Eur. J. Immunol. 2008. 38: 2548–2556 lung mast cells (HLMC) is closed by both salbutamol and adenosine via the b2-adrenoceptor and A2A adenosine receptors, respectively [9, 10], which are both Gs-coupled G protein-coupled receptors (GPCR). In keeping with a critical role for KCa3.1 in HLMC migration, adenosine also inhibits HLMC chemotaxis via the A2A receptor [10]. PGE2 is a prostanoid with four speciﬁc GPCR, designated EP1–4. EP2 and EP4 couple to Gs, EP3 couples predominantly to Gi although two isoforms also couple to Gs, while EP1 receptors mobilise intracellular Ca21, probably through Gq [11]. PGE2 therefore has diverse biological activities depending on the receptors it interacts with and the cells expressing them. With respect to mast cells, mouse mast cells express the EP3 receptor whose activation induces both degranulation and chemotaxis [12, 13]. Human cord blood-derived mast cells, express both EP2 and EP3 receptors. In these cells, PGE2 enhances degranulation and PGD2 production in cells primed by IL-4, an effect mediated through the EP3 receptor. However, following FceRI-dependent activation PGE2 inhibits human cord blood-derived mast cell LTC4, PGD2, IL-5 and TNF-a production via the EP2 receptor but has no effect on degranulation [14]. In contrast, several studies have shown that PGE2 consistently inhibits degranulation and eicosanoid production by HLMC, an effect mediated via the EP2 receptor [15–17]. This receptor also appears to dominate PGE2- dependent signalling on mast cells within the human asthmatic lung as PGE2 markedly attenuates both the early and late phase airway response to allergen challenge [18]. Since KCa3.1 in HLMC is closed by Gs-coupled b2-adrenoceptors and A2A adenosine receptors, we hypothesised that activation of the Gs-coupled EP2 receptor would also close KCa3.1 in these cells. Furthermore, if PGE2 was to close KCa3.1, then it should inhibit HLMC chemotaxis. To test this hypothesis we have used the patch- clamp technique to investigate the effects of PGE2 on HLMC ion channel function, and investigated the effect of PGE2 on HLMC migration induced by asthmatic ASM-conditioned medium. Results PGE2 alone does not open KCa3.1 We initially examined whether PGE2 opens KCa3.1 in HLMC under resting baseline conditions. HLMC are typically electrically silent at rest and it is possible that PGE2 could open either KCa3.1 or another channel as reported previously for adenosine [10]. No signiﬁcant change in current amplitude was observed in seven cells following the addition of 10 5 M PGE2 (current changed from 1.2270.54 to 1.4270.85 pA at 140 mV; p 5 0.678). PGE2 closes KCa3.1 in the presence of the KCa3.1 opener 1-EBIO We then examined whether PGE2 closes KCa3.1. Because PGE2 might potentially inhibit many IgE-dependent cell activation & 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Leukocyte signalling 2549 pathways that could reduce cytosolic-free Ca21, and thus reduce KCa3.1 activity indirectly, we concentrated on studying the effects of PGE2 on KCa3.1 currents that were induced by the KCa3.1 opener 1-EBIO [8–10]. This compound opens KCa3.1 with a half- maximal value of about 30 mM for heterologously expressed KCa3.1, with a maximal effect at about 300 mM [19]. 1-EBIO is speciﬁc for KCa3.1 in HLMC and opens it by enhancing the channels sensitivity to [Ca21]i [19]. Thus at 100 mM EBIO, maximal K1 currents are achieved in the presence of 100 nM free Ca21, which is below the resting [Ca21]i of most cell types including HLMC [8]. In cells in which KCa3.1 had been activated by 1-EBIO, addi- tion of PGE2 (10 8–10 5 M) produced a rapid (within 30 s) dose- responsive inhibition of channel activity with an associated positive shift in membrane potential (Fig. 1A–E). PGE2 10 5 M suppressed the KCa3.1 current in 490% of cells (Fig. 1B). Thus, addition of 10 5 M PGE2 reduced KCa3.1 membrane current at 140 mV from 155.4720.9 to 92.6713.7 pA (p 5 0.0001, n 5 22 cells)(Fig. 1B), with a corresponding shift in reversal potential (Vm) from 69.572.8 to 56.373.9 mV (p 5 0.0004) (Fig. 1C). Half maximal suppression (IC50) of KCa3.1 by PGE2 occurred at approximately 4.0 10 7 M (calculated from six cells). Impor- tantly, the effect of PGE2 was partially reversed within 1 min by removing it from the recording solution (current post PGE2 49.6720.8 pA, post wash 103.4734.5 pA, p 5 0.046; Vm post PGE2 44.1711.7 mV, post wash 72.176.81 mV, p 5 0.028, n 5 5) (Fig. 1D and E), indicating that non-speciﬁc ‘‘rundown’’ was not responsible for the effects seen. KCa3.1 modulation by PGE2 is mediated via EP2 receptors To examine whether the effects of PGE2 were mediated via EP2 prostanoid receptors we examined the effects of EP2 receptor agonists/antagonists. First, we examined the effects of the selective EP2 receptor agonist butaprost. Butaprost mimicked the effects of native PGE2 in a dose-dependent manner (Fig. 2A–D). At a concentration of 10 5 M, butaprost reduced the KCa3.1 current from 146.0718.3 pA to 61.276.1 pA (p 5 0.00006, n 5 20) (Fig. 2B) with a corresponding shift in reversal from 67.571.4 to 54.473.2 mV (p 5 0.00006) (Fig. 2C). Half maximal suppression (IC50) of KCa3.1 by butaprost occurred at approximately 2.1 10 7 M (n 5 6 cells) (Fig. 2D). The suppression of KCa3.1 by 10 5 M PGE2 was partially reversed by the competitive EP1 and EP2 receptor antagonist AH6809 (Fig. 3A–C). Thus, in experiments studying AH6809 at a concentration of 10 5 M, current at 140 mV was 112714.3 pA post PGE2, increasing to 160.9723.2 pA post AH6809 (p 5 0.011, n 5 10 cells) (Fig. 3B). There was however no signiﬁcant shift in reversal potential in these experiments explained by the fact that signiﬁcant KCa3.1 currents remained following PGE2 application (Vm post PGE2 60.972.1 mV, post AH6809 63.671.8 mV, p 5 0.17) (Fig. 3C). We also examined the effect of AH6809 on the selective EP2 agonist butaprost. The suppression of KCa3.1 by www.eji-journal.eu