FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2035798975> ?p ?o ?g. }

• W2035798975 endingPage "12256" @default.
• W2035798975 startingPage "12249" @default.
• W2035798975 abstract "To maintain Ca2+ entry during T lymphocyte activation, a balancing efflux of cations is necessary. Using three approaches, we demonstrate that this cation efflux is mediated by Ca2+-activated K+ (KCa) channels, hSKCa2 in the human leukemic T cell line Jurkat and hIKCa1 in mitogen-activated human T cells. First, several recently developed, selective and potent pharmacological inhibitors of KCa channels but not KV channels reduce Ca2+ entry in Jurkat and in mitogen-activated human T cells. Second, dominant-negative suppression of the native KCa channel in Jurkat T cells by overexpression of a truncated fragment of the cloned hSKCa2 channel decreases Ca2+ influx. Finally, introduction of the hIKCa1 channel into Jurkat T cells maintains rapid Ca2+ entry despite pharmacological inhibition of the native small conductance KCa channel. Thus, KCachannels play a vital role in T cell Ca2+ signaling. To maintain Ca2+ entry during T lymphocyte activation, a balancing efflux of cations is necessary. Using three approaches, we demonstrate that this cation efflux is mediated by Ca2+-activated K+ (KCa) channels, hSKCa2 in the human leukemic T cell line Jurkat and hIKCa1 in mitogen-activated human T cells. First, several recently developed, selective and potent pharmacological inhibitors of KCa channels but not KV channels reduce Ca2+ entry in Jurkat and in mitogen-activated human T cells. Second, dominant-negative suppression of the native KCa channel in Jurkat T cells by overexpression of a truncated fragment of the cloned hSKCa2 channel decreases Ca2+ influx. Finally, introduction of the hIKCa1 channel into Jurkat T cells maintains rapid Ca2+ entry despite pharmacological inhibition of the native small conductance KCa channel. Thus, KCachannels play a vital role in T cell Ca2+ signaling. The human leukemic T cell line Jurkat is widely used as a model system to study intracellular signaling cascades during lymphocyte activation. These studies have revealed the critical requirement for two signaling pathways to complete lymphocyte activation. In the first pathway, activation of protein kinase C, particularly protein kinase Cθ, leads to the phosphorylation of several cytoplasmic proteins and the triggering of transcription via the assembly of the Fos/Jun transcription factor complex on AP1 elements in several genes (1Flanagan W.M. Corthésy B. Bram R.J. Crabtree G.R. Nature. 1991; 352: 803-807Crossref PubMed Scopus (946) Google Scholar, 2Northrop J.P. Ullman K.S. Crabtree G.R. J. Biol. Chem. 1993; 268: 2917-2923Abstract Full Text PDF PubMed Google Scholar, 3Monks C.R.F. Freiberg B.A. Kupfer H. Sciaky N. Kupfer A. Nature. 1998; 395: 82-86Crossref PubMed Scopus (1954) Google Scholar, 4Acuto O. Cantrell D. Annu. Rev. Immunol. 2000; 18: 167-184Crossref Scopus (222) Google Scholar). In the second cascade, the sustained entry of Ca2+ from the external milieu raises the cytoplasmic Ca2+ concentration, leading to gene transcription mediated by the nuclear factor of activated T cells (NF-AT) 1The abbreviations used are: NF-ATnuclear factor of activated T cellsILinterleukinChTXcharybdotoxinCRACCa2+ release-activated Ca2+Dapdiaminopropionic acidGFPgreen fluorescent proteinIKCaintermediate conductance KCaKVvoltage-gated K+KCaCa2+-activated K+SKCasmall conductance KCaTgthapsigargin (5Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2203) Google Scholar, 6Crabtree G.R. Cell. 1999; 96: 611-614Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar). Production of the key T cell cytokine IL-2 requires the simultaneous activation of both pathways, with Ca2+ being absolutely required for the process.In human T lymphocytes and in Jurkat T cells, Ca2+ influx is mediated by the opening of voltage-independent Ca2+release-activated Ca2+ (CRAC) channels (7Zweifach A. Lewis R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6295-6299Crossref PubMed Scopus (691) Google Scholar, 8Partiseti M. Le Deist F. Hivroz C. Fischer A. Korn H. Choquet D. J. Biol. Chem. 1994; 269: 32327-32335Abstract Full Text PDF PubMed Google Scholar, 9Fomina A.F. Fanger C.M. Kozak J.A. Cahalan M.D. J. Cell Biol. 