Eur. J. Immunol. 2008. 38: 2548–2556 Leukocyte signalling 2551 Figure 3. The effect of the EP1/2 receptor antagonist AH6809 on PGE2- and butaprost -dependent closure of KCa3.1. (A) Current–voltage curve demonstrating reversibility of KCa3.1 suppression by PGE2 following administration of the EP1/2 receptor antagonist AH6809. (B) KCa3.1 current measured at 140 mV after addition of 1-EBIO, suppression by 10 5 M PGE2 and then reversibility of suppression following addition of AH6809 (n 5 10 cells). (C) Whole-cell current reversal potential (Vm) after the addition of 1-EBIO, a depolarising positive shift in response to 10 5 M PGE2, and then following addition AH6809 (n 5 10 cells). (D) Current–voltage curve demonstrating reversibility of KCa3.1 suppression by butaprost following administration of AH6809. (E) KCa3.1 current measured at 140 mV after addition of 1-EBIO, suppression by 10 5 M butaprost and then reversibility of suppression following addition of AH6809 (n 5 12 cells). (F) Whole-cell current reversal potential (Vm) after the addition of 1-EBIO, a depolarising positive shift in response to 10 5 M PGE2, and then reversibility following addition AH6809 (n 5 12 cells). 1-EBIO (current at 140 mV post 1-EBIO 128.3794.9 pA, post 17-phenyl trinor PGE2 127.8790.4 pA, p 5 0.9567, n 5 6) and did not open KCa3.1 in resting cells (baseline current at 140 mV 5.7070.88 pA, current post 17-phenyl trinor PGE2 5.3670.949 pA, p 5 0.202, n 5 5). These results exclude a role for EP1, EP3 or EP4 receptors in KCa3.1 modulation. Taking the data together, it can be concluded that suppression of KCa3.1 by PGE2 is mediated by the EP2 receptor. PGE2 closes KCa3.1 following IgE-dependent activation 5.871.3 pA, baseline Vm 17.873.2 mV; post anti-IgE current 62.473.0 mV). PGE2 (10 5 M) suppressed 54.277.6 pA, Vm the current to 34.374.9 pA post PGE2, p 5 0.005) (Fig. 5A and B) and produced an associated positive shift in Vm to 52.274.1 mV, p 5 0.051) (Fig. 5C). This suppressive effect of PGE2 was partially reversed by AH6809 (current 48.376.9 pA, p 5 0.011; Vm 66.674.0 mV, p 5 0.041) (Fig. 5A and C). PGE2 suppresses HLMC migration through the EP2 receptor Lastly, we conﬁrmed whether PGE2-dependent regulation of KCa3.1 was relevant to KCa3.1 channels that had been opened by anti-IgE-dependent activation. Anti-IgE (1:1000 dilution) opened KCa3.1 in 5/5 cells tested (baseline current at 140 mV Conditioned medium from asthmatic ASM, which has been activated with TNFa, IFNg and IL-1b, mediates HLMC chemotaxis predominantly via the CXCL10/CXCR3 pathway with additional contributions from ligands for CXCR1 and CXCR3 [20]. Inhibition & 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu