- Open Access
Orai/CRACM1 and KCa3.1 ion channels interact in the human lung mast cell plasma membrane
Cell Communication and Signaling volume 13, Article number: 32 (2015)
Orai/CRACM1 ion channels provide the major Ca2+ influx pathway for FcεRI-dependent human lung mast cell (HLMC) mediator release. The Ca2+-activated K+ channel KCa3.1 modulates Ca2+ influx and the secretory response through hyperpolarisation of the plasma membrane. We hypothesised that there is a close functional and spatiotemporal interaction between these Ca2+- and K+-selective channels.
Activation of FcεRI-dependent HLMC KCa3.1 currents was dependent on the presence of extracellular Ca2+, and attenuated in the presence of the selective Orai blocker GSK-7975A. Currents elicited by the KCa3.1 opener 1-EBIO were also attenuated by GSK-7975A. The Orai1 E106Q dominant-negative mutant ablated 1-EBIO and FcεRI-dependent KCa3.1 currents in HLMCs. Orai1 but not Orai2 was shown to co-immunoprecipitate with KCa3.1 when overexpressed in HEK293 cells, and Orai1 and KCa3.1 were seen to co-localise in the HEK293 plasma membrane using confocal microscopy.
KCa3.1 activation in HLMCs is highly dependent on Ca2+ influx through Orai1 channels, mediated via a close spatiotemporal interaction between the two channels.
The aberrant activation of mast cells via the high affinity IgE receptor FcεRI results in the release of preformed granule-derived mediators such as histamine and tryptase, the synthesis and release of lipid mediators such as leukotriene (LT)C4 and prostaglandin (PG)D2, and the generation of numerous cytokines, including IL-4, IL-5 and IL-13 . This process contributes to the pathophysiology of allergic diseases such as asthma, rhinitis, urticaria and anaphylaxis .
The influx of extracellular Ca2+ is an essential requirement for the FcεRI-dependent release of mast cell mediators . Members of the recently discovered Orai (also known as CRACM) ion channel family provide the major pathway for this influx in both rodent and human lung mast cells (HLMCs) [3–5]. These channels carry Ca2+ selective currents (ICRAC) that are activated when endoplasmic reticulum (ER) Ca2+ stores are emptied. The family contains three members Orai1, Orai2 and Orai3 [6, 7]. They show a high degree of sequence homology but have distinct functional properties [6, 7]. The channel pore is contained within the Orai channel subunit [8–11]. An ER membrane protein, STIM1, acts as the sensor of the ER Ca2+ concentration and transmits this information to the channel pore . Human and rodent mast cells express all three Orai subunits at the mRNA level, although Orai1 is the dominant channel which is activated following cross-linking of FcεRI [3–5]. In HLMCs pharmacological block of Orai channels reduces Ca2+ influx, degranulation, LTC4 release and cytokine secretion . Similarly, Ca2+ influx, degranulation, LTC4 release and TNFα production are all greatly reduced in foetal liver-derived mast cells from an Orai1 knockout mouse .
Both human and rodent mast cells also express functional intermediate conductance Ca2+ activated K+ channels (KCa3.1) [13–15]. In human mast cells KCa3.1 constitutes the major K+ selective conductance [13, 14]. KCa3.1 channels are activated in mast cells by a rise in the cytosolic free Ca2+ concentration following FcεRI-dependent activation. Calmodulin, which is tightly bound near the C-terminus of the KCa3.1subunit, acts as the Ca2+ sensor. The key consequence of KCa3.1 opening during cell activation is the hyperpolarisation of the cell membrane. This maintains the electrical driving force for Ca2+ influx through store operated channels such as Orai, and enhances Ca2+ influx through Orai channels because they are strongly inwardly rectifying and therefore conduct larger currents at negative membrane potentials . Thus, activation of KCa3.1 channels using the KCa3.1 channel opener 1-ethyl-2-benzimidazolinone (1-EBIO) enhanced IgE-dependent Ca2+ influx and degranulation in HLMCs . In contrast IgE-dependent Ca2+ influx and degranulation was significantly reduced in bone-marrow derived mast cells isolated from a KCa3.1 knockout mouse . KCa3.1 has also been shown to be important for HLMC migration .
KCa3.1 channel activity disappears when extracellular Ca2+ is removed from cultured human mast cells that have been activated through FcεRI , and channel activity is not induced by several stimuli that release Ca2+ from intracellular stores but which do not stimulate Ca2+ influx . In addition, KCa3.1 currents were not elicited in HLMCs activated by the Ca2+ ionophore A23187 . This suggests that the activation of KCa3.1 channels by Ca2+ under physiological conditions relies on a tight spatiotemporal Ca2+ signal provide by Ca2+ influx channels. We therefore hypothesised that there is a close functional relationship between KCa3.1 and Orai channels in HLMCs, and that the two channels may interact physically to maintain tight spatiotemporal control of their activity. To test this hypothesis we have examined the effects of Orai channel inhibition in HLMCs on KCa3.1 channel activity and used co-immunoprecipitation and confocal microscopy to examine the physical interaction between KCa3.1 and Orai channels in co-transfected HEK293 cells.
