Direct Modulation of Calcium- and Voltage-Gated Potassium Channels of Large Conductance by Leukotrienes

Abstract

Calcium/voltage-gated, large-conductance potassium (BK) channels critically control smooth muscle contractility. Modulation of BK by direct interaction between lipids and channel protein has received increasing attention. Leukotrienes (LTA4-E4) are lipid mediators of inflammation. We performed patch-clamp in Xenopus oocytes that co-expressed BK channel-forming (cbv1; AY330293) and accessory β1 (FJ154955) subunits cloned from rat vascular myocytes. Leukotrienes were applied at 0.1 nM-10 μM to either leaflet of cell-free membranes at a range of [Ca2+]i and transmembrane voltage. LTB4 reversibly increased BK steady-state activity (EC50=1nM; Emax ≈10nM), with physiological [Ca2+]i and voltages favoring this activation. Cbv1 or cbv1+β2 were LTB4-resistant. Computational docking placed LTB4 onto the cholane steroid-sensing region in the BK β1 transmembrane domain 2. Consistent with the idea that cholanes and LTB4 share the docking region, co-application of LTB4 and cholane did not further increase LTB4-induced BK channel activation. LTB4 failed to activate cholane-insensitive cbv1+β1T169A. Moreover, LTB4 failed to activate BK channel containing β1A176S or β1K179I. Ala176 and Lys179 are computationally predicted to participate in LTB4-docking but were proven irrelevant for cholane-sensing. Thus, although cholanes and LTB4 share a docking region, LTB4 fit onto the sensing region is tighter and may explain higher potency of LTB4 to activate BK. Co-application of LTB4 with LTA4,LTC4,LTD4 or LTE4 suppressed LTB4-induced channel activation. Inactive leukotrienes only dock onto a portion of the site likely preventing LTB4's tight docking. In summary, we first document that two endogenous lipids from different chemical families (heterocyclic vs. linear) share their site on a channel protein. Thus, cross-talk between leukotrienes and cholanes might converge on regulation of smooth muscle contractility via BK β1. Moreover, identification of LTB4 as a potent ligand for BK channels may be important for developing β1-specific BK channel activators.