2000; 150: 1435-1444Crossref PubMed Scopus (89) Google Scholar). Movement of ions through open channels in the plasma membrane is driven by an electrochemical gradient. Upon T cell stimulation and opening of CRAC channels, the electrochemical gradient supporting Ca2+entry is large, resulting in significant Ca2+ influx. However, Ca2+ entry could result in depolarization of the plasma membrane, limiting further influx. Therefore, to maintain Ca2+ entry over the time scale required for gene transcription, a balancing cation efflux is necessary. Efflux of K+ ions through K+ channels is thought to provide the electrochemical driving force for Ca2+ entry via regulation of membrane potential (10Lewis R.S. Cahalan M.D. Annu. Rev. Immunol. 1995; 13: 623-653Crossref PubMed Scopus (445) Google Scholar). We have directly tested this idea and identified the functionally important K+ channel subtypes in Jurkat T cells and activated normal human T cells.Jurkat T cells express two distinct K+ channels. The first is a voltage-gated K+ (KV) channel encoded by the Kv1.3 gene, and the second is a small conductance Ca2+-activated K+ (KCa) channel recently shown to be encoded by the hSKCa2 gene (11Grissmer S. Lewis R.S. Cahalan M.D. J. Gen. Physiol. 1992; 99: 63-84Crossref PubMed Scopus (116) Google Scholar, 12Grissmer S. Nguyen A.N. Aiyar J. Hanson D.C. Mather R.J. Gutman G.A. Karmilowicz M.J. Auperin D.D. Chandy K.G. Mol. Pharmacol. 1994; 45: 1227-1234PubMed Google Scholar, 13Jäger H. Adelman J.P. Grissmer S. FEBS Lett. 2000; 469: 196-202Crossref PubMed Scopus (44) Google Scholar, 14Desai R. Peretz A. Idelson H. Lazarovici P. Attali B. J. Biol. Chem. 2000; 275: 39954-39963Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Human T cells possess a different KCa channel encoded by hIKCa1, in addition to Kv1.3, but do not expressSKCa2 (15Grissmer S. Nguyen A.N. Cahalan M.D. J. Gen. Physiol. 1993; 102: 601-630Crossref PubMed Scopus (220) Google Scholar, 16Ishii T.M. Silvia C. Hirschberg B. Bond C.T. Adelman J.P. Maylie J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11651-11656Crossref PubMed Scopus (510) Google Scholar, 17Logsdon N.J. Kang J.S. Togo J.A. Christian E.P. Aiyar J. J. Biol. Chem. 1997; 272: 32723-32726Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 18Joiner W.J. Wang L.Y. Tang M.D. Kaczmarek L.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11013-11018Crossref PubMed Scopus (314) Google Scholar). Earlier work investigating the roles of K+ channels in lymphocyte activation was hampered by the lack of sufficiently specific blockers, leading to conflicting results and divergent interpretations (see, for example, Refs. 19Gelfand E.W. Or R. J. Immunol. 1991; 147: 3452-3458PubMed Google Scholar and 20Freedman B.D. Price M.A. Deutsch C.J. J. Immunol. 1992; 149: 3784-3794PubMed Google Scholar). Expression levels of KV and KCa channels are similar in Jurkat and in mitogen-activated human T cells, although the molecular identity of the KCa channel differs in these two cell types (15Grissmer S. Nguyen A.N. Cahalan M.D. J. Gen. Physiol. 1993; 102: 601-630Crossref PubMed Scopus (220) Google Scholar). This difference in expression pattern, the advent of new and highly specific blockers of all three channels, and the potential for genetic manipulation of functional expression levels provide an opportunity to examine the contributions of K+channels in regulating membrane potential and Ca2+signaling in lymphocytes. Our results emphasize the importance of KCa channels in the modulation of Ca2+signaling.EXPERIMENTAL PROCEDURESCell Culture and ChemicalsJurkat E6–1 and COS-7 cells were obtained from ATCC (Manassas, VA). Jurkat E6–1 cells were grown in RPMI medium supplemented with 10% fetal bovine serum, 2 mm glutamine, and 10 mm HEPES at densities of 1–9 × 105 in a 37 °C humidified incubator with 5% CO2. COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 2 mm glutamine and split twice weekly. Human T cells were isolated and cultured as described (21Hess S.D. Oortgiesen M. Cahalan M.D. J. Immunol. 