KCa3.1 requires extracellular Ca2+ for activation by FcεRI cross-linking in HLMCs
HLMC activation via FcεRI stimulates the release of Ca2+ from intracellular stores followed by the influx of extracellular Ca2+ through Orai channels. Influx of extracellular Ca2+ is essential for HLMC degranulation and lipid mediator synthesis .
We showed previously that KCa3.1 currents elicited in human mast cells by FcεRI-dependent activation disappear on removal of extracellular Ca2+, demonstrating the need for extracellular Ca2+ influx for the maintenance of KCa3.1 activity . Here, activation of HLMCs by cross-linking FcεRI in the presence of extracellular Ca2+ evoked typical KCa3.1 currents as previously described in 81 % of cells studied, with a mean whole cell current at +40 mV in responding cells increasing from 6.8 ± 0.8 pA (reversal potential −31.4 ± 3.2 mV) at baseline to 75.6 ± 7.6 pA post anti-FcεRI (reversal potential −72.0 ± 9.3 mV) (n = 27 cells) (p < 0.0001 and p < 0.0001 for current and reversal potential respectively compared to baseline) (Fig. 1a). In contrast, in the absence of extracellular Ca2+, no KCa3.1 currents developed:current at +40 mV pre and post anti-FcεRI 4.5 ± 1.2 pA and 5.5 ± 1.1 pA respectively (p = 0.42), reversal potential pre and post anti-FcεRI −24.2 ± 1.8 mV and −22.0 ± 2.0 mV respectively (p = 0.71)(n = 9 cells)(Fig. 1b). Subsequent addition of extracellular Ca2+ (2 mM) to these cells following FcεRI-dependent activation induced only small KCa3.1 currents (11.0 ± 5.4 pA at +40 mV, p = 0.017 compared to no Ca2+) and a significant shift in reversal potential (−43.8 ± 7.4 mV, p = 0.015 compared to no Ca2+)(Fig. 1b). The small KCa3.1 currents seen here on subsequent addition Ca2+ here are in keeping with the known desensitisation of signalling pathways that occur when FcεRI is activated in the absence of extracellular Ca2+ [18, 19].
In summary, Ca2+ influx from the extracellular fluid is a critical requirement for the initial opening of KCa3.1 after FcεRI-dependent activation in HLMCs, as well as its maintenance following activation as described previously .
Orai channel block attenuates KCa3.1 activation
To assess whether Ca2+ influx through Orai channels contributes to KCa3.1 opening, we used the selective Orai channel blocker, GSK-7975A (a kind gift GlaxoSmithKline) [3, 20]. KCa3.1 currents elicited in response to FcεRI-dependent HLMC activation were significantly attenuated with the subsequent addition of 1 μM GSK-7975A, a concentration that suppresses Orai currents by >90 % in our hands  (Fig. 2a). Thus baseline whole cell currents of 6.8 ± 0.8 pA at +40 mV increased to 75.6 ± 7.6 pA post anti-FcεRI and were reduced to 40.9 ± 4.7 pA following addition of GSK-7975A (p < 0.0001, n = 27 cells). A small but significant positive shift in reversal potential was also evident with the addition of GSK-7975A (post anti-FcεRI −72.0 ± 9.3 mV, post GSK-7975A −61.3 ± 6.7 mV, p = 0.009) (Fig. 2a).
1-EBIO opens KCa3.1 by enhancing its sensitivity to [Ca2+]i. Thus at 100 μM EBIO, maximal K+ currents are achieved in the presence of 100 nM free Ca2+ , which is below the resting [Ca2+]i of most cell types including HLMCs . Interestingly, 1-EBIO-dependent KCa3.1 currents were also attenuated although to a lesser degree by GSK-7975A (Fig. 2b). Thus baseline whole cell currents of 7.4 ± 0.8 pA at +40 mV increased to 76.8 ± 8.8 pA post 1-EBIO, and were reduced to 59.4 ± 7.2 pA following addition of GSK-7975A (p < 0.0001, n = 29 cells). The reduction in KCa3.1 current induced by GSK-7975A following FcεRI-dependent HLMC activation was significantly greater than following 1-EBIO-dependent KCa3.1 activation (p = 0.039).
To confirm that GSK-7975A does not directly block KCa3.1 currents, GFP-KCa3.1 was overexpressed in HLMCs. This generated large (nA) constitutively active KCa3.1 currents that were not blocked by GSK-7975A (1 μM)(Fig. 2c).
