (at 19.5-20.5 h) of transfection competency without concomitant cytokine production or cell proliferation (20,21). Normal human peripheral blood MNC, isolated by density sedimentation (Accuspin System-Histopaque-1077 tubes; Sigma Diagnostics), were grown (3 ϫ 10 6 cells/ml) for ϳ19.5 h in complete RPMI medium (RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, 1 mM Na ϩ pyruvate, 1% nonessential amino acids, 100 units/ml penicillin, 100 g/ml streptomycin, 50 M ␤-mercaptoethanol) and 1 g/ml PHA to induce transfection competence (20). They were re-counted to determine the number of living cells, centrifuged, and re-suspended in fresh medium at 2 ϫ 10 7 cells/ ml. Aliquots of 0.25 ml were electroporated at room temperature with 25 g of DNA of each IKCa1 construct in a Bio-Rad Gene Pulser at 250 volts and 960 microfarads (21), transferred to 10 ml of medium and allowed to rest for 1-2 h at 37°C. Viable cells were counted and re-suspended in fresh medium at 3 ϫ 10 6 cells/ml.
Luciferase Assays-Luciferase activity was measured in triplicate in aliquots of transfected peripheral blood human T-cells (ϳ3 ϫ 10 5 cells in 100 l) or human Jurkat T-cells (Ͼ8 ϫ 10 5 in 1 ml) at various times after transfection and/or mitogen (5 g/ml PHA or 40 nM PMA, or both in combination) stimulation. Cells were lysed in Reporter Lysis Buffer (Promega), harvested, and cleared of debris by centrifugation. 40 l of supernatant was mixed with Luciferase Assay Reagent (Promega) and the reaction monitored for 10 s in a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). To monitor equal transfection efficiency of deletion constructs, in initial experiments we co-transfected the pTRACER construct (containing green fluorescent protein) and counted fluorescent cells. These experiments showed that all constructs were transfected with approximately equal efficiency of ϳ5%.
Electrophysiological Analysis-COS-7 cells were transiently transfected with N-terminal green fluorescent protein-tagged hIKCa1 cDNA with FuGene TM 6 (Roche) according to the manufacturer's protocol. For other experiments, RBL cells were microinjected with IKCa1 cRNA as described previously (22). All experiments were carried out in the whole cell configuration of the patch clamp technique with a holding potential of Ϫ80 mV. An internal pipette solution consisting of (in mM): 145 potassium aspartate, 10 K 2 EGTA, 8.5 CaCl 2 , 2.0 MgCl 2, 10 HEPES, pH 7.2, 290 -310 mOsm, with a calculated free [Ca 2ϩ ] i of 1 M was used to activate the IKCa1 channel. Data were corrected for a liquid junction potential of Ϫ13 mV caused by an aspartate-based internal solution, with normal Ringer as the bath solution containing (in mM) 160 NaCl, 4.5 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 HEPES, pH 7.4. Currents during voltage ramps from Ϫ160 to ϩ40 mV over 200 ms were recorded every 10 s. In other experiments, ramp currents were elicited by 225-ms voltage ramps from Ϫ120 to ϩ30 mV every 5 s before and during the application of K ϩ -Ringer or Rb ϩ -Ringer.
Human MNCs were either nylon-wool purified and then activated with 5 g/ml PHA, 40 nM PMA, 10 nM PMA ϩ 175 nM ionomycin, or 175 nM ionomycin; or activated with 5 ng/ml anti-CD3 Ab and then nylonwool purified directly before the experiments. The same aspartatebased pipette solution as above was used with Na ϩ -aspartate Ringer as an external solution (in mM: 160 Na ϩ aspartate, 4.5 KCl, 2 CaCl 2 , 1 MgCl 2 , 5 HEPES, pH 7.4). Voltage ramps from Ϫ120 to ϩ40 mV over 200 ms were applied every 30 s. Kv1.3 currents in activated T lymphocytes were measured in normal Ringer with an internal pipette solution containing (in mM) 134 KF, 2 MgCl 2 , 10 HEPES, 10 EGTA. 200-ms depolarizing pulses to 40 mV were applied every 30 s and K d values were determined by fitting the Hill equation to the reduction of peak current.