1993; 150: 2620-2633PubMed Google Scholar). T cells were preactivated by addition of 4 μg/ml phytohemagglutinin or phorbol myristate acetate (33 nm) + ionomycin (1 μm) (Calbiochem) for 18–72 h prior to use. Unless otherwise specified, all reagents were obtained from Sigma and all optical filters from Chroma (Brattleboro, VT). The syntheses of bis-quinolinium cyclophane compounds UCL 1530 (8, 19-diaza-1,7(1,4)-diquinolina-3,5(1,4)-dibenzenacyclononadecanephanedium tetratrifluoroacetate hydrate), UCL 1684 (6, 10-diaza-1,5(1,4)-diquinolina-3(1,3),8(1,4)-dibenzenacyclodecaphanedium tritrifluoroacetate hydrate), UCL 1848 (8,14-diaza-1,7(1,4)-diquinolinacyclotetradecaphanedium ditrifluoroacetate), and UCL 2079 (8, 14-diaza-1,7(1,4)-di(6-trifluoromethylquinolina) cyclotetradecaphanedium ditrifluoroacetate) have been previously described (22Campos Rosa, J., Dunn, P. M., Galanakis, D., Ganellin, C. R., Yang, D., and Chen, J. Q. (1997) PCT Int. Appl. WO 97/48705.Google Scholar, 23Benton D.C. Roxburgh C.J. Ganellin C.R. Shiner M.A. Jenkinson D.H. Br. J. Pharmacol. 1999; 126: 169-178Crossref PubMed Scopus (10) Google Scholar, 24Campos Rosa J. Galanakis D. Piergentili A. Bhandari K. Ganellin C.R. Dunn P.M. Jenkinson D.H. J. Med. Chem. 2000; 43: 420-431Crossref PubMed Scopus (90) Google Scholar, 25Chen J.Q. Galanakis D. Ganellin C.R. Dunn P.M. Jenkinson D.H. J. Med. Chem. 2000; 43: 3478-3481Crossref PubMed Scopus (54) Google Scholar). UCL 1684 is very stable in aqueous solution, showing no significant degradation after 24 h in culture medium at 37 °C (data not shown). The stability of other drugs was not tested over long periods of time. ShK-Dap22 and ChTX-Glu32 were obtained from BACHEM (King of Prussia, PA).Transfection of Constructs into Mammalian CellsIn each electroporation cuvette (gap of 0.4 cm), 107 Jurkat cells and 10 μg of the DNA of interest were electroporated at 960 μF, 250 V, and then resuspended in 15 ml of fresh culture medium and returned to the incubator for 36–60 h prior to use. COS-7 cells (5 × 105 cells/chamber) were plated in culture chambers and transiently transfected using the Lipofectin transfection reagent (Life Technologies, Inc.) with the DNA of interest following the manufacturer's recommended protocol in OptiMEM medium (Life Technologies, Inc.). Following an 8–12-h transfection, the cells were placed in fresh growth medium in the incubator for 48 h. Typical transfection efficiencies using this protocol were 18–33%. The DNA vectors used for transfection were prepared using the Qiagen (Valencia, CA) endotoxin-free plasmid maxi-prep kit.DNA ConstructsThe N-terminal GFP fusion protein of human IKCa1 (GFP-IKCa1) was a gift from J. Aiyar (AstraZeneca Pharmaceuticals, Wilmington, DE) and was generated by subcloning hIKCa1 into the pEGFP-C1 vector (CLONTECH, Palo Alto, CA) as aBamHI/BglII-Xho fragment. This cloning strategy introduced 12 extra amino acids between GFP and the initiation codon ofIKCa1. The expressed sequence tag clone IMAGE: 2248 (GenBankTM accession number AI810558), corresponding to nucleotides 491–2193 of the SKCa2 sequenceAF239613, was isolated from the pT7T3 Pac Vector (Amersham Pharmacia Biotech) using NotI and EcoRI restriction sites and subcloned into pBluescript and from there into theSacI restriction site of the pGFP-C1 expression vector. The truncated hSKCa2 dominant negative construct was generated by removing a 1.24-kilobase pair BclI fragment from the GFP-SKCa2 construct, leaving a 564-base pair insert encoding the hSKCa2 N-terminal proximal region terminating in the S3 transmembrane domain. The human SKCa3 clone (GenBankTM accession number AJ251016) containing 19 polyglutamines in the N terminus was cloned in frame to GFP in the GFP vector as an EcoRI/BamI fragment. HEK-293 cells expressing the skeletal muscle sodium channel hSKM1 (SCN4A) were a gift from Dr. F. Lehmann-Horn (University of Ulm, Germany) (26Peter W. Mitrovic N. Schiebe M. Lehmann-Horn F. Lerche H. J. Physiol. (Lond.). 1999; 518: 13-22Crossref Scopus (6) Google Scholar). The murine Kv1.3 channel is stably expressed in L929 cells as previously described (12Grissmer S. Nguyen A.N. Aiyar J. Hanson D.C. Mather R.J. Gutman G.A. Karmilowicz M.J. Auperin D.D. Chandy K.G. Mol. Pharmacol. 