The Orai1 E106Q dominant-negative mutant ablates KCa3.1 currents
To further investigate the role of Orai channels in the regulation of KCa3.1 in HLMCs, the effect of a dominant-negative mutant of Orai1 (E106Q) was assessed . While 7/7 GFP-transduced control HLMCs expressed robust KCa3.1 currents following exposure to 1-EBIO (net current at +40 mV 40.9 ± 19.5 pA, n = 7 cells), no KCa3.1 currents could be elicited in cells transduced with Orai1-E106Q (net current at +40 mV −1.1 pA, n = 6 cells, p = 0.04 compared to GFP) (Fig. 3a and b).
Similarly 8/8 GFP-transduced control HLMCs expressed robust KCa3.1 currents following exposure to anti-FcεRI (net current at +40 mV 121.6 ± 34.2 pA, n = 8 cells), but no FcεRI-dependent KCa3.1 currents could be elicited in cells transduced with Orai1-E106Q (net current at +40 mV 4.1 ± 3.2 pA, n = 9 cells, p = 0.0023 compared to GFP)(Fig. 3b).
Orai1 but not Orai2 co-immunoprecipitates with KCa3.1
The proposed functional interaction between Orai and KCa3.1 channels in HLMCs led us to investigate whether these channels interact physically. We therefore expressed myc epitope-tagged Orai1 and Orai2 and FLAG epitope-tagged KCa3.1 in HEK293 cells and tested for potential interactions by co-immunoprecipitation. Expression of epitope tagged channels was confirmed by Western blotting using antibodies raised against the appropriate epitope tag (Fig. 4a). Multiple bands were observed on blotting for KCa3.1-FLAG protein using an anti-FLAG antibody, with band sizes of 48 kDa (the predicted size of KCa3.1), and less. A similar band pattern has recently been observed on Western blotting for KCa3.1 in human fibrocytes, lung fibroblasts and airway smooth muscle cells, and may reflect differential splicing modification [22–24]. When an anti c-myc antibody was used to immunoprecipitate Orai1-myc protein, KCa3.1-FLAG was found to be co-immunoprecipitated (Fig. 4b). Co-immunoprecipitation of KCa3.1-FLAG was observed only from lysates of cells expressing both Orai1-myc and KCa3.1-FLAG proteins. Similarly when an anti-FLAG antibody was used to immunoprecipitate KCa3.1-FLAG protein, Orai1-myc protein was co-immunoprecipitated (Fig. 4c). Again co-immunoprecipitation was dependent on co-expression of both proteins in HEK293 cells.
In contrast, under identical reaction conditions, no co-immunoprecipitation of KCa3.1-FLAG protein was observed when the anti c-myc antibody was used to immunoprecipitate Orai2-myc protein (Fig. 5a and b). Similarly in the reverse experiment using the anti-FLAG antibody to immunoprecipitate KCa3.1-FLAG, no Orai2-myc protein was co-immunoprecipitated (Fig. 5c). We were unable to test for Orai3-myc and KCa3.1-FLAG co-immunoprecipitation, since we were unable to satisfactorily demonstrate co-expression of Orai3-myc and KCa3.1-FLAG protein in HEK293 cells (data not shown).
Orai1 and KCa3.1 co-localise in the plasma membrane of HEK-293 cells
To assess whether Orai1 and KCa3.1 co-localise in plasma membranes, we dually transfected HEK-293 with FLAG-tagged KCa3.1and myc-tagged Orai1 or Orai2 (HLMCs cannot be transfected efficiently). Subsequent analysis of the immunostained tags using confocal microscopy showed clear co-localisation of Orai1 and KCa3.1 in the HEK293 plasma membrane (Fig. 6a and b), but minimal co-localisation of KCa3.1 with Orai2 (Fig. 6c and d).
The release of mast cell mediators following cell activation requires the influx of extracellular Ca2+. We and others have shown that members of the Orai channel family play a major role in this Ca2+ influx in both human and rodent mast cells [3, 4]. The intermediate conductance Ca2+ activated K+ channel KCa3.1 is also a key regulator of Ca2+ influx into mast cells [13–15], as activated KCa3.1 channels maintain the driving force for Ca2+ entry by hyperpolarising the plasma membrane. Here we show for the first time that KCa3.1 activation has an obligate dependency on Ca2+ influx through Orai1 channels, which appears to be mediated via an intimate spatiotemporal interaction.