[ 3 H]Thymidine Incorporation Assay-Resting or 2-day activated (5 ng/ml anti-CD3 Ab) cells were washed 3 times, re-suspended and seeded at 2 ϫ 10 5 cells/well in culture medium in flat-bottom 96-well plates (final volume 200 l). These cells were preincubated with drug (60 min), and then stimulated with mitogen (5 ng/ml anti-CD3 Ab) for 48 h. [ 3 H]Thymidine (1 Ci/well) was added for the last 6 h. Cells were harvested onto glass fiber filters and radioactivity measured in a scintillation counter.
Intracellular Fluorescence Activated Cell Sorter Assay for IL-2 and Interferon-␥ (IFN-␥)-MNCs were washed 3 times in complete RPMI medium, re-suspended at a concentration of 3 ϫ 10 6 cells/ml, and allowed to rest overnight in an upright costar T-75 tissue culture flask. Cells were placed in small Falcon tubes (1 ϫ 10 6 /ml) and stimulated with 10 nM PMA, 10 nM PMA ϩ 175 nM ionomycin, PMA ϩ ionomycin ϩ 25 nM CsA, or PMA ϩ ionomycin ϩ 1 M TRAM-34. After 48 h stimulation, cells were treated with brefelden A (Golgi Plug, Pharmingen BD) for 12 h to inhibit intracellular transport. Cells were pelleted at 1200 ϫ g, vortexed, fixed, and permeablized with Cytofix/Cytoperm solution (Pharmingen BD) and washed 2 times in Perm/Wash solution (Pharmingen BD). The cells were then stained with anti-CD4-PE antibodies along with either anti-IL-2-fluorescein isothiocyanate or IFN-␥fluorescein isothiocyanate antibodies, re-washed 3 times and then analyzed using a Becton Dickinson FACScan flow cytometer. The number of CD4ϩ T-cells (red channel) that expressed intracellular IL-2 or IFN-␥ (green channel) was determined. The green/red channel compensation and gain were set using singly stained samples and isotype matched controls.

Pharmacological Profile of the Cloned IKCa1 Channel
Matches the IK Ca Channel in Human T Cells-We initially compared characteristics of the cloned intermediate-conductance calcium-activated channel, IKCa1, expressed in COS or RBL cells, with native IK Ca currents in resting and activated human T lymphocytes. Cloned IKCa1 channels and native IK Ca current both exhibit a P Rb /P K permeability ratio of 1.2 ( Fig. 1A) and are blocked in a voltage-dependent manner by 10 mM Ba 2ϩ (Fig. 1B) and 16 mM Cs ϩ (data not shown) (14). In Fig. 1C we show that the cloned IKCa1 channel is also blocked by peptides ChTX and ShK, and by a ChTX analog, ChTX-Glu 32 , designed to target the IKCa1 channel specifically (23). Several structurally diverse small molecules also block the cloned channel ( Fig.  1C), including clotrimazole, TRAM-34, nitrendipine, nimodipine, nifedipine, econazole, ketoconazole, and tetraethylammonium chloride, with potencies similar to that of the endogenous channel in T-cells. The close similarity in ion selectivity and pharmacological characteristics, here using 11 channel blockers spanning 7 log units of potency, strongly suggests that the native channel is a homotetramer of IKCa1 subunits, in agreement with previous reports (10,14,22,24).