1994; 45: 1227-1234PubMed Google Scholar). The hSlo construct was the gift of Dr. Ligia Toro (University of California, Los Angeles, CA) and is expressed following injection into Xenopus oocytes (27Stefani E. Ottolia M. Noceti F. Olcese R. Wallner M. Latorre R. Toro L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5427-5431Crossref PubMed Scopus (120) Google Scholar,28Rauer H. Lanigan M.D. Pennington M.W. Aiyar J. Ghanshani S. Cahalan M.D. Norton R.S. Chandy K.G. J. Biol. Chem. 2000; 275: 1201-1208Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar).Patch Clamp ExperimentsFor all KCaexperiments, electrophysiological recordings were made in the whole-cell mode with a holding potential of –40 mV and an internal solution consisting of 130 mm potassium aspartate, 10 mm K2EGTA, 8.55 mmCaCl2, 2.08 mm MgCl2, and 10 mm HEPES, pH 7.2, with a calculated free [Ca2+] of ∼1 μm. In KVexperiments, whole-cell recordings were made with a holding potential of –80 mV and an internal solution identical except that it contained 2.28 mm CaCl2, resulting in a calculated free [Ca2+] of ∼50 nm. KVexperiments also used a leak subtraction regimen in which the leak pulse was applied after each voltage pulse. External solutions consisted of normal Ringer solution (155 mm NaCl, 4.5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mmd-glucose, and 5 mm HEPES, pH 7.4), K+ Ringer solution (with identical ingredients except that all NaCl was substituted by KCl, resulting in a final KCl concentration of 159.5 mm), or 40 mm K+ Ringer solution, in which these two solutions were mixed to yield a final Na+ concentration of 119.5 mm and a final K+ concentration of 40 mm. Whole-cell patch clamp recordings were performed using the equipment and techniques described previously, and all data were corrected for a liquid junction potential of −13mV for aspartate-based solutions (29Fanger C.M. Ghanshani S. Logsdon N.J. Rauer H. Kalman K. Zhou J. Beckingham K. Chandy K.G. Cahalan M.D. Aiyar J. J. Biol. Chem. 1999; 274: 5746-5754Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar).Cytokine Expression AssaysJurkat cells were stimulated as described above for T cells but in 96-well tissue culture plates with 105 cells in 200 μl of growth medium. Cytokine production was assayed using the OptEIA enzyme-linked immunosorbent assay kit (BD-Pharmingen, San Diego, CA) and a fluorescence plate reader (Molecular Devices, Sunnyvale, CA) to quantify production of IL-2 and IL-8.Confocal MicroscopyConfocal fluorescence images were taken with a Bio-Rad MRC-600 equipped with an argon laser (488 nm) and a fluorescein isothiocyanate filter set (500–530 nm). All images were acquired under a 63× oil objective on a Zeiss Axiovert 35 microscope. Z-series sections were captured at 0.5-μm intervals, and optical section thickness was estimated to be ∼0.45-μm.Ca2+ Imaging and Membrane Potential MeasurementsCells were loaded in 1 μm fura-2/AM ester (Molecular Probes, Eugene, OR) at 21–24 °C for 30 min, washed, and stored in the dark until use (within 3 h). Intracellular Ca2+ concentrations were estimated, and experiments were performed utilizing a complete video microscopic, ratiometric Ca2+ imaging system (Videoprobe, ETM Systems), as previously described (30Fanger C.M. Neben A.L. Cahalan M.D. J. Immunol. 2000; 164: 1153-1160Crossref PubMed Scopus (95) Google Scholar). At the beginning of data collection, the extracellular solution was normal Ringer solution. Store depletion in the presence of Tg required perfusion with a 0-Ca2+ version of normal Ringer solution in which CaCl2 was replaced by additional MgCl2 to keep divalent concentrations constant and in which 1 mm EGTA was used to chelate residual Ca2+. During measurements on transfected cells, individual GFP-positive, and thus successfully transfected, cells were identified and marked for analysis using a fluorescein isothiocyanate filter set consisting of a 480 ± 20 nm exciter, 505 nm dichroic mirror, and 520 nm long-pass emission filter. By comparing Ca2+ responses of GFP-hSKCa2Δ-expressing or GFP-hIKCa1-expressing cells with GFP-expressing vector control cells, we eliminated the risk of inaccurate quantification due to small amounts of contamination of the fura-2 signal by GFP fluorescence bleed-through. Data processing and statistical analysis were carried out using IgorPRO (Wavemetrics, Lake Oswego, OR) and Excel (Microsoft, Redmond, WA) software. The bis-oxonol dye bis-(1, 3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3), Molecular Probes) was used as an indicator of membrane potential in imaging experiments using the