We have demonstrated previously that KCa3.1 opens in HLMCs following FcεRI-dependent activation, disappears on removal of extracellular Ca2+, and does not open after stimulation with other agonists which globally raise intracellular Ca2+ in HLMCs such as chemokines, PAF, LPA and the Ca2+ ionophore A23187 [13, 17, 25]. This suggested that tight Ca2+ microdomains regulate KCa3.1 activity. Here we found that activation of FcεRI-dependent KCa3.1 currents required the presence of extracellular Ca2+, were attenuated by a selective Orai channel blocker, and were ablated by transduction of a dominant-negative Orai1 mutant. This implies that Ca2+ engages KCa3.1 at the point of entry through Orai channels. The ability of KCa3.1 to co-immunoprecipitate and co-localise with Orai1, the dominant Orai channel in HLMCs, suggests the presence of a tight Orai1-KCa3.1 signalplex in the HLMC plasma membrane.
It is perhaps surprising that Orai1 interacted physically with KCa3.1 but Orai2 did not. However, Orai1 contains a C-terminal SHYA sequence which may act as a PDZ binding site. Although it does not conform to the class II or class III binding sites which are characterised by X-Hydrophobic-X-Hydrophobic or X-Neg charged-X-V/I/A, there is variation and other sequences will bind as well. If this is a PDZ binding site, then there is the potential for KCa3.1 and Orai1 to form part of a macromolecular complex organised by PDZ-containing scaffolding proteins. Orai1also interacts with TRPC family channels via a direct physical interaction at the N- and C-termini, but this is also true for Orai2 and Orai3 , so it seems unlikely that this is the mechanism mediating the interaction of Orai1 with KCa3.1. However, the N-terminus of Orai1 but not Orai3 forms a membrane-delimited signalplex with NFAT via the scaffolding protein AKAP79 . Further work will be required to establish the exact mechanism of the KCa3.1-Orai1 interaction.
Orai1 currents are only activated following store depletion by STIM1, and our data do not directly address whether STIM1 plays a role in the co-regulation of Orai1 and KCa3.1. However, the co-immunoprecipitation and confocal imaging experiments were performed in resting cells following over-expression, while the functional interaction is evident in lung mast cells following IgE-dependent activation, so it is likely that the interaction occurs independently of Ca2+ store depletion. This would be in keeping with previous studies showing that another Ca2+ activated K+ channel, KCa1.1 (bKCa), forms a signalling complex consisting of the β2-adrenoreceptor, the L-type calcium channel Cav1.2 and the A-kinase anchoring proteins AKAP79 and AKAP150 . The Ca2+required for activation of KCa1.1 is provided by voltage-gated Ca2+ channels, either Cav1.2, Cav2.1 or Cav2.2 . There is also limited evidence that KCa3.1 may form a signalplex with the β2-adrenoreceptor , so it will be interesting to assess whether a similar complex utilises KCa3.1 and Orai1.
A close association of KCa1.1 with voltage-gated Ca2+ channels is thought to be necessary because activation requires a relatively high intracellular Ca2+ concentration of 10 μM or more. Such concentrations are thought only to be found in so called Ca2+ nanodomains in the immediate vicinity of the open Cav channels. In contrast, KCa3.1 has a far higher affinity for Ca2+, with a value for half maximal activation (EC50) of approximately 0.3 μM . This high affinity has been considered to obviate the requirement for a close interaction with a Ca2+ source . However, it is clear that if KCa3.1 is coupled closely to a particular Ca2+ entry pathway and signalplex, there will be greater sensitivity and specificity with regards to KCa3.1 channel activation. Furthermore, Orai channels have a very low single channel conductance, estimated to be more than 100-fold lower than for voltage-gated Ca2+ channels [33, 34]. As a consequence the increase in Ca2+ concentration in the vicinity of an open Orai channel is likely to be relatively small. In mast cells the Ca2+ concentration has been estimated to be of the order of 2 μM at a distance of 10 nm from an open Orai channel, falling steeply with increasing distance . While clustering of (open) Orai channels in mast cells would lead to considerably higher local Ca2+concentrations, a close association of Orai1 with KCa3.1 in mast cells may therefore be required to ensure efficient activation of KCa3.1 channels and so regulation of Ca2+ influx.
