Orai/CRACM1 and KCa3.1 ion channels interact in the human lung mast cell plasma membrane

Abstract

Background: Orai/CRACM1 ion channels provide the major Ca(2+) influx pathway for FcεRI-dependent human lung mast cell (HLMC) mediator release. The Ca(2+)-activated K(+) channel KCa3.1 modulates Ca(2+) influx and the secretory response through hyperpolarisation of the plasma membrane. We hypothesised that there is a close functional and spatiotemporal interaction between these Ca(2+)- and K(+)-selective channels.

Results: Activation of FcεRI-dependent HLMC KCa3.1 currents was dependent on the presence of extracellular Ca(2+), 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.

Conclusion: KCa3.1 activation in HLMCs is highly dependent on Ca(2+) influx through Orai1 channels, mediated via a close spatiotemporal interaction between the two channels.

Fig. 1

FcεRI-dependent HLMC K_Ca3.1 currents require the presence of external Ca²⁺. a Current–voltage curves from HLMCs activated via FcεRI in the presence external Ca²⁺ (2 mM). b Current–voltage curves from HLMCs activated via FcεRI in the absence of external Ca²⁺. Typical K_Ca3.1 currents appear in the presence of Ca²⁺ (2 mM) but not when it is absent. Small K_Ca3.1 currents appear in (b) when Ca²⁺ is then introduced. Data presented as mean ± SEM

Fig. 2

FcεRI- and 1-EBIO-dependent HLMC K_Ca3.1 currents are attenuated by an Orai channel blocker. a K_Ca3.1 currents induced following FcεRI-dependent activation are attenuated in HLMCs following addition of the Orai channel blocker GSK-7975A (n = 27 cells). b K_Ca3.1 currents induced following 1-EBIO-dependent activation are attenuated following addition of the Orai channel blocker GSK-7975A (n = 29 cells). Data presented as mean ± SEM. c Overexpressed K_Ca3.1 channels in HLMCs were constitutively active and were not blocked by GSK-7975A (1 μM)(n = 4 cells, p = 0.43 at +40 mV)

Fig. 3

FcεRI- and 1-EBIO-dependent HLMC K_Ca3.1 currents are inhibited by expression of an Orai1-E106Q dominant-negative mutant. Transduction of HLMCs with an Orai1-E106Q dominant-negative mutant ablated (a) 1-EBIO- dependent and (b) FcεRI-dependent K_Ca3.1 currents. For clarity, data are presented as the subtracted net activation-dependent currents (activation minus baseline) for each condition, expressed as mean ± SEM

Fig. 4

Orai1 and K_Ca3.1 proteins co-immunoprecipitate. a Western blots using either an antibody recognising the myc epitope (left) or an antibody recognising the FLAG epitope (right) of HEK293 cell lysates. Lysates expressed either myc epitope tagged Orai1, FLAG epitope-tagged K_Ca3.1, or both as indicated in the panel above. b Lysates of HEK293 cells expressing the indicated proteins were immunoprecipitated with an anti-myc antibody. Immunoprecipitates were then Western blotted using either an anti-Orai1 antibody (left) or an anti-FLAG antibody (right). c As (b) except cell lysates were immunoprecipitated with an anti-FLAG antibody and then Western blotted with an anti-FLAG antibody (left) or an anti-myc antibody (right). Blots shown are representative of 3 independent experiments

Fig. 5

Orai2 and K_Ca3.1 proteins do not co-immunoprecipitate under the conditions used to co-immunoprecipitate Orai1 and K_Ca3.1. a Western blots using either an antibody recognising the myc epitope (left) or an antibody recognising the FLAG epitope (right) of HEK293 cell lysates. Lysates expressed either myc epitope tagged Orai2, FLAG epitope-tagged K_Ca3.1, or both as indicated in the panel above. b HEK293 cell lysates expressing the proteins indicated in the panel above were immunoprecipitated with an anti-myc antibody. Immunoprecipitates were then Western blotted using either an anti-Orai2 antibody (left) or an anti-FLAG antibody (right). Control HEK293 cell lysate expressing K_Ca3.1-FLAG protein. c As (b) except cell lysates were immunoprecipitated with an anti-FLAG antibody and then Western blotted with an anti-Orai2 antibody (left) or an anti-FLAG antibody (right). Control HEK293 cell lysate expressing Orai2-myc protein. Blots shown are representative of 3 independent experiments

Fig. 6

Orai1 and K_Ca3.1 co-localise in the plasma membrane. a HEK293 cells, dually transfected with FLAG-tagged K_Ca3.1 and myc-tagged Orai1 and then immunostained, show co-localisation in the plasma membrane by single plane confocal microscopy (top panels). Dually transfected HEK293 show negative staining for appropriate isotype controls (bottom panels): rabbit IgG control, dual stained with anti-myc, and mouse IgG1 control dual stained with anti-FLAG. b Fluorescence intensity plot shows increased fluorescence at the plasma membrane. myc-Orai1 is shown in green and FLAG-K_Ca3.1 in red. Arrows indicate increased fluorescence where the region of interest (ROI) intersects the plasma membrane. c HEK293 cells, dually transfected with FLAG-tagged K_Ca3.1 and myc-tagged Orai2 and then immunostained, show poor co-localisation in the plasma membrane by single plane confocal microscopy (top panels). Dually transfected HEK293 show negative staining for appropriate isotype controls (bottom panels): rabbit IgG control, dual stained with anti-myc, and mouse IgG1 control dual stained with anti-FLAG. d Fluorescence intensity plot shows poor co-localisation of K_Ca3.1 and Orai2 signals. myc-Orai2 is shown in green and FLAG-K_Ca3.1 in red. Scale bars are 10 μm