same software and hardware described for Ca2+ imaging, and using the fluorescein, filter set described above. Cells were preequilibrated with 125 nmDiBAC4(3) for 5–10 min prior to the start of each experiment, and dye concentration was maintained in all external solutions throughout the experiment.RESULTSSpecific K+ Channel Blockers Define the Role of KV and KCa Channels in Jurkat Ca2+SignalingUsing the patch clamp technique, we characterized highly potent and specific KCa and KV channel blockers in Jurkat T cells and activated human T lymphocytes. In Jurkat cells, whole-cell recording with Ca2+ maintained at a low concentration inside the pipette revealed only KV currents (Fig. 1 A). These currents inactivated during repetitive pulsing and were blocked by ShK-Dap22, a sea anemone peptide modified to gain specificity for the Kv1.3 channel (IC50 = ∼25 pm) (31Kalman K. Pennington M.W. Lanigan M.D. Nguyen A. Rauer H. Mahnir V. Paschetto K. Kem W.R. Grissmer S. Gutman G.A. Christian E.P. Cahalan M.D. Norton R.S. Chandy K.G. J. Biol. Chem. 1998; 273: 32697-32707Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Application of 10 nmShK-Dap22 blocked ∼97% of the KV current. The voltage dependence, inactivation kinetics, and pharmacological properties of Jurkat KV currents are consistent withKv1.3 encoding this channel, as reported previously (32Grissmer S. Dethlefs B. Wasmuth J.J. Goldin A.L. Gutman G.A. Cahalan M.D. Chandy K.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9411-9415Crossref PubMed Scopus (169) Google Scholar).Elevation of [Ca2+] inside the pipette to 1 μm activated an additional small, voltage-independent current. By increasing extracellular K+ concentrations to 40 mm, the inward component of this current could be observed. The current reversed near the predicted Nernst potential for a K+-selective current in 40 mm K+extracellular solution (Fig. 1 B). Application of 10 nm apamin, a peptide from bee venom, completely and irreversibly blocked this KCa current (data not shown), in agreement with a previous study (11Grissmer S. Lewis R.S. Cahalan M.D. J. Gen. Physiol. 1992; 99: 63-84Crossref PubMed Scopus (116) Google Scholar), whereas Kv1.3 blockers ShK-Dap22 (250 nm, Fig. 1 B) and charybdotoxin (10 nm, data not shown) had little or no effect. Recently, a group of bis-quinolinium derivatives developed by the group of C. R. Ganellin and D. H. Jenkinson at University College London were found to block with high affinity the apamin-sensitive small conductance KCa channel in rat superior cervical ganglion cells (33Campos Rosa J. Galanakis D. Ganellin C.R. Dunn P.M. Jenkinson D.H. J. Med. Chem. 1998; 41: 2-5Crossref PubMed Scopus (71) Google Scholar). We tested these same compounds on the apamin-sensitive KCa current in Jurkat T cells. The bis-quinolinium cyclophane UCL 1684 (see under “Experimental Procedures” for full name) at a concentration of 10 nmblocked 95 ± 5% of the KCa current but had no effect on the KV current even at 250 nm (Fig. 1,A and B). Blocking was rapid (usually complete within 1 min) and, in contrast to apamin, rapidly reversible. The dose-response curve in Fig. 1 D shows that UCL 1684 blocks the Jurkat KCa channel with subnanomolar affinity (IC50 = 180 pm), making it the most potent inhibitor of this channel yet described. The molecular identity of the KCa channel in Jurkat T cells was verified by measuring the efficacy of UCL 1684 in blocking current through the cloned humanSKCa2 and SKCa3 channels expressed in COS-7 cells (IC50 values of 280 pm and 9.5 nm, respectively). UCL 1684 selectively blocks Jurkat KCachannels and SKCa2 channels over the closely relatedSKCa3 channel and several other more distantly related channels (Fig. 1 D; Table I). Other bis-quinolinium cyclophanes (UCL 2079, UCL 1848, and UCL 1530; see under “Experimental Procedures” for full names) also blocked Jurkat KCa currents with high affinity (Fig.1 C). Table I summarizes the selectivity of all compounds and demonstrates that UCL 1684, UCL 1848, and UCL 2079 are all ∼104-fold more effective at blocking SKCa currents than any other channels tested. Because these drugs are highly specific for the KCa channel found in Jurkat T cells and appear equally potent in blocking the expressed human SKCa2channel, our results provide a confirmation