The importance of Ca2+ microdomains for the regulation of specific Ca2+-dependent cell responses was highlighted recently by Parekh [35, 36]. For example there is evidence of distinct Orai channel signalling domains regulating the activity plasma membrane-associated enzymes such as Syk and cPLA2, and transcription factors such as NFAT [27, 37, 38]. A close KCa3.1-Orai1 interaction would therefore appear to make sense from several perspectives. The activation of KCa3.1 independently of global intracellular Ca2+ will fine-tune signals requiring Orai1 activation, with rapid and selective feedback ensuring that the Ca2+ signal is maintained, supporting Ca2+-dependent local membrane-associated signalling events. Furthermore, modulation of KCa3.1 by de-phosphorylation for example , or β2-adrenoceptor signalling , has the potential to feedback on Orai1 and limit Ca2+ entry. In resting cells where KCa3.1 and perhaps Orai1 expression may be relatively low , their co-localisation would ensure that efficient signalling could occur during an initial cell response. In addition, in cells expressing numerous KCa3.1 and Orai channels, there would be enhanced sensitivity to low grade stimuli, thus increasing the dynamic range, specificity and fidelity of the response to an external stimulus. It is also evident that KCa3.1 and Orai channels are localised to specific regions of activated cells. For example, localised KCa3.1 channel activity regulates shrinkage of the uropod in migrating cells even although the channels are distributed throughout the plasma membrane , while both Orai1 and KCa3.1 co-localise at the immunological synapse in activated T cells [42, 43]. Their ability to form a close physical interaction would ensure that they interact specifically in these regions. It is also clear that Orai1-3 have distinct physiological roles and electrophysiological characteristics [6, 44, 45]. A selective interaction between Orai1 and KCa3.1 would ensure that unwanted KCa3.1 activation does not occur in Orai2 or Orai3-dependent cell responses. The physiological relevance of this interpretation is supported by previous work in HLMCs. These cells express both Orai1 and Orai2 protein, but FcεRI-dependent degranulation and leukotriene release is driven largely by Orai1 .
There is a close spatio-temporal and functional interaction between KCa3.1 channels and Orai1 channels in HLMCs with evidence of a physical interaction leading to a restricted membrane-delimited signalplex. This is likely to facilitate the selective activation and fine-tuning of KCa3.1-Orai1-dependent cell processes.
Human mast cell purification and cell culture
HLMCs were purified from enzymatically dispersed healthy human lung obtained within one hour of surgery for lung cancer [46, 47]. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) containing 10 % foetal calf serum and 100 ng/ml stem cell factor, 50 ng/ml IL-6 and 10 ng/ml IL-10 as previously described . All human subjects donating lung tissue gave written informed consent and the study was approved by the Leicestershire Research Ethics Committee. Final mast cell purity was >99 %.
HEK293 cells were cultured in DMEM containing 10 % FCS.
Orai and KCa3.1 protein expression and co-immunoprecipitation
The construction of vectors directing the expression of c-Myc epitope-tagged Orai1 and Orai2 has been described previously . Briefly full length Orai1 and −2 cDNAs were cloned in frame immediately following the c-Myc epitope tag in vector pCruz Myc (Santa Cruz Biotechnology Inc). For the expression of FLAG epitope tagged KCa3.1, a PCR fragment containing the entire human KCa3.1open reading frame (ORF), an EcoR1 site immediately adjacent to the ATG initiation codon and an Xho1 site immediately adjacent to the stop codon was cloned into the EcoR1/Xho1 sites of vector pcDNA3 (Invitrogen). The stop codon was then removed by site directed mutagenesis. Oligonucleotides 5′ TCGAGGACTACAAAGACGATGACGACAAGTAGC 3′ and 5′ TCGAGCTACTTGTCGTCATCGTCTTTGTAGTCC 3′ encoding the FLAG eiptope (DYKDDDDK) were annealed together and inserted at the Xho1 site of the mutant KCa3.1 construct. The resulting vector directs the expression of KCa3.1 with a FLAG epitope in frame with the last codon of the KCa3.1 ORF. The construct was verified by DNA sequencing.
HEK293 cells were transiently transfected with vectors directing the expression of c-Myc epitope tagged Orai1 and −2 and/or FLAG epitope-tagged KCa3.1 using GeneJuice transfection reagent (Merck Bioscience Ltd). Cells were harvested for lysis 24 h later and lysed in a buffer containing 50 mM Tris pH8.0, 150 mM NaCl, 1 % Triton X-100 and a protease inhibitor cocktail (Sigma). Lysates were centrifuged at 13,200 rpm for 15 min at 4 °C and supernatants used for co-immunoprecipitation experiments.
For the immunoprecipitation of Orai1 or Orai2, supernatants were pre-cleared with protein G-sepharose beads and then incubated with a mouse monoclonal anti-c-Myc antibody (clone 9E10, Sigma) for 16–18 h at 4 °C. Immune complexes were recovered by incubation with protein G-sepharose beads for 15 min at 4 °C and washed 5 times with lysis buffer. Immunoprecipitation of KCa3.1 was as above except rabbit polyclonal anti-FLAG antibody (Sigma) and protein A-agarose were used.
