Activation of calcium - and voltage - gated potassium channels of large conductance by leukotriene B 4 * Anna

Calcium/voltage-gated, large-conductance potassium (BK) channels control numerous physiological processes, including myogenic tone. BK channel regulation by direct interaction between lipid and channel protein sites has received increasing attention. Leukotrienes (LTA4,LTB4,LTC4,LTD4, LTE4) are inflammatory lipid mediators. We performed patch-clamp studies in Xenopus oocytes that co-expressed BK channel-forming (cbv1) and accessory 1 subunits cloned from rat cerebral artery myocytes. Leukotrienes were applied at 0.1 nM10 M to either leaflet of cell-free membranes at a wide range of [Ca]i and voltages. Only LTB4 reversibly increased BK steady-state activity (EC50=1 nM; Emax reached at 10 nM), with physiological [Ca]i and voltages favoring this activation. Channels lacking 1 or containing 2 proteins were LTB4-resistant. Computational modeling predicted that LTB4 docked onto the cholane steroid-sensing site in the BK 1 transmembrane domain 2 (TM2). Co-application of LTB4 and cholane steroid did not further increase LTB4-induced activation. LTB4 failed to activate 1 subunitcontaining channels when 1 carried T169A, A176S, or K179I within the docking site. Coapplication of LTB4 with LTA4, LTC4, LTD4, or LTE4 suppressed LTB4-induced activation. Inactive leukotrienes docked onto a portion of the site, likely preventing tight docking of LTB4. In summary, we document the ability of two endogenous lipids from different chemical families to share their site of action on the channel protein. Thus, cross-talk between leukotrienes and cholane steroids might converge on regulation of smooth muscle contractility via BK 1. Moreover, identification of LTB4 as a highly potent ligand for BK channels is critical for the future development of 1-specific BK channel activators. http://www.jbc.org/cgi/doi/10.1074/jbc.M114.577825 The latest version is at JBC Papers in Press. Published on November 4, 2014 as Manuscript M114.577825 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from