that the SKCa2gene encodes the KCa channel in Jurkat T cells (13Jäger H. Adelman J.P. Grissmer S. FEBS Lett. 2000; 469: 196-202Crossref PubMed Scopus (44) Google Scholar,14Desai R. Peretz A. Idelson H. Lazarovici P. Attali B. J. Biol. Chem. 2000; 275: 39954-39963Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar).Table IIon channel selectivity of UCL drugsJurkat SKCahSKCa2hSKCa3mKv1.3T-cell hIKCa1hSlo (maxiK)hskm1(Na+ channel)UCL 16840.180.289.54900>5000>5000>5000UCL 153030NDND21004700NDNDUCL 18480.24NDNDNDNDNDNDUCL 20790.35NDND>5000>5000NDNDIC50 values (in nm) of bis-quinolinium cyclophane derivatives were determined using whole cell recordings from cell lines expressing native or transfected ion channels. hSKCa2 and hSKCa3 are the human SKCa2 and SKCa3genes, respectively, transiently expressed in COS-7 cells. mKv1.3 is the mouse lymphocyte voltage-gated K+channel stably expressed in L929 cells (12Grissmer S. Nguyen A.N. Aiyar J. Hanson D.C. Mather R.J. Gutman G.A. Karmilowicz M.J. Auperin D.D. Chandy K.G. Mol. Pharmacol. 1994; 45: 1227-1234PubMed Google Scholar). hSlo is the large-conductance human KCa channel, and hskm1 is a human skeletal muscle Na+ channel (see under “Experimental Procedures”). IC50 values for drugs were estimated based on fits to the Hill equation. ND, not determined. Open table in a new tab We used a pharmacological approach to discern the contributions ofKv1.3 and SKCa2 channels to calcium signaling in Jurkat T-cells. Ca2+ signaling can be induced by thapsigargin (Tg), a specific inhibitor of the sarco-endoplasmic reticulum Ca2+ ATPase that enables study of Ca2+ entry independent of Ca2+ release from internal stores (34Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (2986) Google Scholar). Depletion of intracellular Ca2+stores with Tg in the absence of extracellular Ca2+activates CRAC channels in the plasma membrane, permitting Ca2+ entry upon reintroduction (35Fanger C.M. Hoth M. Crabtree G.R. Lewis R.S. J. Cell Biol. 1995; 131: 655-667Crossref PubMed Scopus (164) Google Scholar). In Jurkat T cells, the KCa channel inhibitor UCL 1684 reduced Ca2+influx, but not the Tg-induced release of intracellular Ca2+ stores (Fig.2 A), in a dose-dependent manner that closely paralleled the dose-dependent block of the native KCa current (Fig. 2 B). Maximal inhibition of the [Ca2+]i plateau was achieved at 10 nmUCL 1684, a dosage that blocked 95 ± 5% of Jurkat KCa channels. This dosage of UCL 1684 had no effect on IL-2 production in Jurkat cells but inhibited IL-8 production by 30% (data not shown). In contrast, application of the Kv1.3 blocker ShK-Dap22 (up to 10 nm) did not affect the Ca2+ response or cytokine production (Fig. 2 Aand data not shown). These results demonstrate that KCachannels but not KV channels help maintain Jurkat cell Ca2+ entry.Figure 2KCa (SKCa2) but not KV block reduces Ca2+ influx in Jurkat T cells. In Ca2+ imaging experiments, fura-2-loaded Jurkat cells and human T cells were stimulated with 1 μmTg in 0-Ca2+ Ringer solution (see bars above panels) in the presence or absence of UCL 1684 or ShK-Dap22. After ∼8 min, normal Ringer solution (2 mm Ca2+) was reintroduced, causing a rapid, sustained Ca2+ influx. A, Ca2+responses of Jurkat cells stimulated with Tg in the presence of varying doses of UCL 1684 (numbers), of 10 nmShK-Dap22, or no drug (control). Eachtrace represents the average response of ∼100 cells from a typical experiment. Combined addition of ShK-Dap22 and UCL 1684 had little or no added effect. B, comparison of the dose-response curve from patch clamp experiments shown in Fig.1 C (solid line) with the plateau Ca2+values from A (filled circles). C,membrane potential was monitored during stimulation in the presence (dotted trace, 64 cells) or in the absence (solid trace, 45 cells) of UCL 1684 (10 nm). Increasing DiBAC4(3) fluorescence intensity indicates depolarization of the membrane potential. Tg (1 μm) was used in a stimulation protocol identical to that used in A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The most likely mechanism by which UCL 1684 inhibits Ca2+entry is depolarization of the membrane potential resulting from Ca2+ entry through CRAC channels in the absence of KCa channel function. To test this possibility, the membrane potential of Jurkat T cells was