Proteins were separated on 12 % Bis-Tris Nu-Page gels (Invitrogen) and then blotted onto polyvinylidene fluoride membranes. Membranes were blocked with 5 % nonfat milk in phosphate buffered saline. Blots were then probed with rabbit polyclonal antibodies recognising either Orai1 or Orai2 (both Alomone Labs Ltd) , anti-c-Myc antibody and/or anti-FLAG antibody as required. Blots were subsequently probed with Clean-Blot IP-horseradish peroxidase conjugate (Thermo Scientific). Immunoreactive bands were visualised using Pierce ECL Western Blotting Substrate (Fisher Scientific, Loughborough, United Kingdom).
HLMCs were transduced with adenoviruses expressing Orai E106Q and GFP control as described previously . HLMCs were also transduced with GFP-KCa3.1 using the same methodology.
Patch clamp electrophysiology
The whole-cell variant of the patch-clamp technique was used [3, 13]. Patch pipettes were made from borosilicate fibre-containing glass (Harvard Instruments, UK), and their tips were heat polished, typically resulting in resistances of 4–6 MΩ. The standard pipette solution contained (in mM): KCl (140), MgCl2 (2), HEPES (10), NaATP (2) GTP (0.1); pH 7.3 with KOH. The standard external solution contained (in mM), NaCl (140), KCl (5), CaCl2 (2), MgCl2 (1), HEPES (10) and glucose (5); pH 7.3 with NaOH. For recording, mast cells were placed in 35 mm dishes containing standard external solution.
Whole cell currents were recorded using an Axoclamp 200A amplifier (Axon Instruments, Foster City, CA, USA), and currents usually evoked by applying voltage commands to a range of potentials in 10 mV steps from a holding potential of −20 mV. The currents were digitised (sampled at a frequency of 10 kHz), stored on computer and subsequently analysed using pClamp10 software (Axon Instruments). Capacitance transients were minimised using the capacitance neutralisation circuits on the amplifier. Correction for series resistance was not routinely applied. Experiments were performed at 27 °C, temperature being controlled by a Peltier device. Drugs were added directly to the recording chamber as required.
HEK-293 cells (3×105) were seeded on 25 mm cover glasses and 24 h later dual-transfected with FLAG-tagged KCa3.1 and myc-tagged Orai1 or Orai2 using GeneJuice Transfection Reagent (Novagen). After 24 h, cells were methanol fixed and immunostained. The myc tag was detected with anti-myc 9E10 (gift from ADAS, Nottingham, UK) plus rabbit anti-mouse FITC (F0313, DAKO) alongside anti-mouse IgG1 (X0931, DAKO) isotype control. The FLAG tag was detected by anti-FLAG (F7425, Sigma) plus sheep anti-rabbit RPE (STAR35, AbD Serotec) alongside anti-rabbit IgG (550875, BD Pharmingen) isotype control. Fluorescence microscopy was performed using a Leica TCS SP5 confocal microscope and analysed with ImageJ software. Identical exposures were used for isotype controls.
Data were compared using paired or unpaired t test as appropriate. P < 0.05 was considered statistically significant.
Human lung mast cells
- IP3 :
Moiseeva EP, Bradding P. Mast cells in lung inflammation. Adv Exp Med Biol. 2011;716:235–69.
Church MK, Pao GJ, Holgate ST. Characterization of histamine secretion from mechanically dispersed human lung mast cells: effects of anti-IgE, calcium ionophore A23187, compound 48/80, and basic polypeptides. J Immunol. 1982;129:2116–21.
Ashmole I, Duffy SM, Leyland ML, Morrison VS, Begg M, Bradding P. CRACM/Orai ion channel expression and function in human lung mast cells. J Allergy Clin Immunol. 2012;129:1628–35.
Vig M, DeHaven WI, Bird GS, Billingsley JM, Wang H, Rao PE, et al. Defective mast cell effector functions in mice lacking the CRACM1 pore subunit of store-operated calcium release-activated calcium channels. Nat Immunol. 2008;9:89–96.
Ashmole I, Duffy SM, Leyland ML, Bradding P. The contribution of Orai(CRACM)1 and Orai(CRACM)2 channels in store-operated Ca2+ entry and mediator release in human lung mast cells. PLoS One. 2013;8:e74895.
DeHaven WI, Smyth JT, Boyles RR, Putney Jr JW. Calcium inhibition and calcium potentiation of Orai1, Orai2, and Orai3 calcium release-activated calcium channels. J Biol Chem. 2007;282:17548–56.
Lis A, Peinelt C, Beck A, Parvez S, Monteilh-Zoller M, Fleig A, et al. CRACM1, CRACM2, and CRACM3 are store-operated Ca2+ channels with distinct functional properties. Curr Biol. 2007;17:794–800.
Prakriya M, Feske S, Gwack Y, Srikanth S, Rao A, Hogan PG. Orai1 is an essential pore subunit of the CRAC channel. Nature. 2006;443:230–3.