Mitogen-induced Up-regulation of IKCa1 Channels in Human T-cells-To determine the effect of mitogen stimulation on
IKCa1 expression, whole cell patch clamp measurements were performed on lymphocytes pre-stimulated to activate either the calcium signaling cascade, PKC-dependent events, or both. As an example, Fig. 2 illustrates up-regulation of IKCa1 currents in T-cells pre-stimulated through the T-cell receptor by the anti-CD3 Ab to trigger both calcium signaling and PKC. Two components of K ϩ current can be observed during voltage ramps in T-cells dialyzed with 1 M free Ca 2ϩ in the pipette. At potentials more negative than Ϫ40 mV, IKCa1 currents are induced rapidly upon break-in to achieve whole cell dialysis with 1 M free Ca 2ϩ in the pipette, as illustrated by changes in slope conductance with a reversal potential of Ϫ80 mV ( Fig A, selectivity sequence of monovalent cations for the IKCa1 channel expressed in RBL cells. B, Ba 2ϩ block of IKCa1. IKCa1 channels were activated as in A and ramp currents recorded with the bath solution changed from K ϩ -Ringer to a K ϩ -Ringer solution containing 10 mM Ba 2ϩ . C, dosedependent block of IKCa1 current in RBL or COS-7 cells by inhibitors: , and tetraethylammonium chloride (Ⅺ, K d ϭ 24 mM; RBL). IKCa1 currents were activated as in A and ramp currents were elicited every 10 s in normal Ringer solution and then in the presence of varying amounts of each blocker. K d values for each blocker (n ϭ 3, mean Ϯ S.D.) were determined from the reduction of slope conductance at Ϫ80 mV. 2C), pharmacologically confirming the channels' identity in resting and activated T-cells. In cells activated with anti-CD3 Ab for 2 days, the increased slope conductance near Ϫ80 mV indicates a dramatic enhancement in IKCa1 conductance, compared with resting cells (Fig. 2D). A similar enhancement of IKCa1 current is observed in cells pretreated with PMA for 48 h (Figs. 2, E and F). Kv1.3 currents are also enhanced in both anti-CD3 Ab-and PMA-activated cells (Fig. 2, D and F) in agreement with previous results (13,14). Acute treatment of resting T-cells with PMA (1-4 h) did not augment IKCa1 conductance (0.022 Ϯ 0.029 nS; 0.009 Ϯ 0.009 nS/pF; mean Ϯ S.D.) compared with resting T-cells (Fig. 3), suggesting that the enhanced IKCa1 conductance is most likely due to an increase in channel number induced by the activation stimulus, rather than modulation of existing channels. Fig. 3 summarizes experiments with a variety of stimuli, assaying the expression of IKCa1 channels. The number of channels per cell was computed by dividing the whole cell conductance by the measured single-channel conductance of 11 pS (14). Resting T-cells have an average IKCa1 conductance of ϳ0.1 nS, corresponding to an average of 8 channels/cell. As described previously (14), the mitogenic lectin PHA augments IKCa1 expression dramatically (second and third columns). The average IKCa1 conductance 24 h after PHA stimulation is 0.37 nS (34 channels/cell), representing a 4-fold increase that is statistically significant (p ϭ 0.006). By day 2, the IKCa1 conductance increases substantially to ϳ5.7 nS, corresponding to 520 channels/cell (a 65-fold increase). In comparison, following stimulation with anti-CD3 Ab, the conductance increases more rapidly, to ϳ1.25 nS (113 channels/cell) on day 1 and to ϳ5.69 nS (516 channels/cell) on day 2 (fourth and fifth columns, Fig.  3). Since lymphocytes enlarge during activation, we measured membrane capacitance to determine each cell's surface area and surface density of IKCa1 channels. The surface area of T-cells increases 3-fold following PHA or anti-CD3 Ab stimulation (Fig. 3). When normalized for membrane capacitance, the normalized IKCa1 conductance in resting cells is 0.05 nS/ pF, representing a very low channel density of 0.04 channels/  The channel density increases 15-20-fold following PHA or anti-CD3 Ab stimulation. We conclude that the up-regulation of IKCa1 channel expression more than compensates for the increased membrane surface area, resulting in a substantial increase in surface density.