monitored using a voltage-sensitive dye during the same Tg stimulation protocol used in Fig. 2 A. Treatment with Tg in the absence of extracellular Ca2+ caused a moderate depolarization of most cells, followed by a marked hyperpolarization upon Ca2+ readdition (Fig. 2 C, solid line). This hyperpolarization must be caused by the opening of SKCa2 channels because cells treated with UCL 1684 instead showed profound membrane depolarization following Ca2+ reintroduction (Fig. 2 C, dotted line). Similar results were observed in perforated-patch current clamp recordings (data not shown). These results demonstrate the tight link between KCa channel opening, modulation of membrane potential, and regulation of Ca2+ entry.The Role of KCa Channels in Activated Normal Human T CellsActivated human T cells present an excellent system to further test the role of KCa channels in Ca2+regulation. Mitogen-activated human T cells and Jurkat cells express similar numbers of KV and KCa channels, suggesting that they may regulate calcium signaling in a similar manner. The KV channel in human lymphocytes and in Jurkat T cells is encoded by the Kv1.3 gene (32Grissmer S. Dethlefs B. Wasmuth J.J. Goldin A.L. Gutman G.A. Cahalan M.D. Chandy K.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9411-9415Crossref PubMed Scopus (169) Google Scholar, 36Cai, Y., Osbourne, P., North, R., Dooley, D., and Douglass, J. (1992)11, 163–172.Google Scholar). However, the KCa channel in human T cells is the product of theIKCa1 gene that is phylogenetically related to theSKCa2 gene found in Jurkat cells. Both KCachannels share a common calmodulin-dependent mechanism for calcium-dependent gating (29Fanger C.M. Ghanshani S. Logsdon N.J. Rauer H. Kalman K. Zhou J. Beckingham K. Chandy K.G. Cahalan M.D. Aiyar J. J. Biol. Chem. 1999; 274: 5746-5754Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 37Xia X.M. Fakler B. Rivard A. Wayman G. Johnson-Pais T. Keen J.E. Ishii T. Hirschberg B. Bond C.T. Lutsenko S. Maylie J. Adelman J.P. Nature. 1998; 395: 503-507Crossref PubMed Scopus (725) Google Scholar, 38Khanna R. Chang M.C. Joiner W.J. Kaczmarek L.K. Schlichter L.C. J. Biol. Chem. 1999; 274: 14838-14849Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), and both exhibit a conserved genomic organization (39Ghanshani S. Wulff H. Miller M.J. Rohm H. Neben A. Gutman G.A. Cahalan M.D. Chandy K.G. J. Biol. Chem. 2000; 275: 37137-37149Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar).To test whether IKCa1 and SKCa2 serve similar functions in sustaining Ca2+ signaling, we used agents that are selective for the block of the KCa channels found in mitogen-activated human T cells, including the charybdotoxin mutant ChTX-Glu32 and the clotrimazole analogue TRAM-34 (28Rauer H. Lanigan M.D. Pennington M.W. Aiyar J. Ghanshani S. Cahalan M.D. Norton R.S. Chandy K.G. J. Biol. Chem. 2000; 275: 1201-1208Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 40Wulff H. Miller M.J. Hansel W. Grissmer S. Cahalan M.D. Chandy K.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8151-8156Crossref PubMed Scopus (526) Google Scholar). We first verified that these blockers are selective forIKCa1 by testing them on this channel in an expressed system. As shown in Fig. 3 A, IKCa1 channels are blocked by ChTX-Glu32(IC50 = ∼30 nm) (28Rauer H. Lanigan M.D. Pennington M.W. Aiyar J. Ghanshani S. Cahalan M.D. Norton R.S. Chandy K.G. J. Biol. Chem. 2000; 275: 1201-1208Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Neither the UCL compounds (10 nm) nor ShK-Dap22 (250 nm) affected the IKCa1 channel (Fig.3 A and data not shown). The triarylmethane blocker ofIKCa1, TRAM-34 (IC50 = ∼20 nm), also blocks native or expressed IKCa1 current with high selectivity (data not shown) (40Wulff H. Miller M.J. Hansel W. Grissmer S." @default.
• W2035798975 created "2016-06-24" @default.
• W2035798975 creator A5005335169 @default.
• W2035798975 creator A5007974371 @default.
• W2035798975 creator A5008223387 @default.
• W2035798975 creator A5012380551 @default.
• W2035798975 creator A5017121030 @default.
• W2035798975 creator A5026660883 @default.
• W2035798975 creator A5043567383 @default.
• W2035798975 creator A5056296835 @default.
• W2035798975 creator A5076121043 @default.
• W2035798975 creator A5090685833 @default.
