Eur. J. Immunol. 2008. 38: 2548–2556 Leukocyte signalling 2553 Figure 6. Inhibition of HLMC chemotaxis through EP2 receptor activation. (A) HLMC migration using conditioned medium from asthmatic airway smooth muscle as the chemotactic stimulus. (B) HLMC migration is attenuated signiﬁcantly by butaprost (n 5 4) but not PGE2 (n 5 3) in the presence of ASM-conditioned medium. po0.05 compared with control (no PGE2 or butaprost). (C) PGE2 is chemotactic when present in isolation. po0.05 compared to control (no PGE2), n 5 4. (D) PGE2-dependent chemotaxis is enhanced in the presence of the EP1/2 receptor antagonist AH6809 (10 5 M). po0.05 compared to control (no AH6809), n 5 4. quence masks the potential chemotactic activity of PGE2 on HLMC, which is particularly evident when the EP2 receptor is blocked pharmacologically. PGE2 closed KCa3.1 reversibly following IgE-dependent mast cell activation demonstrating physiological relevance. PGE2 also closed KCa3.1 channels that had been activated by the KCa3.1 opener 1-EBIO. The inhibition of KCa3.1 by PGE2 was reversed both by removing it from the recording solution, and by the addition of the competitive EP1 and EP2 receptor antagonist AH6809 indicating a receptor-mediated mechanism. Since the effects of PGE2 were mimicked by the speciﬁc EP2 receptor agonist butaprost but not the EP1 agonist 17-phenyl trinor PGE2, and no effects of native PGE2 were seen in the presence of the EP1/2 receptor antagonist AH6809, we have ﬁrm evidence that the suppression of KCa3.1 by PGE2 is mediated via the EP2 receptor. Because 1-EBIO opens KCa3.1 directly in resting cells by increasing its afﬁnity for Ca21 [19], it suggests that there is tight coupling between the EP2 receptor and the channel rather than modulation of intracellular signalling pathways. The ability of PGE2 to close KCa3.1 is in keeping with our previous observations that this channel is closed by Gs-coupled b2-adrenoceptors [9] and Gs-coupled adenosine A2A receptors [10]. These effects on KCa3.1 are not mimicked by cAMP analo- gues or the activator of adenylate cyclase forskolin [9], and considering they are seen in whole-cell conﬁguration of the patch-clamp technique, indicate that the most likely mechanism of action is membrane-delimited involving the Gas or bg subunits of these GPCR. This view is further supported by the observation that the b2-adrenoceptor inverse agonist ICI-118551 actually opens KCa3.1 [9], but Gi agonists such as CXCL10 and PAF, which lower intracellular cAMP, do not directly activate this channel [6]. & 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim The ability of GPCR to modify KCa3.1 appears to be limited to Gs-coupled receptors as we have not found any evidence that Gi, Gq, or G12/13-coupled receptors modify KCa3.1 activity. In parti- cular blockade of EP1 and EP2 in this study did not uncover any Gi-coupled EP3 effects, and GPCR agonists active on human mast cells such as CXCL10 (Gai) [6], PAF (Gi), LPA (Gq, 12/13 and i), and UTP (Gaq P2Y2 receptor) do not open or close KCa3.1 in HLMC. It is well established using cell attached, inside-out and outside patch-clamp recording that most classes of GPCR including Gas couple directly to ion channels through membrane- delimited mechanisms [21,22]. This may lead to either channel opening or channel closing depending on the channel and receptor, and may utilise either the Ga component or speciﬁc combinations of bg subunits [21, 22]. A further level of speciﬁcity between receptor and channel is likely to be achieved by the close approximation of these proteins in tight membrane-restricted signalling complexes. For example, the b2 adrenoceptor that can modify both KCa1.1 and voltage-gated Ca21 channel gating is associated with these two channels in a macromolecular complex held together by A-kinase-anchoring proteins [23]. It is therefore interesting to speculate that KCa3.1 also localises with the various Gas receptors that modify its function. This and the exact mechanism by which Gas modiﬁes KCa3.1 function will be an important area for future research. Several molecules that attenuate HLMC secretion including PGE2, adenosine and b2-adrenoceptor agonists increase intracel- lular cAMP [24]. The generally held view is that this increase in intracellular cAMP couples to inhibition of secretion, supported by the observation that cAMP analogues and non-speciﬁc inhi- bitors of adenylate cyclase can also attenuate secretion from HLMC [24]. However, no mechanism has been identiﬁed in mast cells that explains how increases in cAMP inhibit the secretory pathway, and the exclusive role of cAMP in the inhibition of www.eji-journal.eu