T-cells were treated with either the phorbol ester PMA (triggers PKC pathway) or with ionomycin (activates calcium cascade) for 1 and 2 days and then analyzed by whole cell patch clamp to determine if either pathway alone is sufficient for IKCa1 up-regulation. PMA dramatically enhances IKCa1 conductance on days 1 and 2 (sixth and seventh columns, Fig. 3). Within 1 day of PMA activation, the IKCa1 channel number increases to ϳ100 channels/cell (1.12 nS), and by day 2 the number is ϳ370 channels/cell (4.1 nS). Interestingly, PMAinduced up-regulation of IKCa1 on day 1 is not accompanied by measurable changes in membrane capacitance, although by day 2 the increase in IKCa1 current is accompanied by a modest enhancement in membrane capacitance (Figs. 3 and 4). The selective enhancement of IKCa1 conductance is best illustrated by Fig. 4, demonstrating that PMA can increase conductance values relative to resting T-cells, without increasing membrane capacitance. When normalized for membrane capacitance, PMA augments IKCa1 channel density about 10-fold to 0.5 channels/m 2 (normalized conductance ϭ 0.59 nS/pF) on day 1, increasing further to 1.2 channels/m 2 (normalized conductance ϭ 1.43 nS/pF on day 2). Up-regulation of IKCa1 conductance occurs prior to cell enlargement (Fig. 4), and in the absence of cell DNA synthesis (measured by [ 3 H]thymidine incorporation, Fig. 3), or production of IL-2 (2-day PMA-treated cells ϭ 12% IL-2 ϩ ; resting cells ϭ 15% IL-2 ϩ ) or IFN-␥ (2 day PMA-treated cells ϭ 11% IFN-␥ ϩ ; resting ϭ 10% IFN-␥ ϩ ). Taken together, these results indicate that activation of the PKC-dependent signaling pathway alone leads to an increase in IKCa1 expression equivalent to the augmentation found when both pathways are triggered by anti-CD3 Ab or PHA, and this up-regulation is a relatively early event during T-cell mitogenesis.
New Synthesis Contributes to the Up-regulation of IKCa1 in Mitogen-activated Lymphocytes-Mitogen up-regulation of IKCa1 might be a consequence of new synthesis of the IKCa1 mRNA and/or protein, or due to the recruitment and activation of pre-existing IKCa1 molecules in the cell. In earlier studies, IKCa1 mRNAs measured by Northern blot analysis or RNase protection were found to be increased ϳ10-fold 24 h after activation with PHA (10, 26), suggesting that new synthesis of IKCa1 proteins may underlie the up-regulation of functional IKCa1 channels. To investigate this issue in more detail, we examined the distribution of IKCa1 mRNAs in six different human lymphoid tissues and discovered three IKCa1 mRNA species (2.2, 2.5, 4.5 kb). IKCa1 mRNAs are expressed abundantly in the spleen, lymph node, bone marrow, and fetal liver, while the thymus and peripheral blood leukocytes have lower levels (Fig. 5A). The 2.2-kb mRNA is the major band in the spleen, whereas the 4.5-kb transcript predominates in lymph nodes. Bone marrow and fetal liver, tissues containing immature hematopoietic cells, express roughly equivalent levels of the 2.2-and 2.5-kb mRNAs and very little of the large transcript. All three transcripts are expressed at roughly equivalent levels in thymus and peripheral blood leukocytes. Analysis of other human tissues reveals abundant expression of the 2.2-kb transcript in placenta and smaller amounts in lung and pancreas. Human heart, brain, liver, and skeletal muscle do not exhibit this transcript in any appreciable amount (Fig. 5B). The larger 4.5-kb IKCa1 transcript is detected in some tissues. Several transformed cell lines also express IKCa1 transcripts (Fig. 5C). Thus, IKCa1 has a wide tissue distribution.
In keeping with earlier reports (10,26), IKCa1 transcripts are almost undetectable in resting peripheral blood lymphocytes, while cells stimulated with PHA for 48 h enhance expression of the 2.2-kb IKCa1 mRNA (Fig. 5D). Although equal amounts of mRNA (2 g/lane) were loaded in both lanes, as an additional control, the blot was probed with LEF, a T-cell specific transcription factor that is not significantly up-regulated following T-cell activation (27). Since we observed a ϳ3fold increase in the LEF signal by densitometric scanning in activated versus resting cells (Fig. 5D, bottom), we normalized the LEF signal to be the same in both lanes and obtained a corrected estimate of the IKCa1 mRNA levels. PHA activation for 48 h augments IKCa1 mRNA levels ϳ10-fold compared with resting cells. In separate experiments, cells stimulated with PHA for 24 h had ϳ4-fold more IKCa1 mRNAs than resting cells, while PMA enhanced IKCa1 expression ϳ18-fold (data not shown). These results, in combination with earlier published data (10,26), indicate that new synthesis of IKCa1 channels contribute to the increased IKCa1 channel numbers observed during T-cell activation.
Mitogen-stimulated Transcription Contributes to Enhanced IKCa1 Expression in Activated Cells-The PMA-and PHAstimulated increases in IKCa1 mRNA levels might be a consequence of enhanced transcription of the gene and/or mRNA stability. The presence of ATTTA motifs in 3Ј non-coding regions (NCR) destabilize many transcripts, including those of T-cell cytokine genes (28,29) and the potassium channel Kv1.4 (30), and their removal enhances mRNA stability. The 3Ј NCR of IKCa1 lacks ATTTA motifs indicating that this mechanism does not underlie the mitogen-stimulated increase in IKCa1 mRNA expression. If transcriptional mechanisms are responsible for the up-regulation, both mitogens might be expected to enhance IKCa1 promoter activity to roughly the same extent as the increase in IKCa1 mRNAs and currents. To address this possibility, we determined the genomic organization of the major 2.2-kb IKCa1 transcript (the mRNA that is increased in both PHA-and PMA-stimulated cells), mapped the IKCa1 promoter elements, and ascertained whether promoter activity in transfected human T-cells was augmented by PMA and PHA. Since the known IKCa1 cDNAs (AF033021 and AF022797) are 2226-bp long, roughly the length of the 2.2-kb transcript, the transcription start site for this message must lie at or close to the beginning of the known cDNA sequence. To test this idea, primers close to the 5Ј end of the cDNA were used in primer extension assays to map the IKCa1 transcription start site (Fig.  5E). We used mRNA from the placenta for this purpose since this tissue primarily expresses the 2.2-kb transcript (Fig. 5B). The transcriptional start site lies three nucleotides upstream of the first nucleotide in the published cDNA sequences. An identical start site was found (data not shown) using mRNA from MOLT-4 and HL-60 cells that predominantly express the 2.2-kb mRNA (Fig. 5C). From the transcription start site to the polyadenylation signal the IKCa1 mRNA is 2229 bp long and is composed of 399 bp of 5Ј non-coding sequence, 1284 bp of coding region, and 546 bp of 3Ј NCR.
We next screened a human genomic library with a human IKCa1-specific probe and isolated two overlapping genomic clones. Analysis of these clones shows that IKCa1 is encoded by nine exons (Fig. 6). We also determined the genomic organization of the related human small conductance calcium-activated K ϩ channels, SKCa2/KCNN2 and SKCa3/KCNN3, by BLAST analysis and sequence alignments of known cDNAs with genomic contigs (Fig. 6). The intron-exon structure of SKCa2/ KCNN2 was ascertained by comparing the sequences of the chromosome 5 contigs, AC021415 and AC0121085 with the rat cDNA U69882, while the genomic organization of SKCa3/ KCNN3 was discerned by the sequence alignment of human cDNA AF031815 with chromosome 1 contigs AC034149, AC027645, and AC025385. Comparison of the intron-exon organization of these three genes and that of SKCa1/KCNN1 (31) reveals a conserved intron-exon placement (Fig. 6). Fig. 7 shows the sequences at the seven-conserved intron-exon junctions for IKCa1, SKCa2, and SKCa3 genes. The conservation of the genomic organization of these four genes is unexpected since IKCa1 shares only ϳ40% sequence similarity with the SKCa1-3 channels, has a significantly different pharmacological and biophysical fingerprint, and is located at a different locus in the genome (19q13.2) than SKCa1 (19p13.1), SKCa2 (5q23.1-23.2), and SKCa3 (1q21). SKCa1 has an additional exon, not present in IKCa1, SKCa2, or SKCa3 (Fig. 6), that encodes three additional residues, Ala-Gln-Lys, in the calmodulin-binding segment (22), suggesting that this exon is a relatively recent acquisition. Collectively, these results indicate that the SKCa1-3 and IKCa1 genes have a conserved genomic structure, which must predate the divergence of these two families from a common ancestral gene. Since IKCa1 (Fig. 5, A and D), SKCa2 (32), and SKCa3 (EST accession numbers numbers AA767647 and AA731772) are present in human lymphoid cells, their common intron placement may be a factor in regulating the lymphoid expression of these genes. The genomic organization of the SKCa1-3 and IKCa1 genes differs from that of the Slo gene that encodes the BK Ca channel (33).
The 5Ј NCR and 5Ј-flanking sequences are shown in Fig. 8. No canonical TATA box is present in the 50 bp upstream of the transcript's origin as has been shown for other K ϩ channel genes (30,34,35). To identify the human IKCa1 promoter, 5Ј-flanking fragments in the luciferase-enhancer (pGL2-e) or basic (pGL2-b) vectors were transfected into human T-cells in parallel with negative control plasmids pGL2-e or pGL2-b. Luciferase activity was measured at varying times after transfection.
Sense fragments (Ϫ1877/ϩ395 and Ϫ300/ϩ34) exhibit strong promoter activity in human lymphocytes, while the Ϫ300/ϩ34 antisense fragment is minimally active (Fig. 9A). Two additional deletion fragments (Ϫ205/ϩ34 and Ϫ117/ϩ34) show activities roughly equivalent to the longer fragment, indicating that the promoter lies between nucleotides Ϫ117 and ϩ34. Similar results were obtained when these constructs were expressed in human Jurkat T-cells (data not shown). Since the Ϫ117 to ϩ34 region contains putative sites for the DNA-binding proteins AP1 and Ik-2 (Fig. 8), we mutated each site separately and together. Mutation of either motif individually results in a significant decrease in promoter activity, while the combined deletion reduces activity further (Fig. 9B). Thus, the basal promoter required for transcription in human T lymphocytes lies between nucleotides Ϫ117 and ϩ34, and the AP1 and Ik-2 motifs are both essential for promoter activity.
If transcriptional mechanisms underlie the mitogen-stimulated enhancement in IKCa1 mRNA expression, PMA would be expected to enhance IKCa1 promoter activity more potently than PHA on day-1, paralleling the effects of these mitogens on the expression of IKCa1 mRNAs and currents (Figs. 3 and 5D). Furthermore, since PMA enhances IKCa1 expression prior to an increase in membrane capacitance (Fig. 3), this mitogen would be predicted to augment IKCa1 promoter activity early in the activation cascade. To test these ideas, human lymphocytes were first transfected with the IKCa1 promoter constructs and then stimulated with PMA, PHA, or a combination of these two mitogens for 3-24 h, and luciferase activity measured. Consistent with our expectation, PMA enhances activity of the Ϫ300/ϩ34 and Ϫ1877/ϩ395 sense fragments at the earliest time point measured (3 h), peak levels being detected at 10 -15 h poststimulation, while the antisense Ϫ300/ϩ34 fragment is inactive (Fig. 10A). Activity of all four sense fragments (Ϫ1877/ϩ395, Ϫ300/ϩ34, Ϫ205/ϩ34, and Ϫ117/ϩ34) increases ϳ5-7-fold following PMA stimulation for 10 h (Fig. 10B), which is roughly proportional to the increase in IKCa1 mRNA and IKCa1 channel number/cell measured at 24 h. Similar results were obtained with PHA, the ϳ4 -6-fold augmentation of IKCa1 currents and mRNA levels on day 1 being accompanied by a ϳ3-fold increase in promoter activity (Fig. 10B). A combination of the two mitogens increases promoter activity to a greater extent than either mitogen alone. The parallel increases in IKCa1 conductance, IKCa1 mRNA expression, and IKCa1 promoter activity by both mitogens strongly suggest that transcriptional mechanisms contribute to the channel up-regulation that accompanies T-cell activation.
We next analyzed AP1 and Ik-2 mutants to determine whether they are required for mitogen-dependent up-regulation. As shown in Fig. 10C, the AP1 mutant exhibits substantially diminished PMA responsiveness relative to the wild-type fragment (Ϫ117/ϩ34) and to its activity in resting T-cells. Although the Ik-2 mutant is less effective in reducing PMA inducibility of the promoter than the AP1 mutant, a double AP1/Ik-2 knockout decreases PMA responsiveness to a greater extent than either mutant alone. These results suggest that AP1, and to a lesser extent Ik-2, is essential for PMA inducibility of the IKCa1 promoter. Either mutant alone attenuates the PHA-stimulated enhancement of promoter responsiveness to PMA (Fig. 10C), indicating that the AP1 and Ik-2 sites are required for this effect. Thus, AP1 and Ik-2-dependent transcriptional mechanisms contribute to the IKCa1 up-regulation during human T-cell activation.