• W2035798975 date "2001-04-01" @default.
• W2035798975 modified "2023-10-13" @default.
• W2035798975 title "Calcium-activated Potassium Channels Sustain Calcium Signaling in T Lymphocytes" @default.
• W2035798975 cites W1490915066 @default.
• W2035798975 cites W1556269802 @default.
• W2035798975 cites W1562441478 @default.
• W2035798975 cites W1573590097 @default.
• W2035798975 cites W1601157315 @default.
• W2035798975 cites W1637699187 @default.
• W2035798975 cites W1680442092 @default.
• W2035798975 cites W1970249433 @default.
• W2035798975 cites W1986433722 @default.
• W2035798975 cites W1991976286 @default.
• W2035798975 cites W1997867282 @default.
• W2035798975 cites W2016204104 @default.
• W2035798975 cites W2017610520 @default.
• W2035798975 cites W2019384325 @default.
• W2035798975 cites W2019863974 @default.
• W2035798975 cites W2020477012 @default.
• W2035798975 cites W2027030217 @default.
• W2035798975 cites W2034245104 @default.
• W2035798975 cites W2037263968 @default.
• W2035798975 cites W2041266960 @default.
• W2035798975 cites W2051040059 @default.
• W2035798975 cites W2051231103 @default.
• W2035798975 cites W2052124270 @default.
• W2035798975 cites W2053132428 @default.
• W2035798975 cites W2053546226 @default.
• W2035798975 cites W2063535351 @default.
• W2035798975 cites W2067967075 @default.
• W2035798975 cites W2076882136 @default.
• W2035798975 cites W2082502466 @default.
• W2035798975 cites W2089329518 @default.
• W2035798975 cites W2089991403 @default.
• W2035798975 cites W2091222105 @default.
• W2035798975 cites W2094401453 @default.
• W2035798975 cites W2108442939 @default.
• W2035798975 cites W2108774434 @default.
• W2035798975 cites W2117176042 @default.
• W2035798975 cites W2118593499 @default.
• W2035798975 cites W2125469822 @default.
• W2035798975 cites W2133926860 @default.
• W2035798975 cites W2139312497 @default.
• W2035798975 cites W2140707292 @default.
• W2035798975 cites W2141578443 @default.
• W2035798975 cites W2150769903 @default.
• W2035798975 cites W2152148059 @default.
• W2035798975 cites W2161387630 @default.
• W2035798975 cites W2168393307 @default.
• W2035798975 cites W2949872895 @default.
• W2035798975 cites W2951870536 @default.
• W2035798975 doi "https://doi.org/10.1074/jbc.m011342200" @default.
• W2035798975 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11278890" @default.
• W2035798975 hasPublicationYear "2001" @default.
• W2035798975 type Work @default.
• W2035798975 sameAs 2035798975 @default.
• W2035798975 citedByCount "159" @default.
• W2035798975 countsByYear W20357989752012 @default.
• W2035798975 countsByYear W20357989752013 @default.
• W2035798975 countsByYear W20357989752014 @default.
• W2035798975 countsByYear W20357989752015 @default.
• W2035798975 countsByYear W20357989752016 @default.
• W2035798975 countsByYear W20357989752017 @default.
• W2035798975 countsByYear W20357989752018 @default.
• W2035798975 countsByYear W20357989752019 @default.
• W2035798975 countsByYear W20357989752020 @default.
• W2035798975 countsByYear W20357989752021 @default.
• W2035798975 countsByYear W20357989752022 @default.
• W2035798975 countsByYear W20357989752023 @default.
• W2035798975 crossrefType "journal-article" @default.
• W2035798975 hasAuthorship W2035798975A5005335169 @default.
• W2035798975 hasAuthorship W2035798975A5007974371 @default.
• W2035798975 hasAuthorship W2035798975A5008223387 @default.
• W2035798975 hasAuthorship W2035798975A5012380551 @default.
• W2035798975 hasAuthorship W2035798975A5017121030 @default.
• W2035798975 hasAuthorship W2035798975A5026660883 @default.
• W2035798975 hasAuthorship W2035798975A5043567383 @default.
• W2035798975 hasAuthorship W2035798975A5056296835 @default.
• W2035798975 hasAuthorship W2035798975A5076121043 @default.
• W2035798975 hasAuthorship W2035798975A5090685833 @default.
• W2035798975 hasBestOaLocation W20357989751 @default.
• W2035798975 hasConcept C12554922 @default.
• W2035798975 hasConcept C149614440 @default.
• W2035798975 hasConcept C178790620 @default.
• W2035798975 hasConcept C185592680 @default.
• W2035798975 hasConcept C201571599 @default.

• next