Feske S, Gwack Y, Prakriya M, Srikanth S, Puppel SH, Tanasa B, et al. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature. 2006;441:179–85.
Yeromin AV, Zhang SL, Jiang W, Yu Y, Safrina O, Cahalan MD. Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature. 2006;443:226–9.
Vig M, Peinelt C, Beck A, Koomoa DL, Rabah D, Koblan-Huberson M, et al. CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry. Science. 2006;312:1220–3.
Zhang SL, Yu Y, Roos J, Kozak JA, Deerinck TJ, Ellisman MH, et al. STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature. 2005;437:902–5.
Duffy SM, Lawley WJ, Conley EC, Bradding P. Resting and activation-dependent ion channels in human mast cells. J Immunol. 2001;167:4261–70.
Duffy SM, Berger P, Cruse G, Yang W, Bolton SJ, Bradding P. The K+ channel IKCa1 potentiates Ca2+ influx and degranulation in human lung mast cells. J Allergy Clin Immunol. 2004;114:66–72.
Shumilina E, Lam RS, Wolbing F, Matzner N, Zemtsova IM, Sobiesiak M, et al. Blunted IgE-mediated activation of mast cells in mice lacking the Ca(2+)-activated K(+) channel K(Ca)3.1. J Immunol. 2008;180:8040–7.
Hoth M, Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature. 1992;355:353–6.
Cruse G, Duffy SM, Brightling CE, Bradding P. Functional KCa3.1 K+ channels are required for human lung mast cell migration. Thorax. 2006;61:880–5.
Ishizaka T, Sterk AR, Daeron M, Becker EL, Ishizaka K. Biochemical analysis of desensitization of mouse mast cells. J Immunol. 1985;135:492–501.
Gericke M, Dar O, Droogmans G, Pecht I, Nilius B. Immunological stimulation of single rat basophilic leukemia RBL-2H3 cells co-activates Ca(2+)-entry and K(+)-channels. Cell Calcium. 1995;17:71–83.
Derler I, Schindl R, Fritsch R, Heftberger P, Riedl MC, Begg M, et al. The action of selective CRAC channel blockers is affected by the Orai pore geometry. Cell Calcium. 2012;53:139–51.
Pedersen KA, Schroder RL, Skaaning-Jensen B, Strobaek D, Olesen SP, Christophersen P. Activation of the human intermediate-conductance Ca(2+)-activated K(+) channel by 1-ethyl-2-benzimidazolinone is strongly Ca(2+)-dependent. Biochim Biophys Acta. 1999;1420:231–40.
Cruse G, Singh SR, Duffy SM, Doe C, Saunders R, Brightling CE, et al. Functional K(Ca)3.1 K(+) channels are required for human fibrocyte migration. J Allergy Clin Immunol. 2011;128:1303–9.
Roach KM, Duffy SM, Coward W, Feghali-Bostwick C, Wulff H, Bradding P. The K(+) Channel KCa3.1 as a Novel Target for Idiopathic Pulmonary Fibrosis. PLoS One. 2013;8:e85244.
Chachi L, Shikotra A, Duffy SM, Tliba O, Brightling C, Bradding P, et al. Functional KCa3.1 channels regulate steroid insensitivity in bronchial smooth muscle cells. J Immunol. 2013;191:2624–36.
Duffy SM, Cruse G, Cockerill SL, Brightling CE, Bradding P. Engagement of the EP2 prostanoid receptor closes the K+ channel KCa3.1 in human lung mast cells and attenuates their migration. Eur J Immunol. 2008;38:2548–56.
Liao Y, Erxleben C, Yildirim E, Abramowitz J, Armstrong DL, Birnbaumer L. Orai proteins interact with TRPC channels and confer responsiveness to store depletion. Proc Natl Acad Sci U S A. 2007;104:4682–7.
Kar P, Samanta K, Kramer H, Morris O, Bakowski D, Parekh AB. Dynamic assembly of a membrane signaling complex enables selective activation of NFAT by Orai1. Curr Biol. 2014;24:1361–8.
Liu G, Shi J, Yang L, Cao L, Park SM, Cui J, et al. Assembly of a Ca2 + −dependent BK channel signaling complex by binding to beta2 adrenergic receptor. EMBO J. 2004;23:2196–205.
Berkefeld H, Sailer CA, Bildl W, Rohde V, Thumfart JO, Eble S, et al. BKCa-Cav channel complexes mediate rapid and localized Ca2 + −activated K+ signaling. Science. 2006;314:615–20.
Duffy SM, Cruse G, Lawley WJ, Bradding P. Beta2-adrenoceptor regulation of the K+ channel IKCa1 in human mast cells. FASEB J. 2005;19:1006–8.