Since the putative AP1 site is critical for IKCa1 promoter activity, we examined whether this site could bind AP1 protein.
HeLa cell extracts (Promega, Madison, WI), previously characterized for AP1 binding, interact with a 32 P-labeled commercially available AP1 oligonucleotide probe in gel-shift assays (Fig. 11, lane 2). This binding is competed by 100-fold excess unlabeled AP1 probe (lane 3) and by a 24-bp IKCa1 probe spanning the AP1 site (lane 4), but not by an IKCa1 probe in which the AP1 site is mutated (lane 5). HeLa cell extracts also bind to the IKCa1 AP1 site (lane 7), but not to the mutated site (lane 11). This interaction is specific since it can be competed by 100-fold excess of the AP1 probe (lane 8) and by the IKCa1-AP1 wild-type probe (lane 9), but not by the IKCa1-AP1 mutant probe (lane 10).

DISCUSSION
To investigate the molecular mechanism of IKCa1 channel up-regulation in T lymphocytes, we determined the genomic organizations of IKCa1, SKCa2, and SKCa3, and functionally mapped the promoter of IKCa1. The striking similarity in intron-exon boundaries suggests a common evolutionary origin of IKCa1 and SKCa1-3 genes. IKCa1 functional expression is enhanced by treatment with PHA, anti-CD3 Ab, PMA, or PMA ϩ ionomycin (Fig. 3). This increase is in direct proportion to the increase in IKCa1 transcripts (Fig. 5) and to the enhanced activity of the IKCa1 promoter. The PMA-triggered IKCa1 up-regulation is an early event in the T-cell activation cascade. Enhanced IKCa1 promoter activity is detected as early as 3 h after activation (Fig. 10), and augmented channel expression is observed prior to increase in cell size, onset of DNA synthesis, or cytokine production (Figs. 3 and 4). Thus, transcriptional mechanisms are likely to underlie the increased IKCa1 expression in activated lymphocytes, although post-transcriptional mechanisms (including increased channel trafficking) may also contribute. Within the promoter region of the IKCa1 gene, several potential transcription factor-binding sites were identified and functionally probed by deletion and mutational analysis. Mutagenesis and gel-shift studies suggest that the AP1 and Ik-2 transcription factors, but not NFAT, are required for basal transcription of the IKCa1 gene and mediate the transcriptional augmentation of IKCa1 expression during the T-cell activation response. These results may be relevant to B lymphocytes (16) and T-cell subsets (43)  MNCs were transfected with Ϫ1877/ϩ395 (sense), Ϫ300/ϩ34 (sense), Ϫ300/ϩ34 (antisense), or pGL2-e, and were then left in media or treated with PMA (40 nM). Luciferase activity was measured at various times after stimulation with PMA. Data are representative of one of three experiments with similar results. B, mitogen inducibility of the IKCa1 promoter. MNC were transfected with Ϫ1877/ϩ395 (sense), Ϫ300/ϩ34 (sense), Ϫ205/ϩ34 (sense), or Ϫ117/ϩ34 (sense), and then incubated in media or treated with PHA (5 g/ml), PMA (40 nM), or a combination of the two mitogens for 10 h. Data are representative of one of four experiments with similar results. C, effect of AP1 and Ik-2 mutations on mitogen inducibility of the promoter. Lymphocytes were transfected with Ϫ117/ϩ34 (sense) or mutants of this fragment. Luciferase activity was measured 10 h following stimulation with PMA (40 nM), PHA (5 g/ml), or a combination of both mitogens. Control cells were left in media. Data are representative of one of four experiments with similar results.
blasts (44,45), which may be mediated via the AP1-dependent pathway described below in human T lymphocytes (Fig. 13). Fig. 13 summarizes the signaling pathways that likely contribute to IKCa1 up-regulation in T lymphocytes. Anti-CD3 Ab or PMA augment IKCa1 transcription in an AP1-dependent manner via stimulation of the PKC and downstream Ras and JNK pathways. The resulting AP1 (c-Fos/c-Jun heterodimer) complex binds to the IKCa1 promoter (as shown in Fig. 11) and initiates transcription of the IKCa1 message in conjunction with the transcription factor, Ik-2. Ik-2 is a nuclear factor that sets a threshold for T-cell mitogenesis; in activated T-cells, Ik-2 co-localizes with the DNA replication machinery and modulates cell entry into the S-phase (46). Increased IKCa1 mRNA levels lead to enhanced expression of functional IKCa1 channels on the cell membrane tightly complexed to calmodulin, which serves as the calcium sensor for these channels (22). Interestingly, calmodulin expression is also augmented during human T-cell activation, especially the CAM-III mRNA and protein (47). CsA partially suppresses the mitogen-stimulated increase in IKCa1 expression (Fig. 3), but this is not due to inhibition of the IKCa1 promoter, and may instead result from blockade of a post-transcriptional step. Ionomycin by itself fails to increase IKCa1 expression significantly, most likely due to its inability to stimulate AP1 production, whereas its enhancement of PMA-induced up-regulation of IKCa1 (Figs. 2, 3, and 12) may be due to co-activation of the JNK pathway via an increase in cytoplasmic calcium (Fig. 13). The up-regulation of IKCa1 channels during human T-cell activation parallels the recently described ϳ10-fold increase in numbers of the CRAC channels induced by PHA and PMA, but not ionomycin (48), raising the possibility that these two channels involved in calcium signaling could be coordinately regulated.
Calcium-entry through CRAC channels is promoted by membrane hyperpolarization due to the opening of IKCa1 and Kv1.3 channels (11). Since quiescent human T lymphocytes contain on average roughly ϳ300 -400 Kv1.3 channels/cell and only ϳ8 IKCa1 channels, the membrane potential of quiescent cells is thought to be mainly dependent on the voltage-gated channel with the IKCa1 channels playing a minimal role (11). In keeping with this idea, blockade of Kv1.3 by specific and potent inhibitors attenuates the calcium signaling response and suppresses the activation response of resting human T-cells both in vitro and in vivo (25,38,39,41) (Fig. 12C). In contrast, clotrimazole and TRAM-34, both potent IKCa1 inhibitors, suppress the activation of resting human T-cells (Fig. 12, B and C) (18,26,42) only at concentrations (ϳ5 M) that are 70 -250 times the channel-blocking dose, perhaps through nonspecific mechanisms.
The relative numbers of the two K ϩ channels change in activated human T-cells. T-cells stimulated for 48 -72 h with mitogens have 300 -800 IKCa1 channels along with 400 -500 Kv1.3 channels (Figs. 2 and 3) (13,14). Khanna and colleagues (26), reported that PHA-induced proliferation of PHA preactivated T-cells was potently suppressed by clotrimazole tested at a single dose of 250 nM. We have extended these studies by using a complete range of concentrations of clotrimazole and TRAM-34 and showing that these inhibitors potently suppress reactivation of anti-CD3 Ab-or PMA preactivated lymphocytes at submicromolar concentrations consistent with their channel-blocking dose (Fig. 12, B and D (18)). Our results taken together with earlier studies (10,14,26) suggest that different mitogens (PHA, anti-CD3 Ab, PMA, PMA ϩ ionomycin) augment IKCa1 channel expression in lymphocytes and this induction is functionally important. The parallel enhancement of IKCa1 and CRAC channels might allow the activated T-cell to fine-tune its regulation of membrane potential in response to subtle changes in cytoplasmic calcium, which in turn would modulate calcium entry. Consistent with this notion, previous studies on activated T-cells have shown coupling between IK Ca channels and membrane potential (49,50). More recently, we have found that the IKCa1 peptide inhibitor, ChTX-Glu 32 (23), suppresses thapsigargin-induced calcium entry into activated human T-cells (51). Thus, the concerted action of the two potassium channels regulates the entry of calcium through CRAC channels in quiescent and activated T-cells and thereby modulates the immune response.