Ishii TM, Silvia C, Hirschberg B, Bond CT, Adelman JP, Maylie J. A human intermediate conductance calcium-activated potassium channel. Proc Natl Acad Sci USA. 1997;94:11651–6.
Berkefeld H, Fakler B, Schulte U. Ca2 + −activated K+ channels: from protein complexes to function. Physiol Rev. 2010;90:1437–59.
Zweifach A, Lewis RS. Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores. Proc Natl Acad Sci U S A. 1993;90:6295–9.
Church PJ, Stanley EF. Single L-type calcium channel conductance with physiological levels of calcium in chick ciliary ganglion neurons. J Physiol. 1996;496(Pt 1):59–68.
Parekh AB. Ca2+ microdomains near plasma membrane Ca2+ channels: impact on cell function. J Physiol. 2008;586:3043–54.
Parekh AB, Muallem S. Ca(2+) signalling and gene regulation. Cell Calcium. 2011;49:279.
Ng SW, Di CJ, Singaravelu K, Parekh AB. Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels. J Biol Chem. 2008;283:31348–55.
Chang WC, Parekh AB. Close functional coupling between Ca2+ release-activated Ca2+ channels, arachidonic acid release, and leukotriene C4 secretion. J Biol Chem. 2004;279:29994–9.
Srivastava S, Li Z, Ko K, Choudhury P, Albaqumi M, Johnson AK, et al. Histidine phosphorylation of the potassium channel KCa3.1 by nucleoside diphosphate kinase B is required for activation of KCa3.1 and CD4 T cells. Mol Cell. 2006;24:665–75.
Shepherd MC, Duffy SM, Harris T, Cruse G, Schuliga M, Brightling CE, et al. KCa3.1 Ca2+ activated K+ channels regulate human airway smooth muscle proliferation. Am J Respir Cell Mol Biol. 2007;37:525–31.
Schwab A, Wulf A, Schulz C, Kessler W, Nechyporuk-Zloy V, Romer M, et al. Subcellular distribution of calcium-sensitive potassium channels (IK1) in migrating cells. J Cell Physiol. 2006;206:86–94.
Lioudyno MI, Kozak JA, Penna A, Safrina O, Zhang SL, Sen D, et al. Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation. Proc Natl Acad Sci U S A. 2008;105:2011–6.
Nicolaou SA, Neumeier L, Peng Y, Devor DC, Conforti L. The Ca(2+)-activated K(+) channel KCa3.1 compartmentalizes in the immunological synapse of human T lymphocytes. Am J Physiol Cell Physiol. 2007;292:C1431–9.
Motiani RK, Abdullaev IF, Trebak M. A novel native store-operated calcium channel encoded by Orai3: selective requirement of Orai3 versus Orai1 in estrogen receptor-positive versus estrogen receptor-negative breast cancer cells. J Biol Chem. 2010;285:19173–83.
Gwack Y, Srikanth S, Feske S, Cruz-Guilloty F, Oh-Hora M, Neems DS, et al. Biochemical and functional characterization of Orai proteins. J Biol Chem. 2007;282:16232–43.
Sanmugalingam D, Wardlaw AJ, Bradding P. Adhesion of human lung mast cells to bronchial epithelium: evidence for a novel carbohydrate-mediated mechanism. J Leuk Biol. 2000;68:38–46.
Duffy SM, Lawley WJ, Kaur D, Yang W, Bradding P. Inhibition of human mast cell proliferation and survival by tamoxifen in association with ion channel modulation. J Allergy Clin Immunol. 2003;112:970–7.
This work was supported by project grant no. 087499 from the Wellcome Trust, UK and was also supported in part by the National Institute for Health Research Leicester Respiratory Biomedical Research Unit. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. We thank Dr Malcom Begg for the supply of GSK-7975A. We thank the Advanced Imaging Facility at the University of Leicester and Dr Kees Straatman for support.
PB has received honoraria from GlaxoSmithKline for lectures at educational meetings, and has acted as a consultant for GlaxoSmithKline.
Conception and Design: SMD, IA, ML, PB. Acquisition of data: SMD (patch clamp electrophysiology), IA (molecular biology, immunoprecipitation and western blotting), DS (molecular biology, confocal microscopy). Analysis and interpretation of data: SMD, IA, DS, PB. Drafting of manuscript: IA, PB. Revision of manuscript for critical intellectual content: SMD, IA, DS, ML, PB. All authors read and approved the final manuscript.
S. Mark Duffy and Ian Ashmole contributed equally to this work.
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Duffy, S.M., Ashmole, I., Smallwood, D.T. et al. Orai/CRACM1 and KCa3.1 ion channels interact in the human lung mast cell plasma membrane. Cell Commun Signal 13, 32 (2015). https://doi.org/10.1186/s12964-015-0112-z
- Mast cell